CLÁUDIO AFONSO PINHO LOPES UTILIZAÇÃO DA BIOTÉCNICA … · clÁudio afonso pinho lopes...

UNIVERSIDADE ESTADUAL DO CEARÁ PRÓ-REITORIA DE PÓS-GRADUAÇÃO E PESQUISA

FACULDADE DE VETERINÁRIA PROGRAMA DE PÓS-GRADUAÇÃO EM CIÊNCIAS VETERINÁRIAS

CLÁUDIO AFONSO PINHO LOPES

UTILIZAÇÃO DA BIOTÉCNICA DE MANIPULAÇÃO DE OÓCITOS INCLUSOS EM FOLÍCULOS PRÉ-ANTRAIS PARA A AVALIAÇÃO

IN VITRO DO POTENCIAL DE ANTICORPOS ANTI-ZONA PELÚCIDA PARA A IMUNOESTERILIZAÇÃO DE CADELAS

FORTALEZA-CE 2008




Tese apresentada ao Programa de Pós-Graduação em Ciências Veterinárias da Faculdade de Veterinária da Universidade Estadual do Ceará, como requisito parcial para a obtenção do título de Doutor em Ciências Veterinárias. Área de Concentração: Reprodução e Sanidade Animal. Linha de Pesquisa: Reprodução e sanidade de carnívoros, onívoros, herbívoros e aves. Orientador: Prof. Dr. José Ricardo de Figueiredo

FORTALEZA-CE 2008




Tese apresentada ao Programa de Pós-Graduação em Ciências Veterinárias da Faculdade de Veterinária da Universidade Estadual do Ceará, como requisito parcial para a obtenção do título de Doutor em Ciências Veterinárias.

Aprovada em: _22_/_12_/_2008_ Conceito: Satisfatório “Com Louvor” Nota: 10 (Dez)

Banca Examinadora

_________________________________ Prof. Dr. José Ricardo de Figueiredo

Orientador – UECE

_______________________________ _______________________________ Profa. Dra. Maria Denise Lopes Profa. Dra. Lúcia Daniel Machado da Silva Examinadora-UNESP Examinadora-UECE

________________________________ ______________________________ Prof. Dr. Claudio Cabral Campello Prof. Dr. José Roberto Viana Silva Examinador-UECE Examinador-UFC

Aos animais

DEDICO

AGRADECIMENTOS

Agradeço à Universidade Estadual do Ceará (UECE) por toda a formação

profissional de qualidade proporcionada nos últimos doze anos, que culmina neste

momento com a conclusão do curso de doutorado em Ciências Veterinárias.

Ao Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

pela bolsa de estudos para a realização do curso de doutorado, que foi muito importante

para o êxito deste trabalho.

À Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) pela

bolsa para a realização de treinamento e estudos no exterior, que possibilitaram um

importante aprimoramento desta tese, além do incremento de minha qualificação e

experiência profissionais.

À Universidade de Brasília (UnB, Instituto de Biologia, Laboratório de

Microscopia Eletrônica) e ao Leibniz Institute for Zoo and Wildlife Research (IZW,

Berlim, Alemanha), pela oportunidade de treinamento e de realização de estudos.

Aos professores do Programa de Pós-Graduação em Ciências Veterinárias

(PPGCV) pelos ensinamentos e apoio prestados. Aos funcionários do PPGCV, e, em

especial, às secretárias Adriana Albuquerque e Cristina Sabóia do Nascimento, por toda

assistência sempre provida com atenção e cordialidade.

Agradeço a Deus pelo amor, força e orientação para prosseguir durante esta

jornada que se encerra e naquelas que estão por vir.

Aos meus amados pais Francisco Heine Maia Lopes e Suely Pinho Lopes por

todo o amor e dedicação sempre fundamentais.

À minha amada irmã Cláudia Valéria Pinho Lopes pelo amor, amizade,

orientação e alegria.

Ao meu querido orientador e amigo Prof. Dr. José Ricardo de Figueiredo por

todos os ensinamentos, pela confiança, pela atenção, pela compreensão, pelo apoio

incondicional, pela amizade, pelo exemplo de pesquisador competente e pessoa íntegra.

Aos Professores Dra. Sônia Nair Báo, Dra. Katarina Jewgenow e Dr. Claudio

Cabral Campello, pelos valiosos ensinamentos, pela confiança, pelo apoio e pela

amizade.

Aos Professores Dra. Maria Denise Lopes, Dra. Lúcia Daniel Machado da Silva

e Dr. José Roberto Viana Silva, por terem gentilmente aceito o convite para aprimorar

com sua competência este trabalho.

Aos companheiros do Laboratório de Manipulação de Oócitos e Folículos Pré-

antrais (LAMOFOPA) da UECE pela colaboração e pela amizade, e, em especial, a

Juliana Jalles de Holanda Celestino, Dra. Maria Helena Tavares de Matos, Márcia

Viviane Alves Saraiva, Ana Kelen Felipe Lima, Dra. Ana Paula Ribeiro Rodrigues,

Anelise Maria Costa Vasconcelos Alves, Mônica Aline Parente Melo, Fabrício de Sousa

Martins, Guilherme Ferreira de Sousa Meneses, Karla Daniely dos Santos Bastos, José

Erisvaldo Maia Júnior, Isabel Bezerra Lima-Verde, Dra. Liliam Mara Trevisan Tavares

e Jamily Bezerra Bruno.

Aos companheiros do Laboratório de Microscopia Eletrônica da UnB, Victor

Hugo da Silva Tibúrcio, Bruno Fiorillo, Bruno Arrivabene Cordeiro, Shélida Braz

Vasconcelos, Carolina Aquino Luque, Leonora Tavares Bastos, João Victor, Larissa

Cunha, Suzi, Elaine Porfírio, Débora, Khesller Name e Wellington.

Aos companheiros do IZW, Romy Waurich, Maxi Pöschmann, Birgit Bieber,

Karin Müller, Christiane Franz, Sigrid Holz, Jennifer Ringleb, Mirja Faβbender, Ulrike

Jakop, Sebastian Waldau, Antje Frank, Katrin Paschmionka, Marlies Rohleder, Beate

Braun, Yuyu Niu, Yvonne Meyer-Lucht, Aines Castro-Prieto, Rafael Prieto, Fabiano

Fernandes, Francisca Robles, Jan Axtner, Nina Schwensow, Jennifer Schön, Alexandra

Weyrich, Roland Frey, Simone Sommer, Joseph Saragusty, Heribert Hofer, Silke Ehle,

Beate Peters-Mergner, Wolfgang Richter, Steven Seet, Arne Ludwig, Wolfgang Tauche

e Steffen Berthold.

A todos que não foram aqui mencionados, mas que, direta ou indiretamente,

contribuíram para a realização deste trabalho.

RESUMO

O objetivo deste estudo foi avaliar o potencial de anticorpos anti-zona pelúcida para a

imunoesterilização de cães, através da análise da capacidade de antisoro produzido por

imunização de coelhos com zona pelúcida porcina (pZP) de promover a atresia de

folículos pré-antrais (FOPA). Para este propósito, utilizou-se a biotécnica de

manipulação de oócitos inclusos em folículos pré-antrais para se estabelecer um modelo

experimental in vitro, compreendendo métodos de preservação e cultivo in vitro de

FOPA caninos. Na Fase I, avaliaram-se os efeitos da temperatura (4, 20 ou 38°C), meio

(salina fisiológica ou Meio Essencial Mínimo – MEM) e tempo (2, 6, 12 ou 24 h) de

armazenamento sobre a morfologia e viabilidade folicular. As técnicas de histologia

clássica e microscopia eletrônica de transmissão, e a análise de viabilidade utilizando-se

Azul de Tripan e marcadores fluorescentes (calceína-AM e etídio homodímero-1) foram

utilizadas para essa finalidade. O uso de MEM a 4ºC apresentou-se como o método

mais eficiente, possibilitando a preservação de percentuais de folículos viáveis com

ultraestrutura íntegra similares ao controle fresco por até 12 h. Na Fase II, avaliou-se a

eficiência dos crioprotetores dimetil sulfóxido, etileno glicol, glicerol e 1,3-propanodiol

para a criopreservação de FOPA caninos através de congelação lenta. Observou-se que

o dimetil sulfóxido (DMSO) na concentração de 1,5 M possibilitou a preservação dos

maiores percentuais de FOPA viáveis e com ultra-estrutura intacta após a

descongelação. Na fase III, avaliou-se o efeito de soro anti-pZP sobre a viabilidade de

FOPA caninos cultivados in vitro durante 24 h, e também sobre a ligação de

espermatozóides caninos a óocitos homólogos. Utilizaram-se FOPA frescos ou

criopreservados com 1,5 M DMSO, que apresentaram resultados similares. Observou-se

que a adição de 10% de soro anti-pZP ao meio de cultivo não teve efeito sobre folículos

com diâmetro inferior a 100 µm em relação aos controles (ausência de soro ou adição de

10% de soro pré-imune). Entretanto, o soro anti-pZP causou a atresia de FOPA com

diâmetros superiores a 100 µm, conforme avaliado com calceína-AM e etídio

homodímero-1. Este soro a 10% mostrou-se capaz também de inibir a ligação de

espermatozóides à zona pelúcida canina. Desta forma, demonstrou-se in vitro o

potencial do soro anti-pZP para a imunoesterilização (através da eliminação de FOPA) e

para a imunocontracepção (através do bloqueio da ligação de espermatozóides aos

oócitos) de cães.

Palavras-chave: Imunocontracepção. Zona pelúcida. Folículos pré-antrais. Cães.

ABSTRACT

The aim of this study was do evaluate the potential of anti-zona pellucida antibodies for

immunosterilization in dogs, through analyzing the capability of anti-porcine zona

pellucida (pZP) antibodies to cause atresia of preantral follicles (PF). For this purpose,

the biotechnique of manipulation of oocytes enclosed in preantral follicles was

employed to establish an in vitro experimental model, comprising preservation and in

vitro culture methods for canine PF. In Phase I, the effects of storage temperature (4, 20

ou 38°C), medium (saline solution or Minimum Essential Medium – MEM) and time (2,

6, 12 ou 24 h) on follicular morphology and viability were evaluated. Classical

Histology and transmission electron microscopy, and viability analysis using Trypan

Blue and fluorescent labels (calcein-AM and ethidium homodimer-1) were used.

Storage in MEM at 4ºC was the most efficient method, which enabled preservation of

the percentages of viable follicles with intact ultrastructure similar to the fresh control

for up to 12 h. In Phase II, the efficiency of the cryoprotectants dimethyl sulfoxide,

ethylene glycol, glycerol and 1,2-propanediol for the cryopreservation of canine PF

through slow freezing was assessed. It was observed that dimethyl sulfoxide (DMSO) at

1.5 M provided the preservation of the highest percentages of viable PF with intact

ultrastructure after thawing. In phase III, the effects of anti-pZP serum on viability of

canine PF cultured in vitro for 24 h, and also on canine sperm binding to homologous

oocytes was evaluated. Fresh PF as well as PF cryopreserved in 1.5 M DMSO were

used and results between then were similar. Supplementation of culture medium with

10% anti-pZP serum had no effect on follicles smaller than 100 µm as compared to

controls (absence of serum or supplementation with pre-immune serum). However, anti-

pZP serum led PF with diameter larger than 100 µm to degenerate, as detected using

calcein-AM and ethidium homodimer-1. Anti-pZP serum also blocked sperm binding to

canine ZP. In conclusion, it was demonstrated in vitro the potential of anti-pZP serum

for immunosterilization (through induction of atresia to PF) and for

immunocontraception (through sperm binding blocking) in dogs.

Keywords: Immunocontraception. Zona pellucida. Preantral follicles. Dogs.

LISTA DE FIGURAS

Capítulo I

Figura 1. Esquema representativo do processo reprodutivo. 43

Capítulo II

Figura 1. Design of experiment I. 63

Figura 2. Histological features of stored canine ovarian fragments. 67

Figura 3. Effects of medium, temperature and time of storage on the

percentages of morphologically normal canine preantral

follicles. 68

Figura 4. Electron micrographs of normal follicles from control group and

stored in MEM at 4°C for 12 h. 70

Figura 5. Ultrastructure of follicles stored in MEM at 4°C for 24 h. 71

Figura 6. Electron micrographs of follicles stored in saline solution at 4°C

for 24 h. 72

Figura 7. Viability assessment of canine preantral follicles using trypan

blue dye exclusion test. 73

Figura 8. Viability assessment of canine preantral follicles using

fluorescent probes. 76

Figura 9. Percentages of viable canine preantral follicles in fresh ovaries

and after storage in MEM at 4ºC for 12 h or 24 h as assessed by

trypan blue dye exclusion test and labeling with calcein-AM and

ethidium homodimer. 77

Capítulo III

Figura 1. Percentages of morphologically normal preantral follicles in

ovarian tissue before (control) and after exposure and freezing-

thawing tests in medium containing 1.5 M EG or GLY. 90

Figura 2. Electron micrographs of canine preantral follicles from control

and frozen-thawed using 1.5 M EG or GLY. 93

Capítulo IV

Figura 1. Histological features of canine ovarian fragments before and

after freezing-thawing with 1.5 M DMSO or PROH. 108

Figura 2. Percentages of morphologically normal preantral follicles in

ovarian tissue before (fresh control) and after exposure and

freezing-thawing tests in medium containing 1.5 M DMSO or

PROH or without cryoprotectants (exposure and freezing

controls). 109

Figura 3. Electron micrographs of canine preantral follicles from control

and frozen-thawed using 1.5 M DMSO or PROH. 111

Figura 4. Viability assessment of canine preantral follicles using


Figura 5. Percentages of viable canine preantral follicles in fresh ovaries

and after exposure and freezing-thawing using 1.5 M DMSO as

assessed by trypan blue dye exclusion test and labeling with

calcein-AM and ethidium homodimer. 114

Capítulo V

Figura 1. Percentages of viable canine preantral follicles before (fresh

control time 0 h) and after in vitro culture for 24 h with in

medium containing no serum or 10% pre-immune serum

(control) or anti-pZP. 128

Figura 2. Viability evaluation of canine preantral follicles using


Figura 3. Assessment of anti-pZP antibodies ability to inhibit canine

oocyte-sperm interaction. 130

Figura 4. Assessment of the contraceptive efficiency of anti-porcine zona

pellucida (pZP) antibodies in dogs using an in vitro test for

canine sperm binding to homologous ZP. 131

LISTA DE TABELAS

Capítulo II

Tabela 1. Percentages (viable/total) of viable canine preantral follicles in

control and after storage. 74

Capítulo III

Tabela 1. Degenerated preantral follicles (%) in control and after exposure

and freezing-thawing tests. 91

Capítulo IV

Tabela 1. Percentages of viable follicles in the fresh control and after

exposure to cryoprotectants and slow freezing-thawing as

assessed using trypan blue. 112

LISTA DE ABREVIATURAS E SIGLAS

A Antro

ANOVA Analysis of variance (Análise de variância)

ATP Adenosina trifosfato

bm basement membrane (membrana basal)

BrdU Bromo-deoxiuridina

BSA Bovine serum albumin (Albumina sérica bovina)

CAPES Coordenação de Aperfeiçoamento de Pessoal de Nível Superior

CCZ Centro de Controle de Zoonoses

CG Células da granulosa

CGP Células germinativas primordiais

CNPq Conselho Nacional de Desenvolvimento Científico e Tecnológico

CT Células da teca

DMSO Dimetilsulfóxido

DNA Deoxyribonucleic acid (Ácido desoxirribonucléico)

EG Etilenoglicol

er endoplasmic reticulum (retículo endoplasmático)

FOPA Folículo ovariano pré-antral

FSH Follicle stimulating hormone (Hormônio folículo estimulante)

GC Granulosa cells (Células da granulosa)

GLY Glycerol (glicerol)

GnRH Gonadotrophin releasing hormone (Hormônio liberador de gonadotrofinas)

h Horas

HC Histologia clássica

HE Hematoxilina-eosina

ICC Instituto Central de Ciências

IVM in vitro maturation (maturação in vitro)

IZW Institut for Zoo and Wildlife Reseach

l Lipid droplet (gota lipídica)

L Litro

LAMOFOPA Laboratório de Manipulação de Oócitos e Folículos Ovarianos Pré-

Antrais

LFCR Laboratório de Fisiologia e Controle da Reprodução

M Molar

MEM Meio essencial mínimo

mL Mililitro

mM Milimolar

MNPF Morphologically normal preantral follicles (Folículos pré-antrais

morfologicamente normais)

MOIFOPA Manipulação de oócitos inclusos em folículos pré-antrais

mRNA Ácido ribonucléico mensageiro

n número de amostras

NUBIS Núcleo de Biotecnologia de Sobral

PAS Periodic Acid Schiff (Ácido periódico de Schiff)

PBS Phosphate-buffered saline (Salina tamponada com fosfato)

PROH 1,2-Propanediol (1,2-Propanodiol)

pZP Zona pelúcida porcina

OMS Organização Mundial da Saúde

RT Room temperature (temperatura ambiente)

sc stromal cells (células do estroma)

SEM Standard error of the mean (Erro padrão da média)

SNK Student-Newman-Keuls

TB Trypan Blue (Azul de Tripan)

TEM Transmission Electron Microscopy (Microscopia Eletrônica de

Transmissão)

UECE Universidade Estadual do Ceará

UFC Universidade Federal do Ceará

UnB Universidade de Brasília

v Vesicles (vesículas)

ZP Zona pellucida (Zona pelúcida)

μg Micrograma

μL Microlitro

μm Micrômetro

μM Micromolar

ºC Graus Celsius

SUMÁRIO

1. INTRODUÇÃO 16

2. REVISÃO DE LITERATURA

2.1 A superpopulação de cães 18

2.2 Contracepção para cadelas 19

2.3 Imunocontracepção 20

2.4 Ovário mamífero 23

2.5 Oogênese e foliculogênese 23

2.6 População folicular ovariana 24 2.7 A biotécnica de MOIFOPA 25 2.8 Preservação de FOPA durante o transporte de ovários 26

2.9 Criopreservação 27 2.10 Cultivo in vitro de folículos ovarianos 32

3. JUSTIFICATIVA 35 4. HIPÓTESES CIENTÍFICAS 37 5. OBJETIVOS 38 6. CAPÍTULO I

39

Imunocontracepção em mamíferos com ênfase no controle populacional de cães

7. CAPÍTULO II 58Preservação de folículos pré-antrais caninos por períodos curtos: efeitos da temperatura, meio e tempo

8. CAPÍTULO III 83Criopreservação de folículos pré-antrais caninos utilizando-se etilenoglicol e glicerol

9. CAPÍTULO IV 99Criopreservação bem-sucedida de folículos pré-antrais caninos utilizando-se dimetil sulfóxido e 1,3-propanodiol

10. CAPÍTULO V 121Potencial de anticorpos anti-zona pelúcida para a imunoesterilização de cães: efeitos sobre folículos pré-antrais in vitro

11. CONCLUSÕES 138 12. PERSPECTIVAS 139 13. REFERÊNCIAS BIBLIOGRÁFICAS 140

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1 INTRODUÇÃO

A superpopulação de cães constitui um grave problema em diversas cidades do

mundo, caracterizado pela existência de grandes conjuntos de animais sem

responsáveis, que podem compor um importante reservatório de zoonoses. Neste

contexto, as autoridades sanitárias realizam a eliminação sistemática destes animais

como estratégia de controle populacional. Todos os anos, esta situação resulta na

eutanásia de milhões de animais sadios, além do dispêndio de um elevado volume de

recursos econômicos estimados na ordem de bilhões de dólares (Frank e Carlisle-Frank,

2007). Esta abordagem, no entanto, é estritamente paliativa, por não atuar sobre a

origem do problema, que consiste em elevadas taxas de natalidade dos cães. Além

disso, a eliminação dos animais sem responsáveis, associada à sua má qualidade de

vida, faz emergirem questões éticas de bem-estar animal, que definem a necessidade de

novas estratégias de ação. Assim, um sistema de controle populacional para cães, para

ser efetivo, racional e humanitário, deve basear-se no controle de natalidade.

Apesar dos esforços despendidos nos últimos anos para o desenvolvimento de

métodos farmacológicos e químicos para a esterilização cães, as técnicas cirúrgicas têm

se mantido como os principais procedimentos para este propósito (Howe, 2006).

Entretanto, estes métodos demandam muito tempo e possuem alto custo financeiro para

aplicação em escala populacional (Kutzler e Wood, 2006), sendo assim logística e

economicamente inviáveis para uso em populações caninas numerosas, como no caso

das metrópoles.

Uma nova abordagem para o desenvolvimento de métodos contraceptivos,

denominada imunocontracepção, surge como uma importante perspectiva para o

controle reprodutivo canino em larga escala. Esta modalidade contraceptiva tem como

princípio induzir o sistema imunológico à síntese de anticorpos específicos para

moléculas bioativas do aparelho reprodutor que, uma vez revestidas por tais anticorpos,

têm sua função cessada, interrompendo-se assim o processo reprodutivo. Em

determinadas situações, pode ocorrer a eliminação da população de células germinativas

e conseqüente esterilidade, denominando-se este processo imunoesterilização.

Estudos têm demonstrado que a vacinação com proteínas da zona pelúcida (ZP),

estrutura que circunda os óocitos, resulta em infertilidade prolongada em diversas

espécies de mamíferos. Sugere-se que este efeito decorre do bloqueio da ZP à ligação de

17

espermatozóides pelos anticorpos produzidos. A resposta imunológica aos antígenos da

ZP pode ainda causar a eliminação dos oócitos (Ringleb et al., 2004).

Nesta tese, foi avaliado o potencial de anticorpos anti-ZP para a

imunoesterilização de cadelas, através da análise da capacidade de um antisoro

produzido por imunização de coelhos com ZP porcina (pZP) para promover a atresia de

folículos pré-antrais caninos. Para este propósito, a biotécnica de manipulação de

oócitos inclusos em folículos pré-antrais (MOIFOPA) foi utilizada para se estabelecer

um modelo experimental in vitro. Uma vez que os trabalhos relacionados ao uso desta

técnica em cães são bastante escassos e limitados à avaliação de métodos de maturação,

avaliaram-se neste estudo protocolos de preservação de folículos pré-antrais caninos

durante o período compreendido entre a coleta dos ovários e o uso no laboratório, além

da criopreservação utilizando-se diferentes crioprotetores para o armazenamento de

folículos a longo prazo. Por último, os folículos preservados pelos métodos

estabelecidos neste trabalho foram cultivados in vitro para a avaliação dos efeitos dos

anticorpos anti-ZP.

Para uma melhor compreensão dos estudos realizados nesta tese, segue-se uma

revisão de literatura abordando diversos temas relacionados à superpopulação de cães, à

imunocontracepção e à biotécnica de MOIFOPA.

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2 REVISÃO DE LITERATURA

2.1 A superpopulação de cães

A domesticação do cão ocorreu, segundo estudos arqueológicos, na Eurásia, há

cerca de 14.000 anos. Canídeos selvagens passaram a adentrar a área dos acampamentos

humanos, para se alimentarem com sobras de comida, o que fez com que

estabelecessem, paulatinamente, um convívio harmonioso com o homem. Em

contrapartida, esses animais passaram a auxiliar na defesa dessas comunidades e na

caça. Este vínculo de lealdade fora rompido unilateralmente pelo homem quando, tendo

adotado o padrão urbano de assentamento, passou a permitir a reprodução deliberada de

seus cães, não assumindo a responsabilidade pela vida dos animais das numerosas

ninhadas, que são abandonados nas ruas (World Society for the Protection of the

Animals, 1999).

2.1.1 Dinâmica da população de cães

O conjunto de cães sem responsáveis que vivem nos espaços públicos constitui

um importante reservatório de zoonoses, o que leva as autoridades de saúde a realizarem

a eliminação sistemática destes animais. Entretanto, de acordo com a Organização

Mundial da Saúde (OMS), não existe nenhuma prova de que a eliminação de cães possa

gerar um impacto significativo sobre as densidades populacionais desses animais

(World Health Organization, 1992). Assim, os sistemas de controle populacional de

cães baseados em apreensão sistemática e eutanásia em grande escala têm sido adotados

em várias partes do mundo erroneamente, em função do desconhecimento sobre a

composição e a dinâmica da população canina (World Health Organization, 1990).

Qualquer redução no tamanho da população canina, resultante do aumento da

taxa de mortalidade, é rapidamente compensada pelo aumento da taxa de sobrevivência

das frações remanescentes, que terão melhor acesso aos recursos ambientais disponíveis

(Wandeler et al., 1988). Além disso, a taxa de reposição de animais sobrepõe facilmente

a taxa de eliminação, sendo que, em relação a esta, a mais elevada registrada até hoje é

de aproximadamente 15% da população canina (World Health Organization, 1992).

As populações de cães errantes são mantidas a partir dos filhotes em excesso dos

animais domiciliados (que possuem elevadas taxas de sobrevivência, por receberem

abrigo e alimento antes de serem abandonados às ruas), e não pelas proles dos próprios

19

animais errantes, que apresentam taxas de sobrevivência muito reduzidas (Wandeler et

al., 1988). Desta forma, um sistema de controle populacional de cães, para ser efetivo,

deve basear-se na contenção da natalidade, através de programas de controle

reprodutivo.

Modelos matemáticos de dinâmica populacional demonstram que, para espécies

que se reproduzem através de sistemas poligâmicos de acasalamento, como a canina e a

felina, o controle da reprodução de 65% das fêmeas pode permitir a estabilização de

uma determinada população. Por outro lado, a contracepção direcionada aos machos

deve abranger mais de 95% desses animais para que se obtenha o mesmo efeito, o que é

inviável em termos práticos (Jewgenow et al., 2006). Neste contexto, sistemas de

manejo demográfico para cães devem ter como foco o controle reprodutivo das fêmeas.

2.2 Contracepção para cadelas

Os métodos contraceptivos atualmente disponíveis para o controle reprodutivo

canino não são adequados para utilização em escala populacional por razões

econômicas, operacionais, culturais e/ou de segurança.

O confinamento de cadelas durante o estro é considerado um método bastante

satisfatório, por sua simplicidade e por não envolver custo algum. Contudo, em várias

localidades, sua aplicação é dificultada por questões culturais, não se podendo contar

com a cooperação efetiva das comunidades, sobretudo as de baixa renda (World Health

Organization, 1992). Durante a execução de um programa de controle populacional de

cães e gatos realizado em vilas rurais do noroeste do Estado do Paraná, observou-se a

existência de um contigente substancial de pessoas que demonstraram pouca

preocupação com o controle reprodutivo de seus animais. Cerca de um terço dos

proprietários de animais de companhia dessas localidades não aderiu ao projeto, o que

sugere que a consciência acerca da importância da posse responsável depende do nível

educacional da comunidade (Molento et al., 2005).

O controle reprodutivo de cadelas pode ser realizado também através de métodos

farmacológicos. O uso de progestágenos, dentre os quais se destacam o megestrol, a

medroxiprogesterona e a proligestona, é o recurso mais utilizado. Estes compostos

consistem em esteróides sintéticos, que atuam através de retroalimentação negativa

sobre a secreção do hormônio liberador de gonadotrofinas (GnRH) e controle da

liberação das gonadotrofinas pela hipófise, com supressão da esteroidogênese e da

20

gametogênese (Rodrigues e Rodrigues, 2005). Diversos efeitos adversos têm sido

associados à utilização prolongada de progestágenos. A ocorrência de neoplasias

mamárias, alterações clínico-patológicas características de diabetes mellitus e letargia

foi reportada em cadelas após o uso de megestrol. Por sua vez, uma elevada incidência

de hiperplasia endometrial cística e supressão adrenocortical têm sido relatada em

conseqüência do tratamento com medroxiprogesterona (Kutzler e Wood, 2006). Selman

et al. (1995) descreveram alterações histopatológicas em diversos órgãos de cadelas

tratadas com proligestona, sendo notáveis a atrofia do córtex da glândula adrenal,

tumores mamários e vacuolização das células das ilhotas de Langerhans. Além disso,

Selman et al. (1996) demonstraram a afinidade da proligestona com receptores de

glicocorticóides, o que pode resultar em supressão do eixo hipotalâmico-hiposifário-

adrenocortical e resistência à insulina.

A esterilização cirúrgica de cadelas, comumente realizada através de

ovariohisterectomia, possui um custo financeiro elevado e demanda muito tempo, sendo

assim inadequada em termos operacionais para a aplicação em populações caninas

muito numerosas (Kutzler e Wood, 2006). Ademais, complicações pós-operatórias

como hemorragia, piometra e/ou granuloma do coto, fistulações, adesões peritoniais e

incontinência urinária têm sido relatadas, embora sejam facilmente prevenidas por uma

técnica cirúrgica realizada com rigor (Howe, 2006). Apesar de seu limitado potencial

para a redução das taxas de natalidade caninas, este método ainda se constitui na melhor

alternativa para o controle da reprodução de cães.

Neste contexto, faz-se urgente o desenvolvimento de agentes esterilizantes

inócuos injetáveis, para que seja possível o controle reprodutivo de cães de modo

efetivo (World Health Organization, 1992).

2.3 Imunocontracepção

A imunocontracepção consiste em induzir o sistema imunológico a neutralizar

elementos de importância estrutural e funcional envolvidos na reprodução. Antígenos

presentes na superfície dos espermatozóides, as proteínas da ZP, as gonadotrofinas

(FSH e LH), seus receptores específicos e seu hormônio liberador (GnRH) têm sido

utilizados como alvos para a elaboração de vacinas contraceptivas para carnívoros.

Atualmente, os melhores resultados têm sido obtidos através de imunização com ZP,

21

que tem se mostrado capaz de promover contracepção por longos períodos em diversas

espécies (Jewgenow et al., 2006).

A ZP consiste em uma matriz glicoprotéica extracelular que circunda os oócitos

de mamíferos e desempenha funções fundamentais no processo de fecundação através

da regulação da ligação de espermatozóides, indução da reação acrossômica e do

bloqueio à polispermia (Wassarman, 2008). Ao microscópio eletrônico, a ZP é vista

como uma rede entrelaçada com fenestrações, apresentando uma camada interna

compacta com pequenos poros, e uma camada externa frouxamente arranjada com

grandes poros (Greve e Wassarman, 1989).

A vacinação ativa com proteínas da ZP suprime a fertilidade em diversas

espécies por interferência na interação oócito-espermatozóides durante a fecundação,

além de afetar a população de folículos ovarianos em crescimento (Ringleb et al., 2004).

Diversos estudos têm demonstrado a ocorrência de alterações patológicas em folículos

após a vacinação com antígenos da ZP, sendo encontradas evidências que indicam o

envolvimento da resposta imunológica celular nesses processos (Millar et al., 1989;

Rhim et al.,1992; Lou and Tung, 1993; Jackson et al., 1998; Lou et al. 2000). Millar et

al. (1989), utilizando um anticorpo monoclonal para a ZP3 murina (mZP3) com

reconhecida capacidade de bloquear a fecundação, identificou uma seqüência de sete

aminoácidos na referida proteína, que consistiria de um epítopo para linfócitos B, e o

testou como antígeno imunocontraceptivo. Um peptídeo de 16 aminoácidos

incorporando a referida seqüência foi sintetizado e utilizado para imunizar ativamente

fêmeas de camundongo. Os anticorpos produzidos determinaram contracepção

duradoura na ausência de histopatologia do ovário e citotoxicidade celular. Estes autores

sugeriram que tais resultados deviam-se à ausência de epítopos para linfócitos T na

referida seqüência.

A redução do número de folículos primordiais após imunização ativa contra uma

das glicoproteínas zonárias (ZP3) é uma consequência comum (Aitken, 2002). Apesar

dos diversos indícios de mecanismos imunológicos celulares para a patogênese das

alterações de folículos ovarianos, em nenhum dos estudos em que foi verificada

depleção de folículos primordiais, observou-se a infiltração de linfócitos T (Kerr et al.,

1998). Uma explicação para esse fenômeno consiste no recrutamento acelerado de tais

folículos para compor a população de folículos em crescimento que é eliminada devido

à ação de anticorpos anti-ZP (Skinner et al., 1984). Alternativamente, sugere-se que

uma proporção significativa de folículos primordiais expressa a proteína ZP3, sendo

22

destruídos por anticorpos anti-ZP por fixação de complemento (Grootenhuis et al.,

1996). Gwatkin et al. (1977) localizaram anticorpos e complemento na ZP de oócitos de

camundongo inférteis imunizados com ZP de hamster. Outro possível mecanismo pode

consistir da interrupção da comunicação entre oócitos e células da granulosa via junções

do tipo ‘gap’ obstruídas por grandes quantidades de anticorpos anti-ZP aderidos ( Mahi-

Brown et al., 1988).

Barber et al. (2001) demonstraram através de imunohistoquímica ultra-estrutural

que anticorpos anti-ZP são capazes de se ligar a proteínas zonárias presentes no

aparelho de golgi de oócitos caninos em folículos pré-antrais. Além disso, a expressão

da proteína codificada pelo gene ZPA foi detectada no citoplasma de oócitos caninos

inclusos em folículos primordiais, enquanto em folículos primários e secundários foi

observada a transcrição de mRNA a partir dos genes ZPA, ZPB e ZPC (Blackmore et

al., 2004). Um padrão semelhante de expressão de proteínas da ZP foi descrito por

Jewgenow e Fickel (1999) em felinos. Assim, nestas duas espécies de carnívoros, os

mecanismos descritos acima para eliminação de folículos primordiais podem se

processar, estabelecendo-se assim o potencial de anticorpos anti-ZP para a

imunoesterilização desses animais.

Na maioria dos estudos sobre imunocontracepção baseada na ZP, as

glicoproteínas da pZP têm sido utilizadas devido à sua alta disponibilidade em

matadouros e ao elevado grau de homologia com as proteínas da ZP de diversas

espécies mamíferas, o que garante a reatividade cruzada dos anticorpos produzidas com

a ZP nativa (Niu et al., 2006).

Até o presente, o estudo da ação de anticorpos anti-ZP sobre oócitos tem sido

realizado utilizando-se apenas modelos experimentais in vivo. Entretanto, o uso de um

sistema de cultivo in vitro de folículos ovarianos pode proporcionar uma análise mais

precisa do processo de eliminação dos referidos gametas. Neste contexto, a biotécnica

MOIFOPA pode prover os métodos necessários ao desenvolvimento de um modelo

experimental in vitro para este propósito.

No capítulo I, constituído por um artigo de revisão de literatura sobre métodos

imunocontraceptivos, mais informações detalhadas acerca desta estratégia de

contracepção podem ser obtidas.

23

2.4 Ovário mamífero

O ovário mamífero é composto por muitos tipos de células diferenciadas, que

trabalham em conjunto promovendo um ambiente ideal para o desempenho de suas

funções endócrinas e gametogênica (Bristol-Gould e Woodruff, 2006). O ovário é

constituído por duas regiões: cortical e medular. O córtex ovariano, localizado na parte

mais externa, é circundado pelo epitélio germinal e corresponde à região funcional do

órgão, sendo formado por tecido conectivo (fibroblastos, colágeno e fibras reticulares),

folículos ovarianos e corpos lúteos em vários estádios de desenvolvimento ou em

regressão (Liu et al., 2006). A região medular, localizada mais internamente, é

constituída por tecido conjuntivo, células musculares lisas, nervos, vasos sanguíneos e

linfáticos responsáveis pela nutrição e estruturação do ovário (Hafez, 1996).

2.5 Oogênese e foliculogênese

A oogênese consiste na formação e diferenciação do gameta feminino que se

desenvolve em várias fases e tem início na vida fetal com as células germinativas

primordiais (CGP) e culmina com a formação do oócito haplóide fecundado (Bristol-

Gould e Woodruff, 2006). Em mamíferos, nos estádios iniciais do desenvolvimento

ovariano, ocorre a migração das células germinativas primordiais (CGP) do saco

vitelínico para a gônada primitiva com sua posterior colonização (Eppig et al., 2004).

Imediatamente após a diferenciação das gônadas, ocorre a transformação das CGP em

oogônias mitoticamente ativas e, então, em oócitos primários (Suh et al., 2002). Em

seguida, uma camada de células somáticas planas conhecidas como células da pré-

granulosa, originárias do epitélio celômico, circundam os oócitos primários formando

os folículos primordiais (Magoffin, 2005), iniciando assim a foliculogênese. Após a

formação dos folículos primordiais, as células da pré-granulosa param de se multiplicar

e entram num período de quiescência. Os oócitos primários inclusos nesses folículos

encontram-se na fase de prófase I da meiose. A progressão da divisão meiótica ocorre

somente na puberdade, com a liberação do pico pré-ovulatório de FSH e LH, formação

dos oócitos secundários e outra parada da meiose na fase de metáfase II (Gordon, 1994).

A meiose será retomada novamente somente após a fecundação do oócito pelo

espermatozóide, originando o oócito haplóide fecundado e marcando o fim da oogênese

(Figueiredo et al., 2008).

24

Desta forma, aos processos de formação, crescimento e maturação folicular dá-

se o nome de foliculogênese, que é iniciado com a formação do folículo primordial

culminando com o estádio de folículo pré-ovulatório (van den Hurk e Zhao, 2005). O

folículo ovariano é considerado a unidade funcional do ovário mamífero, cuja principal

função é proporcionar um ambiente ideal para o crescimento e maturação do oócito

(Cortvrindt e Smitz, 2001). Durante a foliculogênese, a morfologia folicular é alterada,

uma vez que o oócito cresce e as células circundantes se diferenciam (Bristol-Gould e

Woodruff, 2006). De acordo com o grau de evolução, os folículos podem ser

classificados em folículos pré-antrais (primordiais, em transição, primários e

secundários) e antrais (terciários e pré-ovulatórios) (Silva et al., 2004). Vale ressaltar

que os folículos pré-antrais (FOPA) representam 90% da população folicular e são

responsáveis pela renovação contínua de folículos antrais no ovário (Katska-

Ksiazkiewicz, 2006).

2.6 População folicular ovariana

Em muitos mamíferos, a população folicular ovariana é estabelecida ainda na

vida intra-uterina (primatas e ruminantes – Knight e Glister, 2006) ou em um curto

período de tempo após o nascimento (roedores – Ojeda et al., 2000). Por outro lado,

recentes trabalhos têm demonstrado mecanismos envolvidos na formação de novas

células germinativas e folículos em mulheres (Bukovsky et al., 2004) e camundongas

adultas (Johnson et al., 2004). Esses autores demonstraram indícios da continuidade da

oogênese e foliculogênese no período pós-natal, pela atuação de células-tronco. Para

Bukovsky et al. (2004), a renovação folicular pós-natal ocorre regularmente em

mulheres, mas a origem das células-tronco para tal não seria a medula óssea, e sim

células germinativas presentes na superfície do epitélio ovariano. No entanto, devido

aos resultados controversos até o momento, mais trabalhos sobre esse assunto são

necessários para esclarecer esse fenômeno.

A população folicular difere entre as espécies, além de ser observada uma

grande variação individual (Katska-Ksiazkiewicz, 2006), sendo de aproximadamente

1.500 na camundonga (Shaw et al., 2000); 35.000 na cabra (Lucci et al., 1999); 114.000

na gata (Lima, 2006); 160.000 na ovelha (Amorim et al., 2000); 235.000 na vaca

(Betteridge et al., 1989) e aproximadamente 2.000.000 na mulher (Erickson, 1986). Ao

longo da vida do animal, ocorre uma redução ordenada do número de FOPA (Shaw et

25

al., 2000). Essa redução deve-se a dois fenômenos que ocorrem naturalmente no ovário,

a ovulação e a atresia ou morte folicular. Somente uma pequena parte (0,1%) dos

folículos primordiais chega à ovulação (Nuttinck et al., 1993), enquanto os demais

tornam-se atrésicos durante as fases de crescimento e maturação oocitária (Otala et al.,

2002).

2.7 A biotécnica de MOIFOPA

A disponibilidade de oócitos é um fator limitante no desenvolvimento de novas

técnicas reprodutivas (Smitz e Cortvrindt, 2002). Os métodos atuais para a produção in

vitro de embriões dependem de uma oferta escassa de oócitos competentes de grandes

folículos antrais ou pré-ovulatórios, que estão presentes no ovário em número

relativamente reduzido (Telfer, 1998). Sabe-se que os FOPA representam cerca de 90%

de toda a população folicular, armazenando assim a grande maioria dos oócitos

presentes em ovários mamíferos (Silva et al., 2003). Dessa forma, a possibilidade de

desenvolver sistemas in vitro que explorem o grande número de oócitos provenientes de

folículos imaturos de ovários mamíferos deve ser considerada, destacando-se portanto a

biotécnica de MOIFOPA.

A MOIFOPA é uma biotécnica da reprodução que vem sendo aprimorada nos

últimos tempos e consiste em uma das principais ferramentas utilizadas atualmente para

a elucidação da foliculogênese inicial. Tal biotécnica consiste no isolamento,

conservação (resfriamento e criopreservação) e/ou cultivo in vitro de FOPA, visando a

estocagem, ativação, crescimento e maturação in vitro do folículo primordial até o

estádio pré-ovulatório (Figueiredo et al., 2008), prevenindo-se, assim, a ocorrência da

atresia. Assim, no futuro, será possível obter, de um único ovário, milhares de FOPA

que, cultivados e submetidos a outras biotécnicas da reprodução como a fecundação in

vitro e a clonagem, viabilizarão a produção in vitro de um grande número de embriões.

Destes, um número de crias saudáveis significativamente maior do que aquele obtido

através da reprodução natural também poderá ser alcançado. Na medicina veterinária, o

principal objetivo é aumentar a produtividade de animais de alto valor genético, ou

mesmo a preservação de espécies ameaçadas de extinção.

A biotécnica de MOIFOPA também representa uma excelente alternativa para

incrementar e auxiliar no desenvolvimento de pesquisas relacionadas à indústria

26

farmacêutica (Figueiredo et al., 2008) e à imunoesterilização utilizando-se anticorpos

anti-ZP.

2.8 Preservação de FOPA durante o transporte de ovários

A preservação da viabilidade e da capacidade de desenvolvimento dos FOPA

durante o período compreendido entre a coleta dos ovários e seu uso no laboratório é

um fator crítico em decorrência da condição de isquemia. Para que isto seja possível,

mesmo após longos períodos de transporte, como ocorre nos casos em que os animais

doadores se encontram em regiões bastante afastadas dos laboratórios de manipulação,

alguns fatores essenciais para a sobrevivência das células devem ser levados em

consideração, tais como o meio, a temperatura e o tempo de preservação (Santos et al.,

2004).

Diferentes meios têm sido utilizados para a preservação de FOPA, podendo ser

citados: TCM 199 (Costa et al., 2005), solução salina 0,9% (Matos et al., 2004;

Celestino et al., 2008), solução salina tamponada com fosfato (PBS) - (Santos et al.,

2002), solução Braun-Collins (Carvalho et al., 2001), solução à base de água de coco

(Lucci et al., 2004a) e Meio Essencial Mínimo (MEM) – (Chaves et al., 2008). Métodos

para armazenamento de ovários por curtos períodos já foram desenvolvidos para

caprinos (Silva et al., 2000), ovinos (Andrade et al., 2002a,b; Matos et al., 2004) e

bovinos (Lucci et al., 2004a; Celestino et al., 2007). Nesses estudos, as temperaturas de

4, 20 e 39°C foram testadas para a preservação de FOPA. Em geral, a temperatura mais

eficiente é a de 4°C, permitindo a preservação da morfologia e capacidade de FOPA

suínos de crescer in vitro após períodos de armazenamento de até 18 h, enquanto a

20°C, isto foi possível por apenas 6 h (Lucci et al., 2007). Em caprinos, apenas

temperatura de 4ºC permitiu preservar, após 4 h de armazenamento, a qualidade de

FOPA inclusos em fragmentos de córtex ovariano, que, após cultivo in vitro por sete

dias, apresentaram percentuais de folículos morfologicamente normais similares ao

controle fresco (Chaves et al., 2008).

Durante o transporte dos ovários para o laboratório, a privação de oxigênio do

tecido resulta em uma mudança do metabolismo aeróbico para anaeróbico, sendo o

produto principal, o ácido láctico, acumulado dentro da célula causando redução do pH

(Wongsrikeao et al., 2005). Com a intenção de se minimizarem os efeitos deletérios da

isquemia, o estabelecimento de hipotermia provoca uma redução do metabolismo

27

celular e, assim, dos requerimentos de energia e nutrientes, aumentando a resistência de

folículos durante a preservação in vitro (Silva et al., 2003). A preservação de FOPA por

períodos indeterminados pode ser obtida, por sua vez, através da utilização de técnicas

eficientes de criopreservação (Santos, 2005).

2.9 Criopreservação

2.9.1 Princípios básicos

A criopreservação consiste em um método de preservação de material biológico

a baixas temperaturas, geralmente em nitrogênio líquido a –196°C, ou em sua fase de

vapor a -150°C. Os únicos estados físicos existentes abaixo de aproximadamente

-130°C são o cristalino e o vítreo e, em ambos, a viscosidade é muito elevada, a difusão

é considerada insignificante (dependendo do tempo de armazenamento), a energia

cinética molecular é muito baixa e reações metabólicas impulsionadas por energia

térmica ocorrem muito lentamente ou são paralisadas completamente (Kartha, 1985).

Portanto, à temperatura do nitrogênio líquido, a viabilidade durante o armazenamento

pode ser estendida por longos períodos de tempo, com manutenção da estabilidade do

material genético (Stushnoff e Seufferheld, 1995). Para a criopreservação, faz-se

necessária a utilização de um ou mais componentes que confiram proteção às células

durante a congelação, conhecidos como agentes crioprotetores (Mullen, 2007). A

capacidade do material biológico de sobreviver ao processo de criopreservação depende

de sua tolerância aos agentes crioprotetores, à desidratação, ao resfriamento e ao re-

aquecimento.

2.9.2 Agentes crioprotetores

Os agentes crioprotetores são componentes utilizados durante a congelação para

proteger as células contra os efeitos deletérios da desidratação, resfriamento e da

redução extrema de temperatura. O modo de ação dos agentes crioprotetores na célula

não é plenamente conhecido e isso se dá provavelmente devido aos inúmeros efeitos

deles na célula. Em geral, esses agentes podem agir (i) penetrando nas células

(crioprotetores intracelulares) e substituindo as moléculas de água, (ii) reduzindo o

ponto de congelação, (iii) protegendo membranas celulares por meio da sua ligação às

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cabeças dos grupos fosfolipídicos, (iv) aumentando a viscosidade do meio, e/ou (v)

diminuindo a concentração de eletrólitos durante a criopreservação, e, assim, o risco de

danos osmóticos. Contudo, agentes crioprotetores podem ser tóxicos, bem como podem

facilitar a entrada de agentes tóxicos nas células (Mullen, 2007; Santos, 2007c).

Os agentes crioprotetores podem ser alcoóis, açúcares, amidas e grandes

polímeros, que atuam por diferentes mecanismos (Fuller, 2004; Acker, 2007), sendo

classificados como intra ou extracelulares. Crioprotetores intracelulares ou penetrantes

possuem baixo peso molecular e a capacidade de substituir a água intracelular. Como

exemplos, podem ser citados o glicerol (GLI), etilenoglicol (EG), dimetilsulfóxido

(DMSO) e propanodiol (PROH). Quando os crioprotetores são adicionados, a água

intracelular (solvente) deixa a célula e os crioprotetores penetrantes (soluto) a penetram,

interagindo com a membrana celular, estabilizando as proteínas intracelulares e

reduzindo o ponto de congelação. Estudos recentes demonstraram que o GLI, DMSO,

PROH e EG nas concentrações de 1,5 e 3,0 M foram utilizados com sucesso para a

criopreservação de tecido ovariano de caprinos (Rodrigues et al., 2004a,b) e ovinos

(Santos et al., 2006) previamente exposto a esses compostos a uma temperatura de 20°C

por um período de 20 minutos (período de equilíbrio).

O GLI tem sido usado largamente para a congelação de embriões bovinos devido

à sua baixa citotoxicidade. Entretanto, o GLI induz a severos danos no citoplasma

devido à baixa permeabilidade da membrana plasmática a esta substância (Széll et al.,

1986). Oócitos de camundongos isolados são menos permeáveis ao glicerol que ao

DMSO, PROH e EG (Paynter et al., 1997). A baixa permeabilidade das células ao GLI

aumenta o risco do estresse osmótico durante a descongelação e diluição, pois a entrada

de água na célula ocorre mais rapidamente do que a saída do GLI. Isso explica as baixas

taxas de sobrevivência e de maturação de oócitos criopreservados em GLI (Wani et al.,

2004).

Crioprotetores extracelulares ou não-penetrantes, entre eles os açúcares como a

sacarose e a trealose, e polissacarídeos como o ficol, agem otimizando o efeito dos

crioprotetores penetrantes. Utilizada com freqüência, a sacarose age como um tampão

osmótico contra o estresse causado durante a remoção dos crioprotetores intracelulares

(Mandelbaum et al., 1988). A sacarose pode ser utilizada com sucesso como

crioprotetor extracelular para folículos pré-antrais murinos, especialmente quando

combinada com EG (Salehnia et al., 2002). Ainda, a sacarose (0,1 M) associada ao

DMSO ou PROH mostrou-se eficiente para criopreservar o tecido ovariano bovino, fato

29

demonstrado pela integridade ultra-estrutural dos folículos pré-antrais criopreservados

(Lucci et al., 2004b).

Crioprotetores extracelulares, também conhecidos como agentes de alto peso

molecular, aumentam a viscosidade da solução (por exemplo, o polivinil álcool) ou se

ligam às cabeças dos grupos fosfolipídicos (açúcares, tais como sacarose e trealose),

protegendo as membranas celulares contra as injúrias do frio. Outra substância

comumente adicionada para melhorar a eficiência do meio de criopreservação consiste

no soro fetal bovino. Contudo, a importância do soro na criopreservação não é

cientificamente comprovada, além do risco de conter agentes infecciosos que não são

destruídos durante os procedimentos de criopreservação (Santos, 2007c).

2.9.3 Criopreservação: métodos e danos

O processo de criopreservação envolve basicamente as seguintes etapas: (1)

adição de agente crioprotetor (período de equilíbrio); (2) resfriamento e indução da

formação de gelo, seguidos por congelação ou vitrificação; (3) estocagem em nitrogênio

líquido; (4) descongelação ou aquecimento e (5) remoção ou diluição do agente

crioprotetor. A criopreservação pode ser realizada por dois métodos básicos: congelação

convencional (lenta) e vitrificação.

A congelação lenta é caracterizada pela exposição das células ou tecidos a baixas

concentrações de agente crioprotetor (∼1,5 M) - (Paynter et al., 2000), por um período

que pode variar de 20 (Rodrigues et al., 2004a,b) a 60 minutos (Candy et al., 1997).

Nesse método, é utilizado um freezer programável, em que o material é resfriado

lentamente a uma velocidade de 2ºC/min até –4 a –9ºC, mantendo-se esta temperatura

por um curto período (10 a 15 min) para a estabilização térmica e indução da

cristalização da solução crioprotetora (seeding) através do contato de um elemento

metálico pré-resfriado em nitrogênio líquido com a parede do recipiente que contém o

as amostras. Este procedimento visa prevenir a ocorrência da cristalização do meio em

temperaturas inferiores ao seu ponto de solidificação, que resulta na liberação de

energia sob forma de calor e elevação da temperatura da solução, seguida de redução

brusca durante o equilíbrio térmico com o freezer (Reichenbach et al., 2008). Em

seguida, o sistema continua sendo resfriado lentamente a uma velocidade de 0,3ºC/min.

Uma vez que a desidratação celular é suficientemente atingida (entre –30 a –140ºC), o

material é estocado em nitrogênio líquido (-196°C). Este é o método mais utilizado para

30

a criopreservação de tecido ovariano (Cox et al., 1996; Candy et al., 1997; Newton et

al., 1998; Liu et al., 2008; Qi et al., 2008; Tsuribe et al., 2008). Utilizando este método,

já foi possível a restauração da fertilidade após transplante em ovinos (Salle et al., 1999;

Almodim et al., 2004) e murinos (Liu et al., 2008), bem como já foram obtidos

nascimentos a partir de tecido ovariano criopreservado de murino (Guenasena et al.,

1997), ovino (Gosden et al., 1994; Salle et al., 2003) e humano (Oktay, 2001; Donnez et

al., 2004; Meirow et al., 2005).

O método de vitrificação consiste em utilizar uma taxa de congelação

extremamente rápida, que leva à formação de um estado vítreo do material

criopreservado, protegendo-se as células da formação de cristais de gelo (Huang et al.,

2008). A vitrificação foi idealizada por Luyet em 1937, e, depois de quase 50 anos, Rall

e Fahy descreveram este método como uma alternativa ao processo de congelação lenta

(Rall e Fahy, 1985). Ao contrário da congelação lenta, a vitrificação envolve a

exposição do material biológico a altas concentrações de agente crioprotetor

(geralmente entre 4 e 6 M) por um curto período de tempo (25 segundos a 5 minutos),

geralmente à temperatura ambiente, seguido de um resfriamento ultra-rápido em

nitrogênio líquido, não sendo necessária a utilização de equipamentos sofisticados e de

alto custo. De acordo com Stachecki e Cohen (2004), a vitrificação possui dois aspectos

básicos a serem levados em consideração. O primeiro consiste no fato de que as altas

concentrações de agentes crioprotetores utilizadas na exposição aumentam os efeitos

tóxicos e, em segundo lugar, apesar desse efeito durante o período de equilíbrio, a

vitrificação, por ser uma congelação altamente rápida, aumenta as taxas de

sobrevivência. Este método de congelação já foi reportado para oócitos maturos (Chian

et al., 2004), embriões (Isachenko et al., 2005) e tecido ovariano de diferentes espécies

como camundongas (Kagabu e Umezu, 2000), ratas (Sugimoto et al., 2000) e ovelhas

(Al-aghbari e Menino, 2002). Assim como na congelação lenta, resultados satisfatórios

com nascimentos foram obtidos após vitrificação de ovários inteiros ou FOPA de

camundongas (Liu et al., 2001; de la Peña et al., 2002; Migishima et al., 2003),

entretanto os resultados, principalmente em outras espécies, ainda são controversos.

Existem dois fatores que podem levar à morte celular durante o processo de

congelação/descongelação: a formação de gelo intracelular e o choque osmótico. Esses

efeitos negativos podem ser reduzidos ou evitados com a utilização de agentes

crioprotetores (de la Vega e Wilde, 1991), bem como modificando-se o processo de

criopreservação. De acordo com Stachecki e Cohen (2004), os efeitos do gelo

31

intracelular e do choque osmótico são fatores letais se as células não são tratadas

apropriadamente.

A prevenção da formação de gelo intracelular é considerada um dos fatores mais

importantes quando se deseja realizar um eficiente protocolo de criopreservação. Na

congelação clássica (lenta), essa formação pode ser evitada com um resfriamento celular

lento, permitindo a progressiva desidratação celular (Mazur et al., 2005). Existem duas

hipóteses para a formação de gelo intracelular. A primeira, postulada por Muldrey e

McGann (1990), sugere que o gelo intracelular seja formado como conseqüência de

danos ou defeitos na membrana plasmática, que permitiriam a passagem do gelo

extracelular através da membrana (teoria do fluxo osmótico). Com o super-resfriamento

celular durante a congelação, a força de efluxo da água para o meio extracelular

atingiria um valor crítico e, consequentemente, causaria danos à membrana, permitindo

a entrada do gelo extracelular para o meio intracelular. Uma outra hipótese é a de que o

gelo extracelular em contato (direto ou indireto) com a membrana plasmática, causaria a

formação de gelo intracelular, levando injúria ao conteúdo celular. Há duas versões para

essa segunda hipótese. Na primeira, Toner et al. (1990) sugerem que o gelo extracelular

provocaria uma mudança conformacional na membrana, que se transformaria em um

nucleador heterogêneo dos conteúdos celulares (contato indireto). Na segunda,

postulada por Mazur (2004), o gelo extracelular cresceria através de poros preexistentes

na membrana, onde tal contato direto levaria à formação de gelo intracelular.

Outra causa de morte celular consiste no efeito solução, que envolve alterações

citoplasmáticas como resultado da desidratação, aumento da concentração e

precipitação de solutos e alterações de pH (Mazur et al., 1984). Por muito tempo,

acreditou-se que a morte celular poderia ser causada pelo aumento da concentração de

soluto fora da célula devido ao resfriamento e a congelação da água extracelular

(Lovelock, 1953). Contudo, estudos mais recentes têm reportado uma alta tolerância

celular ao estresse osmótico. Agca et al. (2000) expuseram oócitos bovinos a crescentes

concentrações de cloreto de sódio para elevar a osmolaridade a concentrações

supravitais (até 4800 mOsm) e observaram que até 2400 mOsm, posteriormente os

oócitos foram capazes de ser fecundados e foram obtidos blastocistos. Outros

pesquisadores expuseram oócitos humanos e murinos a concentrações variáveis de

agente crioprotetor ou açúcares sem resfriamento e observaram considerável tolerância

celular às condições osmóticas impostas (Oda et al., 1992; Hotamisligil et al., 1996).

Os danos supracitados não são observados exclusivamente durante o

32

resfriamento, mas também durante o processo de descongelação/re-aquecimento, uma

vez que as células recuperam seu metabolismo na presença de substâncias tóxicas como

os agentes crioprotetores. Assim, El-Naggar et al. (2006) sugerem que as células ou

tecidos criopreservados devem ser descongelados em uma velocidade alta. Agentes

crioprotetores podem ser removidos em uma (Leibo, 1984), três (Rodrigues et al.,

2004a,b) ou mesmo em seis lavagens (Shelton, 1992). Apesar de uma lavagem ser

suficiente para embriões descongelados, folículos isolados e tecido ovariano devem ser

lavados em três passos para retirada de resquícios de agentes crioprotetores (Lima et al.,

2006; Sadeu et al., 2006; Santos et al., 2006).

2.9.4 Métodos de análise de FOPA criopreservados

Para a aplicação de um protocolo de criopreservação, é necessário determinar

o(s) agente(s) crioprotetore(s) mais indicado(s), sua concentração, tempo de exposição e

método de remoção. Um método prático para se verificar a qualidade folicular após a

congelação/descongelação é a histologia clássica. Contudo, esta análise morfológica não

é suficiente para se avaliar o processo de criopreservação (Schotanus et al., 1997; van

den Hurk et al., 1998; Martinez-Madrid et al., 2004), por permitir apenas a identificação

dos sinais primários da atresia (picnose nuclear, danos citoplasmáticos, desconexão

entre as células da granulosa e o oócito, bem como irregularidades na membrana basal)

- (Jorio et al., 1991; Hulshof et al., 1995; Demirci et al., 2002). A morfologia celular

observada por histologia não está sempre correlacionada com a integridade ultra-

estrutural, que requer o uso da microscopia eletrônica de transmissão para sua análise

(Santos et al., 2006). Além das organelas celulares, a verificação da integridade da

membrana plasmática também é fundamental para a avaliação da viabilidade celular, e

tem sido realizada utilizando-se o corante vital azul de trypan (Santos et al., 2007a,b) ou

o marcador fluorescente etídio homodímero (Schotanus et al., 1997; van den Hurk et al.,

1998). Outro parâmetro utilizado para a análise da viabilidade consiste na atividade

enzimática no citoplasma, detectável nas células foliculares através do composto

calceína-AM (Schotanus et al., 1997; van den Hurk et al., 1998), que é clivado por

enzimas esterase em células vivas, resultando em um produto fluorescente (De Clerck et

al., 1994).

33

2.10 Cultivo in vitro de folículos ovarianos

O objetivo principal do cultivo in vitro de FOPA é permitir o desenvolvimento

folicular assegurando o crescimento e a maturação oocitária, bem como a multiplicação

e posterior diferenciação das células da granulosa inclusas nesses folículos (Figueiredo

et al., 2008). O cultivo de ovários tem sido utilizado com diferentes propósitos como

avaliar a importância da vascularização, apoptose, fatores de crescimento e hormônios

para o desenvolvimento de FOPA dentre outros (Fortune et al., 2000; Erickson, 2001;

Flaws et al., 2001; O`Brien et al., 2003; Matos et al., 2007). Dentre os hormônios

testados e já comumente adicionados ao meio de base para o cultivo de folículos

ovarianos, destaca-se o FSH, sendo demonstrada sua importância para a manutenção da

viabilidade e proliferação das células da granulosa de FOPA (Matos et al., 2007; Choi et

al., 2008)

Em roedores, a pequena dimensão dos ovários possibilita o cultivo do órgão

inteiro, o que tem sido bastante útil para o estudo da foliculogênese inicial em pequenos

mamíferos (Fortune, 2003). Em animais domésticos de médio e grande porte, devido às

grandes dimensões dos ovários, não é possível utilizar este modelo. Para estes animais,

o cultivo de pequenos fragmentos de córtex ovariano, rico em folículos primordiais, tem

sido realizado para o estudo da ativação e crescimento de folículos primordiais caprinos

(Silva et al., 2004), bovinos (Braw-Tal e Yossefi, 1997) e humanos (Scott et al., 2004).

Esse tipo de cultivo in vitro tem a vantagem de manter a integridade estrutural folicular

e as interações entre as células foliculares e células do estroma, facilitando a perfusão

do meio para o tecido ovariano (Telfer, 1996). Já o segundo sistema de cultivo folicular

envolve o isolamento, mecânico ou enzimático, dos FOPA (Abir et al., 2001b),

permitindo o monitoramento diário do crescimento folicular, bem como o efeito in vitro

de hormônios e fatores de crescimento sobre cada classe folicular (Abir et al., 2001a).

Apesar dos métodos enzimáticos de isolamento serem mais práticos, os mecânicos têm

sido mais comumente adotados para o isolamento de FOPA de ovários bovinos (Itoh et

al., 2002), caprinos (Arunakumari et al., 2007), ovinos (Amorim et al., 2000), ratas

(Zhao et al., 2000) e camundongas (Lenie et al., 2004). Tais métodos têm

proporcionado a recuperação de um grande número de FOPA, sendo mais utilizados a

microdissecção para folículos com diâmetro igual ou superior a 150 μm (Lee et al.,

2007) e o isolamento pelo tissue chopper para folículos menores (Martinez-Madrid et

al., 2004) ou ainda a associação de ambos os métodos (Gupta et al., 2008). Além disso,

34

o isolamento mecânico mantém a integridade da estrutura folicular, a membrana basal

permanece intacta após o procedimento e as interações oócito-células da granulosa-teca

são mantidas (Demeestere et al., 2002).

Em relação aos avanços obtidos através da MOIFOPA, em gatas (Jewgenow e

Stolte, 1996), gambás (Butcher e Ullman, 1996) e macacas (Fortune et al., 1998) foi

observado o crescimento de FOPA após cultivo in vitro, porém sem a formação de

antro. Nas espécies bovina (Itoh et al., 2002), caprina (Huanmin e Yong, 2000) e

humana (Roy e Treacy, 1993), os FOPA isolados foram cultivados in vitro e

desenvolveram-se até o estádio antral. Em ovinos, folículos secundários isolados e

cultivados por 6 dias atingiram a maturação nuclear (Tamilmani et al., 2005). Em suínos

e bubalinos, folículos secundários crescidos in vitro chegaram até a ovulação, tiveram

seus oócitos fecundados in vitro, com desenvolvimento até estádio de blastocisto (Wu e

Tian, 2007; Gupta et al., 2008). No entanto, os maiores avanços do cultivo folicular

foram alcançados em camundongos, com o nascimento de crias viáveis a partir da

maturação de oócitos provenientes de FOPA cultivados in situ e isolados, em que o

oócito adquiriu competência meiótica para ser fecundado e completar o

desenvolvimento embrionário (O’Brien et al., 2003). Provavelmente, essa estratégia de

associar o cultivo de folículos in situ e isolados seja necessária para promover o

crescimento de folículos primordiais de espécies domésticas e primatas (Silva et al.,

2006).

Para a espécie canina, poucos estudos com FOPA têm sido realizados,

restringindo-se todos à avaliação de sistemas de maturação (Bolamba et al., 1998, 2002,

2006). Nenhum trabalho foi realizado com o objetivo de estabelecer métodos de

preservação ou de cultivo in vitro.

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3 JUSTIFICATIVA

A superpopulação de cães nas cidades resulta na eutanásia de milhões de

animais sadios, além do dispêndio de um elevado volume de recursos econômicos

estimados na ordem de bilhões de dólares anualmente (Frank e Carlisle-Frank, 2007).

Nos países em desenvolvimento, estes recursos são utilizados, principalmente, para a

execução de programas de controle da raiva urbana, que se baseiam na eliminação de

cães errantes, na vacinação da população animal e no tratamento de pessoas

potencialmente expostas ao vírus rábico (Wandeler et al., 1988).

O tratamento profilático pós-exposição para pessoas agredidas por cães de rua,

instituído obrigatoriamente nestes casos por não ser possível a observação do animal

agressor, onera bastante os cofres públicos. Isto porque o custo médio de um esquema

completo de soro-vacinação pode exceder a quantia de U$ 4.000, quando se utilizam

vacinas produzidas em cultivo celular e imunoglobulinas anti-rábicas homólogas

(Dhankhar et al., 2008), e a freqüência desses eventos de agressão em cidades de países

em desenvolvimento, a exemplo do município de Fortaleza (Ceará, Brasil), é bastante

elevada (Lopes et al., 2001).

Outra consideração importante acerca desses tratamentos é o risco de reações

adversas severas quando se utilizam vacinas produzidas por cultivo em tecido nervoso

de animais, que constituem o único tipo disponível em alguns países (Rupprecht et al.,

2002). Por exemplo, reações de desmielinização central e/ou periférica, algumas das

quais fatais, têm sido associadas ao uso da vacina Fuenzalida & Palácios, obtida a partir

de cultivo em cérebros de camundongos lactentes, sendo reportadas incidências de até

um caso para cada 1.250 tratamentos (Meltzer e Rupprecht, 1998).

O Estado do Ceará possui uma das maiores incidências de raiva canina e raiva

humana transmitida por cães no Brasil. No período de 2002 a 2005, confirmaram-se em

laboratório 175 casos de raiva canina, o que indica uma elevada circulação da doença

em cães (Secretaria de Saúde do Estado do Ceará, 2006). Devido ao fato de que o

número de casos de raiva animal e humana tende a ser maior em áreas com elevada

densidade de cães errantes, onde os contatos entre homens e animais são mais

freqüentes (Haddad et al., 1988; Osman e Haddad, 1988), as autoridades sanitárias do

referido, bem como de outros estados da federação, têm realizado a apreensão

sistemática e eutanásia de cães de rua. Contudo, estudos realizados em diversos países

demonstram que a a eliminação de cães errantes é ineficaz e apresenta maior custo que

36

o investimento no controle da reprodução animal e em programas educativos para a

população (World Health Organization, 1990).

Mais que um dilema de ordem econômica e sanitária, a superpopulação canina e

a forma como é controlada compõem um complexo dilema ético. A qualidade de vida

de cães abandonados é extremamente baixa, sendo marcantes a fome, doenças,

desconforto por condições climáticas adversas, atropelamentos, crueldades, enfim, uma

situação multifatorial de sofrimento intenso. Neste contexto, faz-se fundamental e

urgente a realização de programas de controle da reprodução de cães, bem como o

desenvolvimento de métodos contraceptivos eficientes, seguros e de custo

suficientemente baixo para permitir sua utilização em massa. Assim, será possível

intervir de maneira racional, efetiva e humanitária sobre o problema da superpopulação

de cães, obtendo-se, conseqüentemente, uma economia expressiva de recursos públicos,

além de promoção da saúde pública e do bem-estar animal. O potencial da técnica de

imunoesterilização por anticorpos anti-ZP de reunir todas as características supracitadas

representa uma importante perspectiva para a solução desta questão.

Considerando-se a importância da realização de estudos in vitro preliminares aos

testes in vivo, com o intuito de se estabelecer uma base sólida e precisa de

conhecimentos, minimizando-se a experimentação com animais, utilizou-se no presente

trabalho a biotécnica de MOIFOPA como modelo experimental. Entretanto, uma vez

que ainda não há informações relativas à aplicação desta tecnologia para cães, estudos

foram conduzidos para a definição de métodos de preservação e cultivo in vitro de

folículos pré-antrais caninos. Neste contexto, realizou-se a avaliação de protocolos de

resfriamento e criopreservação utilizados para outras espécies, sendo ainda empregado

um sistema de cultivo em desenvolvimento para possibilitar a análise dos efeitos de

anticorpos anti-ZP sobre os referidos folículos.

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4 HIPÓTESES CIENTÍFICAS

Folículos ovarianos pré-antrais caninos podem ser preservados por meio de

armazenamento hipotérmico durante o período de isquemia compreendido entre a coleta

dos ovários e seu uso no laboratório.

Folículos pré-antrais caninos podem ainda ser criopreservados através de

congelação lenta utilizando-se agentes crioprotetores, mantendo-se a viabilidade e a

integridade ultra-estrutural.

Folículos pré-antrais caninos frescos ou criopreservados podem ser cultivados in

vitro mantendo sua viabilidade. Esta técnica pode ser utilizada como modelo

experimental para o teste de agentes esterilizantes.

Anticorpos anti-ZP são capazes de se ligar especificamente à ZP de oócitos de

caninos, causando bloqueio da ligação de espermatozóides ou atresia de folículos pré-

antrais.

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5 OBJETIVOS

5.1 Objetivo Geral

Avaliar o potencial de anticorpos anti-ZP para a imunoesterilização de cadelas.

5.2 Objetivos Específicos

- Definir um método de preservação de FOPA caninos por períodos curtos,

avaliando-se os parâmetros meio (solução de NaCl a 0,9% - salina fisiológica – ou Meio

Essencial Mínimo – MEM), temperatura (4, 20 ou 38°C) e tempo de conservação

(2,6,12 ou 24 h);

- estabelecer um método de criopreservação de FOPA caninos, avaliando-se a

eficiência dos agentes crioprotetores dimetilsulfóxido, etilenoglicol, glicerol e 1,3-

propanodiol durante a execução da técnica de congelação lenta;

- cultivar in vitro FOPA caninos frescos ou criopreservados de modo a manter-

se sua viabilidade;

- analisar o efeito de anticorpos policlonais anti-pZP sobre FOPA caninos

cultivados in vitro.

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6 CAPÍTULO I


Immunocontraception in mammals emphasizing dog population control

Revista Brasileira de Reprodução Animal

(Publicado, v.29, n.3/4, p.159-166, 2005)

40


(Immunocontraception in mammals emphasizing dog population control)

Cláudio Afonso Pinho Lopes*, Diana Célia Sousa Nunes-Pinheiro, José Ricardo de

Figueiredo

Faculdade de Veterinária, Universidade Estadual do Ceará, CEP 60740-000, Fortaleza,

CE, Brasil.

* Autor para correspondência: [email protected]

Resumo

A superpopulação de cães constitui um sério problema em diversas cidades do

mundo, sendo necessário o desenvolvimento de sistemas de contenção de natalidade

baseados no controle da reprodução. Os métodos contraceptivos atualmente disponíveis

são inadequados para uso em escala populacional. Neste contexto, a imunocontracepção

surge como uma importante perspectiva para a solução desta questão. No presente

trabalho, são abordados diferentes métodos de imunocontracepção em mamíferos,

baseados no uso de hormônios reprodutivos, antígenos do embrião, da zona pelúcida e

do espermatozóide, apresentando seus princípios e os avanços obtidos em seu

desenvolvimento.

Palavras-chave: imunocontracepção, imunoesterilização, mamíferos, cães.

Abstract

Dog overpopulation is a serious problem in many cities throughout the world,

which requires the development of birth control systems based upon reproduction

blocking. Contraception methods available nowadays are not suitable for use in a

population scale. In this context, immunocontraception is an important perspective for

filling this gap. In this article, immunocontraceptive methods for mammals based on use

of reproductive hormones and antigens from embryo, zona pellucida and spermatozoon

are reviewed, focusing on their principles and development advances.

Keywords: Immunocontraception, immunosterilization, mammals, dogs.

41

Introdução

A superpopulação de cães constitui um sério problema em diversas cidades do

mundo, caracterizado pela existência de uma grande quantidade de animais sem

responsáveis que vivem nos espaços públicos. Esses animais compõem um reservatório

de zoonoses, e, assim, um risco de agravo à saúde pública, o que implica o dispêndio

expressivo de recursos. Neste contexto, as autoridades sanitárias realizam a eliminação

sistemática desses animais como estratégia de controle populacional.

Esta abordagem, no entanto, é estritamente paliativa, por não atuar sobre a

origem do problema, que consiste das elevadas taxas de natalidade de cães. Além disso,

a eliminação dos animais sem responsáveis faz emergirem questões éticas que definem

a necessidade de novas estratégias de ação. Assim, um sistema de controle populacional

para cães, para ser efetivo, racional e humanitário, deve basear-se no controle de

natalidade.

As técnicas contraceptivas atualmente disponíveis para cães não são adequadas

para uso em escala populacional. Os métodos cirúrgicos, além do custo elevado e dos

riscos inerentes a qualquer procedimento cirúrgico, são logisticamente inviáveis para

populações grandes. Os métodos farmacológicos, baseados no uso de hormônios, têm

apresentado efeitos adversos. Um novo conceito em métodos contraceptivos, a

imunocontracepção, surge como uma importante perspectiva para o desenvolvimento de

métodos aplicáveis ao controle da reprodução de cães em larga escala.

Além de cães, outras espécies de mamíferos também encontram-se em

superpopulação, como os felinos domésticos em diversas regiões do mundo (Slater,

2001) e, em particular, na Austrália (Bradley et al., 1999), o elefante em alguns países

do sul da África (Stout e Colenbrander, 2004) e o cervo de cauda branca (Rutberg et al.,

2004) na América do Norte. Problemas de animais em excesso também ocorrem em

parques zoológicos, em virtude da reprodução dos animais cativos (Kirkpatrick e

Rutberg, 2001). Assim, o uso da imunocontracepção pode ser útil também no controle

das populações dessas espécies. Neste contexto, no presente trabalho, serão abordados

diferentes métodos de imunocontracepção em mamíferos, baseados no uso de

hormônios reprodutivos e de antígenos do embrião, da zona pelúcida e do

espermatozóide, apresentando-se seus princípios e os avanços obtidos em seu

desenvolvimento.

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A imunocontracepção

A imunocontracepção consiste da ligação de anticorpos a moléculas bioativas do

sistema reprodutor responsáveis por interações fundamentais ao processo reprodutivo,

que, desta forma, é interrompido. Por exemplo, a ligação de anticorpos à superfície da

zona pelúcida (ZP) de oócitos torna estes inacessíveis a espermatozóides, impedindo,

assim, as interações moleculares necessárias ao processo de fertilização. O efeito

contraceptivo é reversível, mantendo-se até que as concentrações (títulos) dos

anticorpos específicos, que gradativamente vão decaindo a níveis abaixo de um limiar

contraceptivo, ou seja, tornem-se insuficientes para o bloqueio da reprodução. A

duração deste efeito varia em função do título inicial de anticorpos contraceptivos,

sendo observados períodos de até cinco anos de infertilidade (Brown et al., 1997).

A imunocontracepção tende a ser destituída de efeitos colaterais adversos, uma

vez que as moléculas a serem bloqueadas devem desempenhar funções limitadas ao

processo reprodutivo. Além disso, a verificação criteriosa do potencial de reatividade

cruzada de anticorpos contraceptivos com outros elementos do organismo, em

decorrência de algum grau de homologia, deve sempre ser procedida.

Em alguns casos, os anticorpos produzidos e/ou células citotóxicas do sistema

imunológico ativadas nas vacinações causam a destruição de elementos não-

regeneráveis do sistema reprodutivo, caracterizando-se, assim, um processo de

esterilização imunológica, também denominado imunoesterilização.

Diversos antígenos do sistema reprodutor são passíveis de ligação por

anticorpos, sendo, portanto, moléculas-alvo em potencial para o desenvolvimento de

métodos de imunocontracepção ou imunoesterilização (Fig. 1). O hormônio liberador de

gonadotrofinas (GnRH), secretado pelo hipotálamo, controla a liberação dos hormônios

folículo estimulante (FSH) e luteinizante (LH), que, por sua vez, controlam a

gametogênese em machos e fêmeas. Assim, a ligação de anticorpos às moléculas desses

hormônios ou aos seus receptores resulta na interrupção da ovulação ou da produção de

espermatozóides. As glicoproteínas da ZP e determinados antígenos da superfície do

espermatozóide, responsáveis pela cascata de reações que constituem o processo de

fertilização, também são passíveis de bloqueio por anticorpos. As gonadotrofinas

coriônicas (GC), responsáveis pela manutenção da gestação até o desenvolvimento

completo da placenta, podem também constituir moléculas-alvo para a

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imunocontracepção. O bloqueio por anticorpos de proteínas bioativas do embrião pode

também ser utilizado como método para interrupção da gestação em seu início.

Imunização ativa

Os anticorpos contraceptivos podem ser produzidos por meio de imunização

ativa (vacinação), utilizando-se como imunógeno a molécula-alvo a ser bloqueada.

Entretanto, em populações com grande diversidade genética, uma parcela significativa

dos indivíduos pode não ter capacidade de resposta imunológica a determinados

antígenos, o que resulta na redução da eficácia do método.

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Imunização passiva

Uma técnica alternativa à vacinação contraceptiva, com potencial de reunir

maiores eficácia, segurança e praticidade, consiste da administração de anticorpos

contraceptivos pré-formados em outros animais, ou seja, a imunização passiva

(soroterapia). A maior eficácia decorre do fato de este método não depender da

capacidade de cada indivíduo de responder imunologicamente ao elemento-alvo a ser

neutralizado. Esta técnica é ainda mais segura que a vacinação, pois é possível analisar a

especificidade dos anticorpos produzidos antes de serem administrados (Talwar, 1999).

Outra vantagem deste método é sua praticidade, uma vez que uma única aplicação de

anticorpos pode resultar em títulos adequados, enquanto os procedimentos de

vacinação, geralmente, requerem diversas aplicações de imunógeno. Os anticorpos de

interesse podem ser produzidos pela hiperimunização de plantéis de animais

selecionados para a produção de soro, do qual se procede a purificação e a concentração

das imunoglobulinas específicas para o antígeno a ser neutralizado, a exemplo dos soros

anti-ofídicos produzidos em eqüinos.

Imunocontracepção baseada em hormônios reprodutivos

GnRH

A molécula de GnRH consiste de um decapeptídeo incapaz de desencadear uma

resposta imunológica quando utilizado como vacina. Elementos com estruturas

pequenas não são capazes de estimular as células do sistema imunológico, ou seja, não

são imunogênicos, sendo denominados haptenos (Janeway et al., 2001). Para que uma

resposta imunológica seja estabelecida para haptenos, é necessário que estes sejam

ligados a proteínas de grandes dimensões, denominadas proteínas carreadoras. Assim, o

GnRH tem sido quimicamente conjugado a diversas proteínas, como os toxóides

tetânico (TT) e diftérico (TD), para seu uso como agente imunocontraceptivo.

Ladd et al. (1994) vacinaram cães com GnRH conjugado ao toxóide tetânico,

obtendo uma redução dos níveis séricos de testosterona a limiares observados em

animais castrados, o que resultou em azoospermia. Miller et al. (2000) analisaram a

eficácia de uma vacina constituída por GnRH para o controle reprodutivo de um grupo

de cervos machos e fêmeas de cauda branca. Após um esquema de vacinação composto

45

por duas aplicações apenas de imunógeno, observou-se uma redução de 88% da

fertilidade das fêmeas, além da interrupção da produção de testosterona pelos machos,

com conseqüente perda da libido. Estes efeitos mantiveram-se por até dois anos, sem a

necessidade de vacinações de reforço. Robbins et al. (2004) vacinaram felinos

domésticos machos e fêmeas com GnRH conjugado à leucotoxina A e observaram a

produção de anticorpos em títulos imunoneutralizantes que se mantiveram por mais de

20 meses. Neste período, os machos apresentaram supressão da produção de

testosterona e da espermatogênese, verificando-se atrofia das células de Sertoli e

ausência de espermátides. As fêmeas mantiveram-se em anestro durante o mesmo

período, não sendo observada a ocorrência de folículos terciários ao exame histológico

dos ovários.

Uma única injeção de anticorpos monoclonais anti-GnRH ao início do proestro

suprimiu a ovulação subseqüente em fêmeas de rato. Quando administrados a cadelas,

estes anticorpos também interromperam o estro, conforme verificado pela avaliação do

comportamento, citologia vaginal e perfis de hormônios sexuais (Talwar et al., 1985).

FSH

Nos machos, a ausência de estimulação por FSH afeta significativamente a

proliferação de espermatogônias e, assim, a produção quantitativa de espermatócitos

primários (Suresh et al., 1995). Além disso, evidências sugerem que a privação de FSH

também afeta o processo de espermiogênese, levando à produção de espermatozóides

de baixa qualidade ou imaturos, resultando em infertilidade. Dessa forma, a

neutralização do FSH por anticorpos pode ser utilizada como técnica

imunocontraceptiva (Moudgal et al., 1997a).

Macacos vacinados com FSH ovino (oFSH) apresentaram oligozoospermia e

infertilidade (Srivastav e Das, 1992). A administração a macacos, de anticorpos para o

FSH ou para o receptor deste hormônio (FSH-R), causou uma inibição da transformação

de espermatogônias em espermatócitos primários, bem como do processo de

espermiogênese (Aravindan et al., 1993; Suresh et al., 1995). Não houve alteração da

produção de testosterona, observando-se apenas oligozoospermia, mas a qualidade dos

espermatozóides foi afetada de forma suficiente ao estabelecimento de infertilidade.

Aravindan et al. (1993) observaram que os títulos de anticorpos anti-FSH em

animais vacinados com o FSH ovino não duram mais de 90-100 dias, pois estes são

46

rapidamente usados para a bioneutralização do FSH, produzido de forma contínua pela

hipófise. Assim, uma alternativa de técnica imunocontraceptiva para se afetar a

espermatogênese consiste do uso de anticorpos para o receptor de FSH. Macacos

vacinados com um fragmento recombinante do receptor do FSH apresentaram títulos

efetivos de anticorpos para o antígeno utilizado, que se mantiveram por mais de 300

dias após apenas duas a três injeções do imunógeno. Estes anticorpos ligaram-se

efetivamente a moléculas do receptor do FSH nos testículos, impedindo a ligação do

FSH, o que resultou em inibição da transformação de espermatogônias em

espermatócitos primários. Os animais vacinados, ao serem submetidos ao teste de

cópula, apresentaram-se inférteis. Não houve qualquer alteração nas concentrações

séricas de testosterona (Moudgal et al., 1997b).

LH

A eficácia de vacinas baseadas em LH ovino (oLH) para a interrupção da função

testicular foi avaliada em coelhos (Jeyakumar e Moudgal, 1996) e macacos (Suresh et

al., 1995). Os anticorpos produzidos foram capazes de se ligar ao LH, resultando em

uma redução intensa (~90%) das concentrações séricas de testosterona. A quantificação

da população de células germinais por citometria de fluxo mostrou que, 15-18 semanas

após a vacinação, a população de espermátides foi reduzida em mais de 90%, e a de

espermatócitos primários em mais de 50%. Os autores sugeriram que a azoospermia

resultante decorreu da interrupção da meiose, que é controlada pela testosterona

testicular. Remy et al. (1996) observaram que a vacinação de ratos machos com

receptor de LH (LH-R) suprimiu a fertilidade de forma proporcional aos títulos de

anticorpos produzidos contra os receptores de LH.

Coelhas vacinadas com receptores de LH bovino produziram anticorpos para

este receptor e apresentaram falha de ovulação após cópula, inseminação artificial ou

injeção de gonadotrofina coriônica (Singh et al., 1995). Essas coelhas mantiveram-se

inférteis por 10 meses após a vacinação, mesmo após repetidas cópulas. Entretanto, a

ovulação foi reestabelecida com o declínio dos anticorpos. De modo semelhante,

babuínos e macacos rhesus vacinados com receptores de LH tornaram-se inférteis, mas,

com o declínio dos anticorpos, retornaram à fertilidade normal (Shukla et al., 1991; Pal

et al., 1992).

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Cadelas vacinadas com receptores de LH bovino apresentaram redução dos

níveis de progesterona, sugerindo-se falha de ovulação e de função do corpo lúteo e não

manifestaram sinais de estro. Com o declínio dos anticorpos anti-receptores de LH cerca

de 500 dias após a vacinação, os perfis hormonais, a citologia vaginal e os sinais de

estro retomaram o padrão normal. Assim, comprovou-se que a vacinação de cadelas

com receptores de LH leva a um estado reversível de infertilidade (Saxena et al., 2002).

Imunocontracepção baseada em proteínas embrionárias

Outra modalidade de técnica imunocontraceptiva consiste da ligação de

anticorpos a moléculas bioativas do embrião, como a proteína transportadora de

riboflavina (RCP). A proteína transportadora de riboflavina é o principal mediador do

suprimento de vitamina do embrião em desenvolvimento. A vacinação de fêmeas de

primatas com a proteína transportadora de riboflavina de galinha resultou na produção

de anticorpos que interromperam a gestação, provavelmente durante o estágio peri-

implantação do embrião no útero (Adiga et al., 1997). A demonstração por

imunohistoquímica da presença da proteína transportadora de riboflavina em oócitos

ovulados e embriões em clivagem de fêmeas de rato sugere que estes podem constituir

um alvo para a citotoxicidade mediada por anticorpos e processos degenerativos.

Imunocontracepção baseada na Zona Pelúcida

A zona pelúcida é uma estrutura extracelular que circunda e protege oócitos e,

subseqüentemente, o embrião (Wolgemuth et al., 1984). A zona pelúcida é composta

por três glicoproteínas denominadas ZP1, ZP2 e ZP3. As duas últimas se unem para

formar filamentos, que são interligados por ZP1 (Greve e Wassarman, 1985). A zona

pelúcida desempenha funções fundamentais no processo de fertilização, que é iniciado

pela ligação dos espermatozóides à sua superfície (Gupta et al., 1997). Evidências

sugerem que, em camundongos, hamsters e no homem, a ZP3 constitui o receptor

primário ao qual se ligam os espermatozóides (Moller et al., 1990; Van-Duin et al.,

1994). Em suínos, sugere-se que a ZP3 e a ZP1 associadas participem do processo de

ligação dos espermatozóides (Yurewicz e Sacco, 1996).

A ligação da ZP3 ao receptor cognato do espermatozóide induz uma cascata de

transdução de sinal que determina a reação acrossômica (Gupta et al., 1997). A reação

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acrossômica resulta na liberação de enzimas proteolíticas necessárias para que os

espermatozóides penetrem na estrutura da zona pelúcida e na remodelação da superfície

dessas células para manutenção da adesão à zona pelúcida e para a fusão com a

membrana do oócito (Yanagimachi, 1994). Após a reação acrossômica, a ZP2 passa a

desempenhar a função de receptor (secundário) para os espermatozóides, mantendo-os

aderidos ao oócito (Gupta et al., 1997).

A ZP2 também desempenha uma função importante no mecanismo de bloqueio

à polispermia. Após a fusão de um espermatozóide ao oócito, ocorre a clivagem da ZP2

(Moller e Wassarman, 1989). Os fragmentos resultantes permanecem não-

covalentemente ligados à zona pelúcida, tornando essa zona resistente à proteólise e,

portanto, prevenindo a penetração espermática.

Assim, devido às funções fundamentais das glicoproteínas da zona pelúcida no

processo de fertilização, estas tornam-se moléculas-alvo em potencial para a

constituição de técnicas imunocontraceptivas. A viabilidade do uso de glicoproteínas da

zona pelúcida para a imunocontracepção foi demonstrada pela primeira vez pela

vacinação de fêmeas de camundongo com zona pelúcida de hamster, que resultou na

produção de anticorpos capazes de se ligar à zona pelúcida endógena, induzindo

infertilidade (Gwatkin et al., 1977). Antisoro de hamsters imunizados com ZPA humana

recombinante foi administrado a fêmeas de hamster, observando-se uma redução

significativa da fecundidade (Koyama e Hasegawa, 2004). Anticorpos produzidos

contra um peptídeo sintético correspondente a um segmento da zona pelúcida felina

(fZP) foram capazes de bloquear a ligação de espermatozóides a oócitos de gata e a

fertilização in vitro (Ringleb et al., 2004).

Estudos iniciais de caracterização imunológica revelaram que os anticorpos

produzidos para as glicoproteínas da zona pelúcida de uma espécie são capazes de se

ligar às glicoproteínas de várias outras espécies (Sacco et al., 1981). Esta reatividade

imunológica cruzada deve-se a graus variáveis de homologia das seqüências protéicas, o

que estabelece a possibilidade de vacinação heteróloga. Devido à grande

disponibilidade de ovários suínos em abatedouros, as preparações de zona pelúcida

suína (pZP) tornaram-se os antígenos de escolha (Gupta et al., 2004). A vacinação com

pZP mostrou-se efetiva para o controle populacional de cavalos selvagens (Kirkpatrick

et al., 1990), várias espécies de animais cativos em zoológicos (Kirkpatrick et al.,

1996), cervos de cauda branca (Rutberg et al., 2004) e elefantes-africanos (Fayrer-

Hosken et al., 1999).

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A vacinação de coelhas com pZP resultou em infertilidade (Wood et al., 1981).

O monitoramento subseqüente dos animais imunizados mostrou que o efeito foi

irreversível. A histologia dos ovários destes animais revelou uma destruição de oócitos

em todos os folículos em crescimento e uma depleção severa da população de folículos

quiescentes (Skinner et al., 1984). A vacinação de primatas não-humanos com pZP

também resultou em infertilidade irreversível associada a alterações de folículos

ovarianos (Gulyas et al., 1983). Estes e outros estudos, empregando modelos animais

diferentes, demonstraram que a vacinação com pZP resulta em infertilidade mais

provavelmente por distrofia do ovário que por um bloqueio da fertilização (Gupta et al.,

2004).

Diversos estudos têm acumulado evidências que indicaram o envolvimento da

resposta imunológica celular na histopatologia dos ovários após a vacinação com

antígenos da zona pelúcida (Naz et al., 1995). Rhim et al. (1992) demonstraram ser

possível transferir a ooforite de fêmeas de camundongo vacinadas com peptídeos que

continham segmentos da mZP3, para animais normais, através da transferência de

linfócitos T CD4+, sem a detecção de anticorpos anti-mZP3 nos receptores. Gu et al.

(2004), ao infectarem coelhas com um vírus myxoma recombinante que expressa a ZP2

de coelho (MV-ZPB), observaram a produção de anticorpos específicos para esta

proteína, que se ligaram à zona pelúcida nativa. Houve uma redução significativa das

quantidades de folículos ovarianos terciários e pré-ovulatórios das fêmeas infectadas,

sendo observada a infiltração de linfócitos T nos ovários de alguns animais.

Apesar dos diversos indícios de mecanismos imunológicos celulares para a

patogênese da redução de folículos ovarianos, em nenhum dos estudos em que foi

verificada depleção de folículos primordiais após vacinação com zona pelúcida,

observou-se a infiltração de linfócitos T (Aitken, 2002). De fato, não se conhece uma

associação entre a ooforite observada após a vacinação com zona pelúcida e a depleção

de folículos primordiais (Kerr et al., 1998).

Uma explicação alternativa para a patologia do ovário após a vacinação com

zona pelúcida é que a perda de folículos primordiais reflete seu recrutamento acelerado

para compor a população de folículos em crescimento que é eliminada devido à ação de

anticorpos anti-zona pelúcida (Aitken, 2002). Outra possibilidade é que uma proporção

significativa de folículos primordiais expressem níveis baixos de ZP3, sendo destruídos

por anticorpos antizona pelúcida por fixação de complemento (Grootenhuis et al.,

1996). Em coelhos, foi demonstrado que a resposta imunológica humoral (de

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anticorpos) pode causar perdas foliculares, provavelmente por meio da ligação de

anticorpos aos oócitos, que bloqueia a comunicação entre estas células e as da granulosa

(Skinner et al., 1984).

Em cadelas, o potencial do uso de técnicas imunocontraceptivas que têm as

glicoproteínas da zona pelúcida como moléculas-alvo tem sido avaliado visando ao

controle populacional de cães nas cidades. Mahi e Yanagimachi (1979) demonstraram

que anticorpos produzidos contra extratos de ovários de cadela são capazes de inibir a

fertilização de oócitos caninos in vitro. Em outro estudo, cadelas vacinadas com zona

pelúcida de suínos produziram altos títulos de anticorpos capazes de se ligar

cruzadamente à zona pelúcida canina (dZP) e apresentaram-se inférteis após

acasalamento (Mahi-Brown et al., 1982). A vacinação de cadelas com ZP3 canina

recombinante conjugada ao toxóide diftérico também resultou em infertilidade

(Srivastava et al., 2002). A análise histológica dos ovários dos animais imunizados

revelou um aumento da taxa de atresia folicular, não sendo observada infiltração por

linfócitos. Nosso grupo está avaliando o potencial da imunização passiva com

anticorpos antizona pelúcida canina para a imunoesterilização de cadelas.

Imunocontracepção baseada em antígenos espermáticos

Outra estratégia para o desenvolvimento de técnicas imunocontraceptivas que

bloqueiam a fertilização consiste da utilização de anticorpos capazes de se ligar às

moléculas bioativas da superfície do espermatozóide responsáveis pelas interações que

constituem este processo. Alternativamente, estes anticorpos, se presentes no muco

cervical, podem causar a aglutinação dos espermatozóides, impedindo-os de acessar o

trato reprodutivo superior feminino (Delves et al., 2002). Casos de ocorrência natural de

anticorpos aglutinadores de espermatozóides em mulheres de casais inférteis indicam

que estas estratégias podem ser efetivas (Van Voorhis e Stovall, 1997) e seguras, pois

não se observam quaisquer outros efeitos, além da infertilidade, devido à presença

destes anticorpos (Aitken, 2002). Potencialmente, estas técnicas podem ser aplicadas a

machos, mas o número de espermatozóides a serem neutralizados (~108-109) torna isso

um desafio maior que fazê-lo sobre um número limitado (dezenas a centenas) destas

células no trato reprodutivo superior feminino (Delves et al., 2002).

A vacinação de cangurus machos com espermatozóides homólogos resultou em

produção de anticorpos capazes de penetrar o trato reprodutivo e de se ligar ao

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acrossoma e à peça intermediária dos espermatozóides, resultando em redução das taxas

de fertilização (Asquith et al., 2006).

Dentre os antígenos espermáticos com potencial para servir como moléculas-

alvo em técnicas imunocontraceptivas, destacam-se as enzimas hialuronidase (PH-20),

lactato desidrogenase C4 (LDH-C4) e o antígeno associado ao espermatozóide-9

(SPAG9). A PH-20 está presente na superfície externa da membrana plasmática que

reveste o acrossoma, possibilitando ao espermatozóide penetrar a massa de células do

cumulus rica em ácido hialurônico (Sabeur et al., 1997). Homólogos desta molécula têm

sido encontrados em uma ampla variedade de espécies e sua eficácia contraceptiva

como imunógeno tem sido demonstrada em porcos-da-índia machos. A LDH-C4 é uma

enzima específica do espermatozóide de várias espécies (Goldberg, 1986). A vacinação

de fêmeas de babuíno com a LDH-C4 resultou em uma redução do índice de fertilidade

de 77% (controle) para 29% (Goldberg e Herr, 1999). O SPAG9 é uma molécula

presente no acrossoma em espermatozóides humanos e de outras espécies de primatas,

que parece participar da adesão destas células a oócitos. Anticorpos produzidos contra

SPAG9 recombinante humano (rhSPAG9) mostraram-se capazes de bloquear in vitro a

ligação entre espermatozóides e oócitos humanos (Jagadish et al., 2005).

Conclusão

A viabilidade do desenvolvimento de técnicas imunocontraceptivas eficazes,

demonstrada pelos estudos compilados neste trabalho, constitui uma importante

perspectiva para a solução do problema de superpopulação de cães. O controle da

reprodução de fêmeas é preferível ao de machos, uma vez que, em programas de

contenção de natalidade, poucos indivíduos machos não-controlados são capazes de

manter os índices de fertilidade de uma população conforme já foi verificado em alguns

casos. Neste contexto, para cadelas, destacam-se as técnicas imunocontraceptivas

baseadas no GnRH, receptor de LH, proteína transportadora de riboflavina, zona

pelúcida e antígenos espermáticos. Dentre estas, o uso da zona pelúcida para a

imunoesterilização através da eliminação dos folículos primordiais é uma técnica

bastante promissora.

52

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58

7 CAPÍTULO II

Preservação de folículos pré-antrais caninos por períodos curtos: efeitos da

temperatura, meio e tempo

Short-term preservation of canine preantral follicles: effects of temperature, medium

and time

Animal Reproduction Science

(Aceito para publicação, 2008)

59

Short-term preservation of canine preantral follicles: effects of temperature, medium and time

Cláudio Afonso Pinho Lopesa,b,*, Regiane Rodrigues dos Santosc, Juliana Jales de

Hollanda Celestinoa, Mônica Aline Parente Meloa, Roberta Nogueira Chavesa, Claudio

Cabral Campelloa, José Roberto Viana Silvad, Sônia Nair Báoe, Katarina Jewgenowb,

José Ricardo de Figueiredoa.

a. Faculty of Veterinary, PPGCV, LAMOFOPA, State University of Ceará, Fortaleza, Brazil.

Postal address: Av. Paranjana 1700, Campus do Itaperi, CEP 60.740-903, Fortaleza, CE,

Brazil.

b. Institute for Zoo and Wildlife Research, Berlin, Germany.

Postal address: Postfach 601103, D-10252, Berlin, Germany.

c. Department of Farm Animal Health, Utrecht University, The Netherlands.

Postal address: Yalelaan 7, 3584 CL Utrecht, Netherlands.

d. Biotechnology Nucleus of Sobral (NUBIS), Federal University of Ceará, Sobral, Brazil.

Postal address: Rua Gerardo Rangel s/n, Campus do Derby, CEP 62042-280, Sobral, CE,

Brazil.

e. Laboratory of Electron Microscopy, Institute of Biology, University of Brasília, Brazil.

Postal address: Universidade de Brasília, Campus Universitário Darcy Ribeiro (Asa Norte),

Instituto Central de Ciências (ICC Sul), CEP 70910, Brasília, DF, Brazil.

* Corresponding author. Tel.: +49 176 2671 8956. Fax +49 30 5126 104. E-mail address:

[email protected] (C.A.P. Lopes).

Correspondence address: Institut für Zoo- und Wildtierforschung (IZW). Postfach 601103,

D-10252, Berlin, Germany.

Resumo

O uso da vasta população de folículos pré-antrais é uma alternativa promissora para a

obtenção de elevados números de oócitos fertilizáveis para a biotecnologia

reprodutiva. Esta questão é particularmente importante para canídeos, uma vez que a

taxas atuais de successo das técnicas in vitro que utilizam oócitos são muito limitadas,

e muitas espécies desta família encontram-se ameaçadas pela extinção. O objetivo

deste estudo foi avaliar os efeitos da temperatura, meio e tempo sobre a morfologia e

viabilidade de folículos pré-antrais caninos durante a preservação por curtos períodos.

Ovários caninos foram divididos em fragmentos, que foram incubados em solução de

NaCl a 0,9% ou em Meio Essencial Mínimo (MEM) a 4, 20 ou 38°C por 2, 6, 12 or 24

h. Os folículos pré-antrais inclusos nos fragmentos foram então analisados por

60

histologia, microscopia eletrônica de transmissão e teste de viabilidade utilizando-se

Azul de Tripan, calceína-AM e etídio homodímero-1. As percentagens de folículos

morfologicamente normais e viáveis foram mantidas similares ao controle (tempo 0 h)

após a incubação em salina a 4ºC ou 20ºC por até 6 h, e a 38°C por 2 h. Utilizando-se

MEM, tal preservação foi possível por 12 h a 4°C ou 20ºC, e por 6h a 38ºC. Estes

resultados indicam que a preservação de folículos pré-antrais caninos é realizada com

maior eficiência através do armazenamento hipotérmico (4 ou 20°C) em MEM, que

possibilita a manutenção da morfologia e viabilidade por até 12 h.

Palavras-Chave: Preservação; Hipotermia; Morfologia; Viabilidade; Folículos pré-antrais;

Canino.

Abstract

The use of the large pool of preantral follicles is a promising alternative to

provide high numbers of fertilizable oocytes to reproductive biotechnology. This issue is

particularly important to canids, since current rates of success of in vitro techniques

using oocytes are very limited, and many species within this family are threatened by

extinction. The aim of this study was to evaluate effects of temperature, medium and

time on morphology and viability of canine preantral follicles during short-term

preservation. Canine ovaries were cut into fragments which were incubated in 0.9%

NaCl solution or in Minimum Essential Medium (MEM) at 4, 20 or 38°C for 2, 6, 12 or

24 h. Afterwards, preantral follicles were analyzed by histology, transmission electron

microscopy and viability testing using trypan blue, calcein-AM and ethidium

homodimer-1. Percentages of morphological normal and viable follicles were

maintained similar to control (time 0 h) after incubation in 0.9% NaCl at 4ºC or 20ºC for

up to 6 h and at 38°C for 2 h. Using MEM, such preservation was possible for 12 h at

4°C or 20ºC, and for 6h at 38ºC. These results indicate that preservation of canine

preantral follicles might be better accomplished through hypothermic (4 or 20°C)

storage in MEM, which ensures maintenance of morphology and viability for up to 12

h.

Keywords: Preservation; Hypothermia; Morphology; Viability; Preantral follicle; Canine.

61

1. Introduction

Reproductive biotechnology has a great potential to contribute for the

conservation of endangered wildlife species. However, a limiting factor for the

development and efficiency of reproductive techniques is the lack of abundant numbers

of fertilizable oocytes. This problem could be addressed by using the large source of

oocytes enclosed in preantral follicles (Telfer, 2001), which exist in ovaries in numbers

of thousands to millions depending on the species. In vitro culture systems capable to

promote growth and maturation of these follicles have been developed for several

species, and birth of healthy offspring after embryo transfer of in vitro fertilized oocytes

derived from primordial follicles has, so far, been achieved in mice (Eppig and

Schroeder, 1989).

Due to the fact that time from ovary collection to the outset of in vitro culture can

be very long, especially when the donor animal is far from the reproductive laboratory,

as is the case with free-ranging individuals in the wild, preservation of viability is a

critical issue since cells are under ischemia. In this context, studies have assessed

short-term preservation of preantral follicles combining different temperatures and

media in several species such as caprine (Silva et al. 2000, 2001; Carvalho et al. 2001;

Ferreira et al. 2001), ovine (Andrade et al. 2001, 2002a,b; Matos et al. 2004), bovine

(Lucci et al. 2004) and swine (Lucci et al. 2007).

Conversely, to our knowledge, there is not to date a study on short term

preservation of canine preantral follicles. Few works have addressed the storage of

canine ovaries, but only effects on in vitro maturation (IVM) of oocytes from antral

follicles have been evaluated and findings are controversial. Taş et al. (2006)

demonstrated that bitch oocytes maintained at 4°C in physiologic saline had higher

maturation rates than those kept at 35-38°C. In contrast, Lee et al. (2006) observed

that viability of canine antral oocytes after in vitro maturation was significantly

decreased when ovaries were stored at 4°C in relation to 38°C.

Thus, methods for preservation of canine oocytes are still to be established, and

more studies are needed to gather consistent data. Once established, in vitro embryo

production in dogs based upon the vast pool of preantral follicles could be adapted to

endangered wild canids, which would contribute enormously to the multiplication of

individuals of such species. Nine canid species are under risk of extinction, which has

already occurred to the Falklands Wolf, Dusicyon australis (IUCN, 2007).

In this context, the aim of this study was to assess the preservation of canine

preantral follicles maintained in physiologic saline solution or in Minimum Essential

Medium (MEM) at different temperatures and times of storage. To this end, evaluation

62

of morphology through histological and ultrastructural analyses, as well as viability

assays based on trypan blue dye exclusion and the fluorescent probes calcein-AM and

ethidium homodimer were performed.

2. Materials and methods 2.1. Source and preparation of canine ovaries

Pairs of ovaries (n=15) were aseptically collected during ovariectomy of healthy

mixed-breed bitches (Canis familiaris) whose reproductive history and age were

unknown, but it was estimated that most were 12-24 months old and believed

nulliparous. After removal from the bursa ovarica, ovaries were rinsed once with 70%

ethanol for 10 seconds, twice in sterile phosphate-buffered saline (PBS) and eventual

corpora lutea were excised using a scalpel.

2.2. Experimental design

2.2.1. Experiment I: morphological evaluation of stored canine preantral follicles

Immediately after collection and preparation, pairs of ovaries (n=5) were divided

each into 25 fragments, from which one was randomly selected as control (time 0 h) and fixed for histology and ultrastructural analysis. The remaining fragments were

placed into 15 ml tubes (Corning Glass Works, Corning, NY, USA) containing 2 ml of

sterile saline solution (0.9% NaCl solution, osmolarity 300 mOsmol/l, pH 7.2) or

HEPES-buffered Minimum Essential Medium (MEM, osmolarity 280 mOsmol/l, pH7.2;

Sigma, St. Louis, MO, USA) at 4, 20 or 38ºC and stored for 2, 6, 12, or 24 h, and

subsequently fixed for morphological analysis as the non-stored control sample (Figure

1). Thermoflasks filled with water were used for maintaining temperatures, which were

monitored throughout the experiment. Each treatment was repeated five times.

63

Figure 1. Design of experiment I: pairs of canine ovaries were divided into 25

fragments, being one selected as non-stored control (time 0 h), whilst the others were

incubated in saline solution or in Minimum Essential Medium (MEM) at 4, 20 or 38ºC

for 2, 6, 12, or 24 h. Afterwards, fragments were submitted to morphological analysis.

2.2.2. Experiment II: assessment of viability of stored canine preantral follicles

In this study, effects of storage on canine preantral follicles were further

analyzed through assessment of viability. Five pairs of canine ovaries were cut each

into 25 fragments, and one was randomly selected and immediately submitted to

follicle isolation followed by trypan blue dye exclusion test as described later. The other

fragments were maintained in saline solution or in MEM at 4, 20 or 38°C for 2, 6, 12 or

24 h, and afterwards evaluated similarly as for the control.

Based on results of experiment I and trypan blue test, a second viability trial

was performed. Pairs of ovaries (n=5) were cut into three portions, from which one was

immediately processed for follicle isolation and testing using trypan blue as well as

calcein-AM and ethidium homodimer, which was employed to provide an additional and

more precise method of viability assessment. The remaining fractions were stored in

MEM at 4°C for 12 and 24 h, and afterwards analyzed similarly as for the control.

64

2.3. Histological analysis

In order to assess the morphology of canine preantral follicles submitted to the

different treatments, fragments of ovarian tissue were fixed in Carnoy for 12 h,

dehydrated in a graded series of ethanol, clarified with xylene, embedded in paraffin

wax and serially sectioned at 7 µm. Every fifth section was mounted on glass slides,

stained with periodic acid Schiff (PAS)-hematoxylin and evaluated by light microscopy

at a 400x magnification (Zeiss, Germany). Preantral follicles were defined as an oocyte

surrounded either by one layer of flattened or cuboidal granulosa cells, or several

layers of cuboidal granulosa cells with no antrum. Follicular morphology was evaluated

based on the integrity of the oocyte, granulosa cells and basement membrane.

Preantral follicles were classified and counted as (i) morphologically normal, when

containing an oocyte with regular shape and uniform cytoplasm, and organized layers

of granulosa cells, or (ii) degenerated, when the oocyte exhibited pycnotic nucleus

and/or ooplasma shrinkage, and occasionally granulosa cells layers became

disorganized, detached from the basement membrane and/or included enlarged cells.

To avoid evaluating and counting a follicle more than once, preantral follicles were

analyzed only in the sections where oocyte nucleus was observed.

2.4. Ultrastructure analysis

In order to better examine follicular morphology, transmission electron

microscopy (TEM) was performed to analyze ultrastructure of preantral follicles from

the control, as well as from treatments that did not differ statistically from control in

histology. A portion with a maximum dimension of 1 mm3 was cut from each fragment

of ovarian tissue and fixed in Karnovsky solution (2% paraformaldeyde and 2%

glutaraldeyde in 0.1 M sodium cacodylate buffer pH 7.2) for 3 h at room temperature

(RT, approximately 25°C). After three washes in sodium cacodylate buffer, specimens

were post-fixed in 1% osmium tetroxide, 0.8% potassium ferricyanide and 5 mM

calcium chloride in 0.1 M sodium cacodylate buffer for 1 h at RT. The samples were

then dehydrated through a gradient of acetone solutions and thereafter embedded in

Spurr’s epoxy resin. Afterwards, semi-thin sections (3 µm) were cut, stained with

toluidine blue and analyzed by light microscopy at a 400x magnification. Ultra-thin

sections (60–70 nm) were obtained from preantral follicles classified as morphologically

normal in semi-thin sections, according to the criteria adopted in histology.

Subsequently, ultra-thin sections were contrasted with uranyl acetate and lead citrate,

65

and examined under a Jeol 1011 (Jeol, Tokyo) transmission electron microscope

operating at 80 kV.

2.5. Assessment of preantral follicles viability

Canine preantral follicles were isolated from control and stored ovarian

fragments using the mechanical method described by Figueiredo et al. (1993). Briefly,

using a tissue chopper (The Mickle Laboratory Engineering Co., Gomshal, Surrey, UK)

adjusted to a sectioning interval of 87.5 µm, samples were cut into small pieces, which

were placed in MEM and suspended 40 times using a large Pasteur pipette (diameter

of about 1600 µm) and subsequently 40 times with a smaller Pasteur pipette (diameter

of approximately 600 µm) to dissociate preantral follicles from stroma. The obtained

material was passed through 500- and 100-μm nylon mesh filters, resulting in a

suspension containing preantral follicles smaller than 100 μm in diameter. This

procedure was carried out within 10 min at RT.

Thereafter, viability of preantral follicles was assessed through trypan blue dye

exclusion test (Jewgenow et al., 1998). Briefly, 10 μl of 0.4% trypan blue (Sigma,

Deisenhofen, Germany) were added to 90 μl of the suspension of isolated preantral

follicles, which were examined using an inverted microscope after incubation for 5 min

at RT. Follicles were classified as viable if the oocyte and <10% granulosa cells

remained unstained, or as non-viable if uptake of the dye by the oocyte and/or ≥10%

granulosa cells occurred.

Preantral follicles were also analyzed using a two-color fluorescence cell

viability assay based on the simultaneous determination of live and dead cells by

calcein-AM and ethidium homodimer-1, respectively. Whilst the first probe detected

intracellular esterase activity of viable cells, the later labeled nucleic acids of non-viable

cells with plasma membrane disruption. Since it is difficult to distinguish the outline of

live cells labeled with calcein-AM within follicle structure, determination of the

percentage of viable granulosa cells was achieved through using Hoechst 33343 to

detect all nuclei, enabling counting of the total number of cells. The test was performed

by adding 4 μM calcein-AM, 2 μM ethidium homodimer-1 (Molecular Probes,

Invitrogen, Karlsruhe, Germany) and 10 μM Hoechst 33342 (Sigma, Deisenhofen,

Germany) to the suspension of isolated follicles, followed by incubation at 37°C for 10

min. After being labeled, follicles were washed three times by centrifugation at 100xg

for 5 min and resuspension in MEM, mounted on a glass microscope slide in 5 μl

antifading medium (DABCO, Sigma, Deisenhofen, Germany) to prevent

photobleaching, and finally examined using an a DMLB fluorescence microscope

66

(Leica, Germany). The emitted fluorescent signals of Hoechst, calcein-AM, and

ethidium homodimer were collected at 350, 488, and 568 nm, respectively. Oocytes

and granulosa cells were considered live if the cytoplasm was stained positively with

calcein-AM (green) and chromatin was not labelled with ethidium homodimer (red).

Preantral follicles were classified as viable when the oocyte and <10 % of granulosa

cells were live, and as non-viable, when the oocyte and/or ≥10 % granulosa cells were

dead.

2.6. Statistical analysis

All experiments were replicated five times. Analysis of variance (ANOVA) of the

data was performed using the GLM procedure of the software SAS (SAS Institute Inc.,

Cary, NC, USA) with a factorial design (medium, temperature and time of storage).

Differences of percentages of morphologically normal preantral follicles (MNPF) and

viable follicles between control and treatments (combinations of medium, temperature

and time of storage) were determined by Duncan’s multiple range test. Differences

were considered statistically significant when P < 0.05.

3. Results

3.1. Experiment 1: Morphological evaluation of stored canine preantral follicles

3.1.1. Histological analysis

A total of 4,045 preantral follicles were examined in histology, with a range of

146 to 179 in each treatment. Proportions of preantral follicles at each developmental

stage were similar among fragments obtained from a pair of ovaries and thus follicular

diameters were homogenous between treatments. Most of the studied follicles were at

the primordial or resting stage and had a diameter of 25-30 µm. Primary or activated

follicles (30-40 µm) and secondary follicles (40-100) were also analyzed in lower

numbers equally found in all experimental groups. MNPF in control as well as after

storage exhibited a spherical or elliptical oocyte with a large central or eccentric

nucleus and uniform cytoplasm. Granulosa cells without pycnotic nuclei were well-

organized in layers surrounding the oocyte and a distinguishable intact basement

membrane could be observed (Fig. 2A-C). Degenerated follicles showed a retraced

oocyte with or without a pycnotic nucleus, and occasionally a strongly eosinophilic

cytoplasm (Fig. 2D). Layers of granulosa cells remained unaltered or became

67

disorganized into a low density mass of cells which were many times swollen and/or

detached from basement membrane. Occurrence of pycnotic bodies in granulosa cells

or rupture of the basement membrane were not observed.

Fig. 2. Histological features of stored canine ovarian fragments. Morphologically normal

primordial (A) and primary (B) follicles comprised an oocyte (o) displaying a large

nucleus (nu) and homogenous cytoplasm surrounded by one layer of flattened or

cuboidal granulosa cells (gc) respectively, whilst in secondary follicles two or more

layers were present without antrum (C). Degenerated preantral follicles often displayed

oocyte retraction (D, arrow) and disorganization of granulosa cell layers. The

arrowhead indicates an intact basement membrane. Scale bars represent 6 µm (A) and

10 µm (B,C and D). PAS-hematoxilin.

68

The effects of medium, temperature and time of storage on the percentages of

MNPF are shown in Fig. 3. Maintenance in saline solution at 4°C for up to 24 h, at 20°C

for up to 12 h or at 38°C for 6 h kept proportions of MNPF similar (P > 0.05) to control

values (time zero). A decrease (P < 0.01) of the percentages of MNPF in relation to the

control was observed after holding in saline solution at 20°C for 24 h and at 38°C for 12

and 24 h. With respect to MEM, similar results were obtained, with exception of the

maintenance of follicles at 20°C for 24 h and at 38°C for 12 h, for which percentages of

MNPF were also similar (P > 0.05) to control values.

Fig. 3. Effects of medium, temperature and time of storage on the percentages of

morphologically normal canine preantral follicles. (*) Differs significantly from the

control (P < 0.05); (a, b, c) different letters for the same medium and time of incubation

denotes significant differences between temperatures (P < 0.05); (A, B, C) different

letters for a given combination of medium and temperature indicates significant

differences among times of incubation (P < 0,05); (X,Y) different letters represent

significant differences between media for fixed time and temperature of incubation.

The effect of storage time on the percentages of MNPF at each temperature

and for each solution was analyzed. At 4°C, percentages of MNPF were not affected by

the incubation time, for both solutions. Similar results were obtained after preservation

in MEM at 20°C. Otherwise, when ovarian fragments were kept at 20°C in saline

solution, there was a decrease (P < 0.01) in the percentages of MNPF after 24 h

compared to 2, 6 or 12 h. A reduction (P < 0.05) in the proportions of MNPF occurred

69

at 38°C with the increase of incubation time to 12 h in saline solution and to 24 h in

MEM.

Regarding the effect of temperature on percentages of MNPF for a defined

period of storage, a decrease (P < 0.05) in percentages of MNPF was observed when

ovarian fragments were maintained in MEM for 24 h at 38ºC in comparison to 4ºC.

The comparison between saline solution and MEM at a same temperature and

storage period showed that at 38°C after 12 h, percentages of MNPF were higher when

ovarian fragments were maintained in MEM.

3.1.2. Ultrastructure analysis

Transmission electron microscopy of stored preantral follicles was performed for

assessment of ultrastructure in comparison to normal follicles from the control group. At

least five follicles per group were analyzed. Normal follicles contained an oocyte

displaying a very homogenous cytoplasm plenty of round shaped mitochondria with

continuous membranes, few peripheral cristae and electron-dense granules. Some

elongated forms with parallel cristae could also be seen. Small Golgi apparatus

cisternae were rarely observed. Smooth and rough endoplasmic reticulum were

present, either as isolated aggregations or as complex associations with mitochondria.

The nuclei of oocytes were large and usually round, well delimited by the nuclear

envelope, the chromatin was uncondensed and a nucleolus could often be identified.

Granulosa cells were small and presented a high nucleus-to-cytoplasm ratio. Their

irregularly shaped nuclei contained loose chromatin in the inner part and small

peripheral aggregates of condensed chromatin. The cytoplasm exhibited a great

number of mitochondria and well-developed smooth and rough endoplasmic reticulum.

The cellular membranes of oocyte and surrounding granulosa cells were closely

justaposed, and sometimes few short microvilli could be observed. A distinct

continuous basement membrane surrounded follicles and was tightly attached to

ovarian stroma (Fig. 4).

70

Fig. 4. Electron micrographs of normal follicles from control group (A) and stored in

MEM at 4°C for 12 h (B). O, oocyte; GC, granulosa cells; ne, nuclear envelope; m,

mitochondria; ser, smooth endoplasmic reticulum; bm, basement membrane; sc,

stromal cells. (A, 5000x, scale bar=5 µm; B, 10000x, scale bar = 2 µm).

The ultrastructural pattern described above was observed in normal follicles

from control as well as after storage in MEM at 4°C or 20°C for up to 12 h and at 38°C

for up to 6 h; in saline solution at 4°C or 20°C for up to 6 h. When ovarian fragments

were incubated at 4°C in MEM or in saline solution for 24 h and 12 h respectively, and

at 38ºC in saline solution for 2 h, discreet changes in ultrastructure suggesting onset of

degeneration could be observed in follicles evaluated as morphological normal in semi-

thin sections by light microscopy. Some mitochondria both in the oocyte and in

granulosa cells were very enlarged, displayed fewer cristae and lower electron density

of the matrix, which may indicate swelling (fig. 5).

71

Fig. 5. Ultrastructure of follicles stored in MEM at 4°C for 24 h. Mitochondria displaying

substantial enlargement, loss of peripheral cristae and an electron-lucent matrix

(arrows) were observed both in oocytes (A) and granulosa cells (B). O, oocyte; GC,

granulosa cells; nu, nucleolus; m, mitochondria; bm, basement membrane. (A, 6000x,

scale bar=5 µm; B, 10000x, scale bar = 2 µm).

More severe alterations of ultrastructure were noticed after storage in saline

solution at 4ºC for 24 h, at 20ºC for 12 h and at 38ºC for 6 h; in MEM at 20ºC for 24 h

and at 38ºC for 12 h. Follicles contained high numbers of vacuoles spread throughout

the cytoplasm of all cells, which often fused creating large vacated areas. Furthermore,

signs of proliferation of the endoplasmic reticulum and damage to mitochondrial

membranes and cristae were observed. Granulosa cells had a swollen aspect and

presented a very low density of organelles. Some of these cells completely

disappeared, resulting in a empty space. In oocytes, swelling of the nuclear cisterna

and disruption of the nuclear envelope were observed along some segments of the

nuclear outline (fig. 6). In some follicles kept in MEM at 20ºC for 24 h, the cytoplasm of

oocyte and granulosa cells seemed to be clotted, possibly due to nuclear content leak.

72

Fig. 6. Electron micrographs of follicles stored in saline solution at 4°C for 24 h. A

primordial follicle containing several vacuoles spread throughout the ooplasma as

indicated by arrows (A, 5000x, scale bar=5 µm); the insert is displayed at a higher

magnification (B, 12000x, scale bar=2 µm), note the occurrence of nuclear cisterna

swelling (arrowhead). Another primordial follicle exhibiting widespread vacuolization

and a granulosa cell with a very large vacated area indicated by an asterisk (C, 8000x,

scale bar=2 µm); a higher magnification of the same follicle (D, 15000x, scale bar=2

µm), observe the discontinuity of the nuclear envelope (thick arrow). O, oocyte; GC,

granulosa cells; nu, nucleolus; ne, nuclear envelope; m, mitochondria; bm, basement

membrane.

73

3.2. Experiment II: assessment of viability of stored canine preantral follicles

Preantral follicles were mechanically isolated from control and stored canine

ovarian fragments and viability was assessed by trypan blue dye exclusion test (fig. 7).

A total of 6,037 follicles were examined, with a range of 171 to 281 in each treatment.

Fig. 7. Viability assessment of canine preantral follicles using trypan blue (TB) dye

exclusion test. Isolated preantral follicles were incubated with 0.04% TB for five

minutes and then classified as viable if the oocyte and <10% granulosa cells remained

unstained (A), or as non-viable if oocyte and/or ≥10% granulosa cells were stained (B).

Scale bars represent 10 µm.

The effects of medium, temperature and time of storage on the percentages of

viable preantral follicles are shown in Table 1. Maintenance in saline solution at 4°C or

20ºC for up to 6 h kept proportions of viable follicles similar (P > 0.05) to control values

(time zero). A decrease (P < 0.01) of the percentages of viable follicles in relation to the

control was observed after holding in saline solution at 4ºC or 20°C for 12 and 24 h,

and at 38°C after all incubation times. With respect to MEM, storage of follicles at 4ºC

or 20ºC for up to 12 h, and at 38°C for up to 6 h preserved viable follicles, whose

percentages were similar (P > 0.05) to control values.

74

Table 1.

Percentages (viable/total) of viable canine preantral follicles in control and after storage.

Medium Temperature

(°C)

Time (h)

2 6 12 24

Control 67 (186/279)

Saline 4º 55% abA

(151/276)

52% abA

(132/255)

41% *abA

(107/259)

37% *abA

(87/234)

20º 54% abA

(123/229)

52% abAB

(145/281)

39% *abB

(97/246)

20% *bC

(35/171)

38º 43% *bA

(96/222)

43% *bA

(115/265)

30% *bB

(69/231)

23% *bB

(45/195)

MEM 4º 67% aA

(172/258)

61% aA

(159/259)

64% cA

(176/275)

48% *aB

(96/200)

20º 65% aA

(171/264)

58% abAB

(160/277)

60% cAB

(162/272)

32% *abB

(59/185)

38º 62% aA

(138/222)

58% abAB

(148/255)

47% *aB

(111/238)

22% *bC

(42/189)

(*) Differs significantly from control (P < 0.05); (a, b, c) different letters denotes

significant difference between values within a column (P < 0.05); (A, B, C) different letters

indicates significant difference between values of a row (P < 0,05).

The effect of storage time on the percentages of viable follicles at each

temperature and for each solution was analyzed. At 4°C, percentages of viable follicles

in saline solution were not affected by the incubation time, but a significant reduction (P

< 0.05) was observed in MEM from 12 to 24 h of holding. When ovarian fragments

were kept at 20°C in saline solution, there was a decrease (P < 0.01) of the

percentages of viable follicles after 12 h, and a further reduction from 12 h to 24 h; in

MEM, this reduction was observed after 24 h only. A decrease (P < 0.05) in the

proportions of viable follicles occurred with the increase of incubation time to 12 h in

both solutions at 38°C. An additional decrease (P < 0.01) was observed in MEM at

38°C from 12 to 24 h.

75

Regarding the effect of temperature on percentages of viable follicles for a

defined period of storage, a reduction (P < 0.05) in the viability of follicles was

observed when ovarian fragments were maintained in MEM at 38°C for 12 h compared

to 4ºC and 20ºC, and for 24 h, in relation to 4ºC only.

The comparison between saline solution and MEM at a same temperature and

incubation period showed that after storage at 38ºC for 2 h, and at all temperatures for

12 h, percentages of viable follicles were higher when ovarian fragments were

maintained in MEM.

Based on the results of morphological evaluation (experiment I) and viability

assessment with trypan blue, which evidenced that storage in MEM at 4ºC is able to

maintain viability of canine preantral follicles for the longer time (12 h) and to preserve

ultrastructure more efficiently, as verified after 24 h of incubation, a second viability trial

using this protocol was performed with five replicates. In addition to trypan blue testing,

a fluorescence cell viability assay based on labeling of live and dead cells by calcein-

AM and ethidium homodimer-1, respectively, was employed. Hoechst 33343 was

integrated to the test to allow counting of total cell number for calculation of the

percentage of viable granulosa cells within each follicle (fig. 8).

76

Fig. 8. Viability assessment of canine preantral follicles using fluorescent probes. (A)

An isolated preantral follicle classified as viable (B) since cells were labeled by calcein-

AM (green fluorescence). (D) A secondary follicle considered non-viable (E) as cells

were marked with ethidium homodimer-1 (red fluorescence). (C, F) The same follicles

in A and D respectively, stained with Hoechst 33342, which was also used in the assay

to allow counting of the total number of cells for calculation of the percentage of dead

cells. Scale bars represent 20 µm.

After storing ovarian fragments in MEM at 4ºC for 12 h, percentages of viable

follicles remained unaltered in comparison to control, as assessed by both trypan blue

and calcein-AM/ethidium homodimer assays, whose values did not differ from each

other (P < 0.01). However, a significant decrease (P < 0.05) in percentages of viable

follicles was observed with the increase of incubation to 24 h according to the two

viability tests, which once more retrieved similar results (fig.9).

77

Fig. 9. Percentages of viable canine preantral follicles in fresh ovaries and after storage

in MEM at 4ºC for 12 h or 24 h as assessed by trypan blue dye exclusion test and

labeling with calcein-AM and ethidium homodimer. (*) Differs significantly from control

(P < 0.05); (a, b) different letters for values of viability assays within the same time of

incubation denotes significant differences (P < 0.05); (A, B) different letters for results

of a same test indicates significant differences between times of incubation (P < 0,05).

4. Discussion

The present study evaluated for the first time the short term preservation of

canine preantral follicles, which can be successfully accomplished using either 0.9%

NaCl solution or MEM, but efficiency of each medium depends on temperature and

time of holding.

Percentages of MNPF remained unaltered, as observed by histological

analysis, after incubation at 4ºC in both media for up to 24 h, and at 20ºC in saline

solution and MEM for up to 12 h and 24 h respectively. Conversely, holding at

physiologic temperature (38ºC) maintained percentages of MNPF for only 6 h in saline

solution and 12 h in MEM. Therefore, hypothermia during storage of canine ovaries

provides more efficient preservation of morphology of preantral follicles. This may be

explained by a reduction of metabolism, followed by a decrease in requirements of

oxygen and nutrients as well as metabolites production, which is critically important

since cells are under ischemia. Indeed, hypothermia has proven to be an exceptional

means of protection of any organ, with metabolic rate falling exponentially by about

78

50% per 6°C drop in organ temperature (Kirklin & Barrat-Boyes, 1993). Our results are

in accordance with other works, which reported that maintenance at 4ºC and 20ºC

preserves morphology of bovine (Lucci et al., 2004), caprine (Carvalho et al., 2001;

Ferreira et al., 2001; Silva et al., 2000, 2001) and ovine (Andrade et al., 2001; Matos et

al., 2004) preantral follicles for longer times than at physiological temperature. In

addition, Wood et al. (1997) showed that maintenance in saline at 4ºC inhibited

degeneration of cat follicles for 48 h after excision. Transmission electron microscopy revealed that, after storage in saline solution

at 4ºC and 20ºC for 12 h, at 38°C for 2 h, in MEM at 4ºC and 20ºC for 24 h and at 38ºC

for 12 h, preantral follicles considered morphologically normal in histology had

alterations in ultrastructure indicative of degeneration. Therefore, electron microscopy

proved to be a valuable tool to detect early morphological modifications due to atresia,

which can be observed only at the ultrastructural level before becoming pronounced

more grossly to be identified through light microscopy. Ultrastructure of ovine (Matos et

al., 2004) and caprine (Silva et al., 2000) preantral follicles could be preserved for up to

24 h at 4ºC. The shorter periods of preservation of ultrastructure observed in the

present study may be due to a higher sensibility of canine preantral follicles to ischemia

and/or to cold storage in relation to follicles of small ruminants.

Follicles in ovarian fragments maintained at 4ºC in MEM and saline solution for

24 h and 12 h respectively, and at 38ºC in saline solution for 2 h displayed the least

degree of damaging, containing some enlarged mitochondria with reduced cristae and

electron-lucent matrix. Silva et al. (2001) observed that damage to mitochondria is one

of the first signs of degeneration in goat preantral follicles during storage in vitro.

Ischemia impairs cellular energetic metabolism which decreases the activity of the

Na+/K+-ATPase pump with a gain in sodium (Bonz et al., 1998). During mild ischemia,

mitochondrial matrix swells moderately due to uptake of sodium (Garlid, 1996).

Incubation in MEM at 20ºC for 24 h and at 38ºC for 12 h, and in saline solution

at 4ºC for 24 h and at 20ºC for 12 h led to more severe changes characterized mainly

by high numbers of vacuoles spread throughout the cytoplasm of both oocyte and

granulosa cells. Silva et al. (2001) demonstrated that structural changes due to

degeneration progress with the increase of preservation temperature and incubation

time. Atresia in vivo is also characterized by cytoplasmic vacuoles in oocytes (van den

Hurk et al., 1998) and granulosa cells (Hay et al., 1976), which may be originated from

endoplasmic reticulum swelling. On other hand, these vacuoles may be derived from

altered mitochondria, as observed in cryopreserved bovine oocytes by Fuku et al.

(1995).

79

Notwithstanding previous studies on preservation of preantral follicles provided

important knowledge on this issue, approaches were limited to analysis of morphology,

which is not always correlated with the viability of follicles (Santos et al., 2007).

Therefore, in the present work, viability assessment using the trypan blue dye

exclusion test was employed. It was observed that storage in MEM at 4ºC and 20ºC

maintained percentages of viable preantral follicles for up to 12 h, whilst at 38ºC such

preservation was possible for up to 6 h. In saline, percentages of viable follicles were

kept at 4ºC and 20ºC for up to 6 h, and at 38ºC, a significant decrease of viability was

observed after only 2 h of incubation. Percentages of viable follicles were much lower

than those of morphologically normal follicles in every treatment. This proves that a

more precise determination of the proportions of follicles with adequate quality for

subsequent applications such as in vitro culture may rely on viability assessment.

We further analyzed viability of canine preantral follicles stored in MEM at 4ºC,

the protocol which maintained proportions of viable follicles for the longer time (12 h)

while preserving ultrastructure more efficiently after 24 h, using a more accurate

method based on fluorescent probes. For this purpose, labeling of non-viable cells with

disruption of plasma membrane was performed again by using ethidium homodimer-1,

which enabled a better individualized visualization of dead cells through fluorescent

staining of nuclei. Concomitantly, identification of viable cells was performed through

detection of esterase activity with calcein-AM. Hoechst 33342 was used to mark all

nuclei in each follicle for total cell number counting and calculation of percentages of

live cells. Results of this assay were similar to values observed with trypan blue testing,

which thus proved to be a reliable, practical and quick method for viability assessment

of preantral follicles. Accordingly, Poeschmann et al. (2008) observed a significant

correlation between both methods while analyzing viability of feline preantral follicles.

In this study, MEM was more efficient than saline solution to preserve viability of

follicles after 12 h of incubation at any tested temperature. In addition, at 38ºC, MEM

was able to maintain viability similar to control for 6 h, whilst, in saline solution,

percentages of viable follicles were decreased after only 2 h of incubation.

Furthermore, after 24 h at 4ºC, ultrastructure of follicles was altered to a lesser extent

in ovarian fragments maintained in MEM. Therefore, composition of medium is critical

for short-term storage of canine preantral follicles. We infer that endogenous nutrient

resources in these follicles are very limited, thus it is necessary to provide

supplementary nutritional compounds in preservation solutions. Although hypothermia

may have reduced metabolic rates, these were possibly high enough to have depleted

own energetic sources of follicles kept in saline at 4ºC and 20ºC after 6 h, leading to

80

degeneration, whereas MEM, which comprises sugars, aminoacids, vitamins and

inorganic salts, supported survival for up to 12 h.

In conclusion, preservation of canine preantral follicles during storage is

achieved more efficiently through hypothermia in a nutritive medium. Maintenance of

viability and ultrastructural integrity can be accomplished at 4°C and 20ºC in saline

solution for 6 h or in MEM for 12 h, being the later solution also adequate at 38ºC for 6

h. We suggest the use of MEM at 4ºC for up to 12 h in order to provide optimal

preservation of quality of canine preantral follicles for subsequent applications such as

in vitro culture.

Acknowledgements

During the period of this work, Claudio A. P. Lopes received financial support

from the National Council for Scientific and Technological Development (CNPq;

doctoral scholarship) and the Coordination for Improvement of Graduate Personnel

(CAPES; fellowship for studies abroad, PDEE, grant 5212/06-5), both institutions of the

Brazilian Government. José Ricardo de Figueiredo was also recipient of a grant from

CNPq. We would like to express our appreciation to the Center for Zoonosis Control

(CCZ) of Fortaleza Municipality (Brazil) and to the veterinary clinics of the animal

shelter Tierheim-Berlin and Tierklinik-Biesdorf (Germany), which gently provided the

dog ovaries. We also thank Dr. Vicente J. F. Freitas (LFCR, UECE) for logistical

support.

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Silva, J.R.V., Lucci, C.M., Carvalho, F.C.A., Báo, S.N., Costa, S.H.F., Santos, R.R.,

Figueiredo, J.R., 2000. Effect of coconut water and Braun–Collins solutions at

different temperatures and incubation times on the morphology of goat preantral

follicles preserved in vitro. Theriogenology 54, 809–22.

Silva, J.R.V., Báo, S.N., Lucci, C.M., Carvalho, F.C.A., Andrade, E.R., Ferreira, M.A.L.,

Figueiredo, J.R., 2001. Morphological and ultrastructural changes occuring during

degeneration of goat preantral follicles preserved in vitro. Anim. Reprod. Sci. 66,

209–23.

Taş, M., Evecen, M., Özdaş, Ö.B., Cirit, Ü, Demir, K., Birler, S., Pabuccuğlu, S., 2006.

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bitch oocytes. Anim. Reprod. Sci. 96, 30–34.

Telfer, E.E., 2001. In vitro development of pig preantral follicles. Reprod. Suppl. 58,

81–90.

van den Hurk, R., Spek, E.R., Hage, W.J., Fair, T., Ralph, J.H., Schotanus, K., 1998.

Ultrastructure and viability of isolated bovine preantral follicles. Hum. Reprod.

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taphonomy in cold-stored domestic cat ovaries. Mol. Reprod. Dev. 46, 190–200.

83

8 CAPÍTULO III

Criopreservação de folículos pré-antrais caninos utilizando-se etilenoglicol e

glicerol

Cryopreservation of canine preantral follicles using ethylene glycol and glycerol

Brazilian Journal of Veterinary Research

(Submetido, 2008)

84

Cryopreservation of canine preantral follicles using ethylene glycol and glycerol C.A.P. Lopes1,3, M.C.A. Luque2, S. B. Vasconcelos2, G.M. Silva1, S.N. Báo2, J.R. Figueiredo1 1 LAMOFOPA, PPGCV, Faculty of Veterinary, State University of Ceará, Brazil. 2 Laboratory of Electron Microscopy, Institute of Biology, University of Brasília, Brazil. 3 Corresponding author. Email: [email protected]

Resumo

O objetivo do presente estudo foi avaliar os efeitos da congelação lenta-

descongelação rápida sobre a morfologia de folículos pré-antrais caninos utilizando-se

glicerol e etilenoglicol. Fragmentos de tecido ovariano canino foram equilibrados por

20 min a 20ºC em Meio Essencial Mínimo contendo um dos crioprotetores

mencionados à concentração de 1,5 M, sendo então congelados, estocados em

nitrogênio líquido (-196ºC) por uma semana e descongelados. A morfologia dos

folículos foi analisada através de histologia e microscopia eletrônica de transmissão.

As percentagens de folículos pré-antrais morfologicamente normais (FPMN) foram

significativamente reduzidas após a exposição ao glicerol em relação ao controle, o

que não foi observado com o etilenoglicol. Após a congelação/descongelação, as

percentagens de FPMN apresentaram-se significativamente inferiores em comparação

ao controle utilizando-se ambos crioprotetores, que não diferiram estatisticamente

entre si. Entretanto, a microscopia eletrônica de transmissão revelou que apenas os

folículos congelados com etilenoglicol possuíam ultra-estrutura normal. Como

conclusão, este estudo demonstrou que a morfologia de folículos pré-antrais caninos

pode ser preservada através de congelação lenta seguida de descongelação rápida

utilizando-se etilenoglicol a 1,5 M. Abstract

The aim of the present study was to evaluate the effects of slow freezing-rapid

thawing using glycerol and ethylene glycol on morphology of canine preantral follicles.

Slices of canine ovarian tissue were equilibrated for 20 min at 20ºC in minimum

essential medium containing either cryoprotectants at 1.5 M, and then frozen, stored in

liquid nitrogen (-196ºC) for a week and thawed. Morphology of follicles was analyzed

through histology and transmission electron microscopy. The results showed that the

percentages of morphologically normal preantral follicles (MNPF) were significantly

85

reduced after exposure to glycerol, but not to ethylene glycol in comparison to the

control. After freezing/thawing, the percentages of MNPF were significantly decreased

as compared to the control using either cryoprotectants, which were statistically similar

in relation to each other. However, transmission electron microscopy revealed that only

follicles frozen with ethylene glycol had normal ultrastructure. In conclusion, this study

demonstrated that morphology of canine preantral follicles can be successfully

preserved through slow freezing-rapid thawing using 1.5 M ethylene glycol.

Keywords: cryopreservation, slow freezing, preantral follicles, canine.

Introduction

Cryopreservation of gametes is potentially an important tool for the conservation

of endangered species and biodiversity maintenance. Significant advances in this field

has been obtained through genome resource banking which has been allowed by the

relative success achieved with semen cryopreservation, in which widely implemented

protocols are used. Conversely, almost all mammalian species oocytes studied have

proven very difficult to successfully cryopreserve (Woods et al., 2004).

The process of cryopreservation can cause degeneration of oocytes,

disassembly of cell microtubules resulting in the disruption of the spindle apparatus

followed by aneuploidy, and zona pellucida (ZP) hardening which thus may impair the

subsequent fertilization process. These effects can be a direct consequence of

intracellular ice-crystal formation, rapid or massive osmotic and temperature change

and the relative toxicity of cryoprotectant under certain conditions (Ko et al., 2008). In

order to overcome these problems, the cryopreservation of preantral ovarian follicles

emerges as a promising alternative for female gamete cryobanking. Immature oocytes

in preantral follicles still have not metaphase spindle fibers and lack a ZP and cortical

granules. Moreover, such oocytes are far smaller and less differentiated than

Metaphase II oocytes. All these characteristics are potentially beneficial for freeze

preservation (Oktay et al., 1998). Together with these qualitative advantages, this

strategy for female gamete banking can also potentially provide a much higher quantity

of fertilizable oocytes, because thousands of these follicles can be obtained from an

ovary (Rutherford & Gosden, 1999).

Cryopreservation of isolated primordial follicles through slow freezing was

successfully achieved in the mouse (Carroll and Gosden, 1993). Follicles were

transplanted to sterilized hosts which could afterwards produce normal offspring after

mating. Live young were also obtained after in vitro culture, maturation and fertilization

86

of mouse oocytes within frozen-thawed secondary follicles (Smitz and Cortvrindt,

1998). More recently, live births after orthotopic autotransplantation of cryopreserved

ovarian tissue were described in a woman (Donnez et al., 2004), as well as in ewes

(Bordes et al., 2005).

Although cryopreservation of preantral follicles from carnivores would be

specially important since many wild species of these are currently threatened by

extinction, few studies have been published on this topic. Jewgenow et al. (1998)

demonstrated that small preantral follicles isolated from cat ovaries survive

cryopreservation, remaining structurally intact and physiologically active after thawing.

In another work, Bosch et al. (2004) reported the survival of cat ovarian tissue

subjected to slow freezing-thawing and transplantation to an immunocompromised

host, which was followed by development of follicles from early up to the antral stage.

With respect to canids, to our knowledge, only one attempt to cryopreserve preantral

follicles is reported. Ishijima et al. (2006) vitrified canine ovarian cortex and

xenotransplanted to immunodeficient mice. Although antral follicle formation did not

occur, grafted tissue was morphological normal and proliferating cell nuclear antigen

immunoreactivity was detected in primary follicles. Nonetheless, detailed morphological

and quantitative analyses on cryopreservation of canine preantral follicles are still to be

performed, and knowledge gathered in this species could be applied to endangered

canids.

The aim of this study was to evaluate the cryopreservation of canine preantral

follicles through slow freezing of ovarian tissue using 1.5 M glycerol (GLY) or ethylene

glycol (EG). For this purpose, morphology of follicles after exposure to the

cryoprotectants and freezing-thawing was analyzed through histology and transmission

electron microscopy (TEM).

Materials and Methods

Collection and preservation of canine ovaries

Ovarian pairs (n=5) were aseptically collected at local veterinary clinics during

ovariohysterectomy of healthy mixed-breed bitches (Canis lupus familiaris), whose

reproductive history and age were unknown, but it was estimated that they were 12-24

months old. After removal from the bursa ovarica and excision of eventual corpora

lutea using a scalpel, ovaries were placed into 50 ml tubes (Corning Glass Works,

Corning, NY, USA) containing 15 ml of HEPES-buffered Minimum Essential Medium

(H-MEM, osmolarity 280 mOsmol/l, pH7.2; Sigma, St. Louis, MO, USA). Then,

87

transportation to the laboratory was carried out at 4°C within one hour in thermoflasks

filled with water. All procedures were performed aseptically.

Exposure test

At the laboratory, five fragments of cortex (approximately 3 x 3 x 1 mm) were

cut from each pair of ovaries, and one of these pieces was randomly selected as

control and fixed for histology and ultrastructural analysis. The remaining samples were

placed into cryovials containing 1.8 ml of freezing medium which consisted of Minimum

Essential Medium (MEM) supplemented with 10% fetal bovine serum (FBS) and 1.5 M

GLY or EG, prepared immediately prior to use under sterile conditions. After incubation

at 20ºC for 20 min (equilibration period), half of the fragments was subjected to

cryoprotectant removal according to the method described by Candy et al. (1997).

Briefly, three washes of 5 min each in MEM with 10% FBS were performed at room

temperature (RT, approximately 25°C). Samples were subsequently fixed for histology

and TEM in order to evaluate effects of exposure to cryoprotectants on preantral

follicles.

Freezing-thawing

The cryovials with the remaining fragments were transferred to a biological

programmable freezer (Freeze Control, CryoLogic Pty Ltd.,Waverley, Australia) pre-set

at 20 °C. The following program was used to freeze the tissue samples: cooling from

20 to -7°C at a rate of 2°C/min; holding at -7°C for 15 min (after 5 min, ice crystal

formation - seeding - was induced manually by touching the vials with a forceps pre-

chilled in liquid nitrogen); temperature was then lowered 0.3°C/min to -30°C, and

further reduced by 0.15°C/min to -33°C; finally, the vials were plunged into liquid

nitrogen (-196°C) and stored for 7 days. Thereafter, ovarian tissue pieces were thawed

through rapid warming by exposing the cryovials to air at RT for 1 min, followed by

immersion in a water bath at 38ºC for 3 min. Cryoprotectant removal was carried as

describe for the exposure test, and pieces of ovarian cortex were fixed for histological

and ultrastructural analyses. This experiment was repeated five times.

Histological analysis



88


wax and serially sectioned at 7 μm. Every fifth section was mounted on glass slides,








of granulosa cells; (ii) degenerated grade 1, when the oocyte exhibited pycnotic

nucleus and/or ooplasma shrinkage; (iii) degenerated grade 2, when in addition to

oocyte damage, granulosa cells layers became disorganized, detached from the

basement membrane and/or included enlarged cells. To avoid evaluating and counting

a follicle more than once, preantral follicles were analyzed only in the sections where

oocyte nucleus was observed. All the slides were coded to prevent observer bias.

Ultrastructural analysis

In order to better examine follicular morphology, TEM was performed to analyze

ultrastructure of preantral follicles from the control as well as from treatments that did

not differ statistically from control in histology. A portion with a maximum dimension of

1 mm3 was cut from each fragment of ovarian tissue and fixed in modified Karnovsky

solution (2% paraformaldeyde and 2% glutaraldeyde in 0.1 M sodium cacodylate buffer

pH 7.2) for 3 h at room temperature (RT, approximately 25°C). After three washes in

sodium cacodylate buffer, specimens were post-fixed in 1% osmium tetroxide, 0.8%

potassium ferricyanide and 5 mM calcium chloride in 0.1 M sodium cacodylate buffer

for 1 h at RT. The samples were then dehydrated through a gradient of acetone

solutions and thereafter embedded in Spurr’s epoxy resin. Afterwards, semi-thin

sections (3 μm) were cut, stained with toluidine blue and analyzed by light microscopy

at a 400x magnification. Ultra-thin sections (60–70 nm) were obtained from preantral

follicles classified as morphologically normal in semi-thin sections, according to the

criteria adopted in histology. Subsequently, ultra-thin sections were contrasted with

uranyl acetate and lead citrate, and examined under a Jeol 1011 (Jeol, Tokyo)

transmission electron microscope operating at 80 kV.

89

Statistical analysis

All experiments were replicated five times. Analysis of variance (ANOVA) of the

data was performed using the GLM procedure of the software SAS (SAS Institute Inc.,

Cary, NC, USA). Differences of percentages of morphologically normal preantral

follicles (MNPF) and degenerated follicles between control and treatments were

determined by t-test. Differences were considered statistically significant when

P < 0.05.

Results

Histological analysis of ovarian fragments before and after exposure to cryoprotectants

and freezing-thawing

Thirty preantral follicles were examined per treatment in each replicate through

histology, resulting in a total of 750 analyzed follicles. MNPF in control as well as after

exposure to cryoprotectants and freezing-thawing exhibited a spherical or elliptical

oocyte with a large central or eccentric nucleus and uniform cytoplasm. Granulosa cells

without pycnotic nuclei were well-organized in layers surrounding the oocyte and a

distinguishable intact basement membrane could be observed.

Degenerated grade 1 follicles showed a retraced oocyte with or without a

pycnotic nucleus, and occasionally a strongly eosinophilic cytoplasm, whilst layers of

granulosa cells remained unaltered. In degenerated grade 2 follicles, along with the

described oocyte alterations, granulosa cells layers became disorganized into a low

density mass of cells which were many times swollen and/or detached from basement

membrane. Occurrence of pycnotic bodies in granulosa cells or rupture of the

basement membrane was not observed.

The percentages of MNPF observed in the control and after the exposure and

freezing-thawing tests are shown in figure 1. Immersion of ovarian fragments into the

solution containing 1.5 M EG had no effect on the percentages of normal follicles as

compared with the control (P>0.05). Conversely, exposure to 1.5 M GLY reduced

significantly (P <0.05) the percentages of MNPF in comparison to the control and to

immersion in 1.5 M EG. After freezing-thawing, the percentages of normal follicles were

significantly decreased as compared to the control (P <0.05) using either

cryoprotectant, which were statistically similar in relation to each other (P >0.05). For

both cryoprotectants, it was observed that post-thawing percentages of MNPF were

90

significantly reduced in relation to the values obtained after the exposure test (P

<0.05).

Fig. 1. Percentages of morphologically normal preantral follicles in ovarian tissue

before (control) and after exposure and freezing-thawing tests in medium containing

1.5 M EG or GLY. (*) Differs significantly from the control (P <0.05); (a, b) different

letters for the same test (exposure or freezing-thawing) denote significant differences

between media (P <0.05); (A, B) different letters for a given cryoprotectant indicates

significant differences among exposure and post-thawing values (P <0.05).

Regarding to the distribution of follicular degeneration among the different

experimental groups, table 2 shows the percentages of grade 1 and grade 2

degenerated preantral follicles in ovarian fragments from the control, exposure and

freezing-thawing tests. A significant (P <0.05) predominance of degenerated grade 1

follicles over those of grade 2 was observed in all treatments. In the exposure test,

percentages of degenerated grade 1 follicles increased significantly as compared to the

control (P <0.05) when GLY was used, but remained unchanged in the presence of EG

(P >0.05). Percentages of degenerated grade 2 were similar to control using either

GLY or EG (P >0.05).

91

Table 2. Degenerated preantral follicles (%) in control and after exposure and freezing-

thawing tests.

Exposure test Freezing-thawing Control

GLY EG GLY EG

Grade 1 35*aAd 16bAd 40*aAd 47*aBd 13d

Grade 2 6aAe 5aAe 17*aBe 12*aBe 4e

*P < 0:05, significantly differs from control.

Different superscripts (a, b) differ significantly between cryoprotectants at a same test.

Different superscripts (A, B) differ significantly between exposure test and

cryopreservation.

Different superscripts (d, e) differ significantly between Grades 1 and 2 of

degeneration.

After freezing-thawing, significantly (P <0.05) higher values of degenerated

grade 1 and 2 follicles in comparison to the control were observed with both

cryoprotectants, which were similar to each other (P >0.05). Percentages of

degenerated grade 1 follicles were significantly higher after cryopreservation as

compared to the exposure test when EG was used (P <0.05), but not with GLY (P

>0.05). Regarding percentages of degenerated grade 2 follicles, a significant (P <0.05)

rise was observed after freezing-thawing relative to exposure to either cryoprotectants.


TEM of frozen-thawed preantral follicles was performed in order to assess the

ultrastructure of follicles classified as normal at the histological level. At least five

follicles per group were analyzed. Normal follicles contained an oocyte displaying a

very homogenous cytoplasm plenty of round shaped mitochondria with continuous

membranes, few peripheral cristae and electron-dense granules. Some elongated

forms with parallel cristae could also be seen. Small Golgi apparatus cisternae were

rarely observed. Smooth and rough endoplasmic reticulum were present, either as

isolated aggregations or as complex associations with mitochondria. The nuclei of

oocytes were large and usually round, well delimited by the nuclear envelope. The

chromatin was uncondensed and a nucleolus could often be identified. A few vacuoles

were also observed. Granulosa cells were small and presented a high nucleus-to-

cytoplasm ratio. Their irregularly shaped nuclei contained loose chromatin in the inner

part and small peripheral aggregates of condensed chromatin. The cytoplasm exhibited

92

a great number of mitochondria and well-developed smooth and rough endoplasmic

reticulum. The cellular membranes of oocyte and surrounding granulosa cells were

closely juxtaposed, and sometimes few short microvilli could be observed. A distinct


ovarian stroma (Fig. 2A-C).


from control and freezing-thawing with EG. When GLY was used for freezing, despite

some follicles displayed normal morphology, changes could be detected in follicles

evaluated as normal in semi-thin sections by light microscopy. The ooplasm presented

a reduced electron density and lower numbers of organelles, heterogeneously

distributed in small clusters. Some large vacuoles could also be seen. In granulosa

cells, loss of cytoplasm content was noticed, leading to the existence of very large

empty spaces in some cases (Fig. 2D).

Discussion

The present study evaluated for the first time the cryopreservation of canine

preantral follicles through slow freezing, employing GLY and EG as cryoprotectants. It

was demonstrated that ultrastructural integrity of such follicles can be successfully

maintained with the use of 1.5 M EG.

Exposure of canine ovarian tissue to 1.5 M GLY at 20ºC during 20 minutes

resulted in a significant reduction of MNPF percentages. An osmotic stress due to the

presence of cryoprotectants in the freezing medium is one of the primary theories of

injury during cryopreservation, whilst the inherent toxicity of these compounds is also

an important consideration (Mullen et al., 2008). GLY induces severe osmotic damage

to the cytoplasm due to its low membrane permeability (Szell et al., 1986). In

accordance to our results, it has been demonstrated that exposure of ovarian tissue to

1.5 GLY results in a substantial reduction of the percentage of normal follicles (caprine:

Rodrigues et al., 2004; ovine: Santos et al., 2006). Therefore, we suggest that the

current method for exposure to GLY is inadequate and some protocol adjustments

such as gradual addition/removal and use of an osmotic buffer like sucrose (Adams et

al., 2003) should be done to minimize osmotic damage.

93

Fig. 2. Electron micrographs of canine preantral follicles from control (a) and frozen-

thawed using 1.5 M EG (b, c) or GLY (d). In Fig. 2a (2,500x, scale bar= 10 µm) and

Fig. 2b (3,000x, scale bar= 10 µm), normal follicles containing an oocyte with

homogenous cytoplasm and a large nucleus well delimited by the nuclear envelope are

displayed. A nucleolus could often be identified (nu). In Fig. 2c (8,000x, scale bar= 2

µm), the same follicle shown in Fig. 2b is displayed at a higher magnification. Note the

great number of intact mitochondria (m) and smooth endoplasmic reticulum (ser) in the

cytoplasm of the oocyte and surrounding granulosa cells cryopreserved with EG. In

Fig. 2d (10,000x, scale bar= 2 µm), observe the reduced electron density of the

ooplasm, the lower numbers of organelles and a large empty space (asterisk) after

freezing-thawing with GLY. O, oocyte; GC, granulosa cells; l, lipid droplet.

On other hand, 1.5 M EG, which has a molecular weight of 62.07 compared

with 92.10 for GLY (Massip, 2001), did not exert any effect on preantral follicles after

94

exposure of ovarian tissue under the same conditions of GLY addition. Amorim et al.

(2003) exposed isolated ovine primordial follicles to EG at different concentrations and

observed that viability was maintained in concentrations up to 2 M. Sommerfeld and

Niemann (1999) showed that bovine embryos are able to survive exposure to

concentrations of 3.6 M EG without appreciable loss of developmental capacity.

Conversely, Lucci et al. (2004) observed that exposure of zebu bovine ovarian tissue to

1.5 M EG resulted in a high decrease of MNPF, which was greater than that observed

after exposure to GLY under the same conditions. This discrepancy of EG cytotoxicity

might reflect a higher sensitivity of bovine preantral follicles to chemical effects caused

by this cryoprotectant compared to canine follicles. This is in accordance with the fact

that cells from different species can respond very differently to a cryoprotectant

(Schellander et al., 1994; Cocero et al., 1996).

After freezing and thawing, the percentages of MNPF were significantly reduced

from values observed after exposure to both cryoprotectants, which showed similar

preservation efficiency. Therefore, the freezing and/or thawing processes resulted in

damage to follicles, probably due to intracellular ice formation (IIF). We hypothesize

that the concentration of cryoprotectants used (1.5 M) was not enough to protect cells

against crioinjury. Permeating cryoprotectants such as GLY and EG are small

molecules that permeate the membranes of cells, form hydrogen bonds with water

molecules and prevent ice crystallization. At high enough concentrations, they inhibit

the formation of the characteristic ice crystal and lead to the development of a solid,

glasslike, so-called vitrified state in which water is solidified, but not expanded (Jain

and Paulson, 2006). Rodrigues et al. (2004) demonstrated that a rise in concentration

of EG from 1.5 to 3.0 M was able to significantly increase percentages of normal

preantral follicles during cryopreservation of caprine ovarian tissue.

In the present study, since follicles proved to not be affected by exposure to EG

at 1.5 M, we suggest that concentration of this cryoprotectant may be increased up to a

threshold on which cytotoxic and/or osmotic effects are still inexistent and protection

against cryoinjuries is maximum. Regarding to freezing with GLY, we also have to

consider that the protocol of cryoprotectant addition might have not been adequate for

complete permeation of cells, which resulted in intracellular concentrations not

reaching sufficient levels for cryoprotection. Candy et al. (1997) showed that the

survival rate of mice primordial follicles after freezing increased in function of exposure

time to GLY, which suggests that the extent of follicular survival is at least in part

determined by the final tissue concentration achieved.

In all treatments, percentages of grade 1 degenerated follicles were higher than

that for grade 2 degenerated follicles, which characterizes that oocytes are more

95

susceptible than granulosa cells to degeneration in vivo and after exposure to

cryoprotectants and freezing-thawing. The increase of grade 1 degenerated follicles

after freezing and thawing observed in this work was also found in studies with non-

human primate (Candy et al., 1995), human (Hovatta et al., 1996), bovine (Lucci et al.,

2004), caprine (Rodrigues et al., 2004a,b) and ovine (Santos et al., 2006) ovarian

tissue. We supposed that oocytes are less resistant to exposure to cryoprotectants and

cryopreservation than granulosa cells because of its lower surface area-to-volume

ratio, which reduces permeability to both water and ACPs (Wood et al., 2004).

TEM performed on frozen-thawed follicles classified as normal in histology

revealed that only those frozen with EG had preserved ultrastructure, whilst follicles

frozen with GLY showed severe ultrastructural damages. Therefore, TEM proved to be

an essential tool to detect damage due to the freezing-thawing process which can be

observed only at the ultrastructural level but not through light microscopy. The most

severe damage observed was made up of large empty spaces not surrounded by

membranes. This feature suggests the occurence of crystallization, and was also

observed in glycerol-frozen ovine embryos (Cocero et al., 2002).

Once established, knowledge on cryopreservation of canine preantral follicles

could be applied to endangered canids in order to lengthen the genetic lifespan of each

rare female. To this end, development of in vitro culture systems capable to promote

growth and maturation of this vast pool of oocytes has to be accomplished. However,

at present, such systems still do not lead to normal full follicle development in non-

rodent species, for which oocyte growth is a long-term, coordinated and complex

process, and the time required to reach the mature stage is thought to be up to several

months. Currently, xenotransplantation into immunodeficient recipients seems to be

one the most promising ways for obtaining oocytes from ovaries for further exploration

of assisted reproduction techniques (Jewgenow and Paris, 2006). Fassbender et al.

(2007) demonstrated the survival of cat ovarian cortex tissue transplanted under the

kidney capsule of athymic nude rats. Follicular development was monitored through

high-resolution ultrasonography, and cumulus oocytes complexes could be collected

and in vitro matured.

In conclusion, preservation of morphology of canine preantral follicles can be

accomplished through slow freezing and rapid thawing using 1.5 M EG. Nevertheless,

further studies are necessary to evaluate developmental capacity of such follicles,

which demands the establishment of in vitro culture systems or use of transplantation

approaches.

96

Acknowledgements

CAP Lopes and JR Figueiredo are recipients of grants from CNPq (Brazil). We

would like to express our appreciation to the Center for Zoonosis Control (CCZ) of

Fortaleza Municipality (Brazil), which gently provided the canine ovaries.

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9 CAPÍTULO IV

Criopreservação bem-sucedida de folículos pré-antrais caninos utilizando-se

dimetilsulfóxido e 1,3-propanodiol

Successful cryopreservation of canine preantral follicles using dimethyl sulfoxide and

1,3-propanediol

Reproduction, Fertility and Development

(Submetido, 2008)

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Successful cryopreservation of canine preantral follicles using dimethyl sulfoxide and 1,3-propanediol C. A. P. LopesA,B,E, A. K. F. LimaA, R. R. SantosC, M. H. T. MatosA, A. P. R. RodriguesA,

S. N. BáoD, K. JewgenowB, J. R. FigueiredoA

ALAMOFOPA, PPGCV, Faculty of Veterinary, State University of Ceará, Brazil. BLeibniz-Institute for Zoo and Wildlife Research Berlin, Germany. CDepartment of Equine Sciences, Pharmaceuticals, Pharmacology and Toxicology

Division, Faculty of Veterinary Medicine, Utrecht University, The Netherlands. DLaboratory of Electron Microscopy, Institute of Biology, University of Brasília, Brazil. ECorresponding author. Email: [email protected]

Resumo

A criopreservação de folículos pré-antrais é uma abordagem potencialmente

importante para a conservação de espécies ameaçadas pela extinção, uma vez que

técnicas têm sido desenvolvidas para a obtenção de oócitos fertilizáveis a partir desta

vasta fonte de gametas. O objetivo deste estudo foi avaliar os efeitos da congelação

lenta-descongelação rápida utilizando-se dimetilsulfóxido (DMSO) ou 1,3-propanodiol

(PROH) sobre a morfologia e viabilidade de folículos pré-antrais caninos. Fragmentos

de tecido ovariano canino foram equilibrados por 20 min a 20ºC em Meio Essencial

Mínimo contendo um dos crioprotetores mencionados à concentração de 1,5 M, sendo

então congelados e descongelados. A morfologia dos folículos foi analisada através de

histologia e microscopia eletrônica de transmissão (MET), e a viabilidade avaliada

utilizando-se Azul de Tripan e marcadores fluorescentes. Os resultados demonstraram

que a exposição de tecido ovariano canino a ambos crioprotetores não apresentou

efeito sobre a morfologia e viabilidade foliculares. Após a congelação e

descongelação, os folículos mantiveram-se histologicamente normais, mas a MET

revelou danos à ultra-estrutura dos folículos congelados em PROH. A viabilidade foi

significativamente reduzida com ambos crioprotetores. Entretanto, altos percentuais de

folículos viáveis (65%) foram matidos com DMSO. Como conclusão, este estudo

demonstrou que a criopreservação de folículos pré-antrais caninos viáveis com ultra-

estrutura intacta pode ser realizada com sucesso através de congelação lenta e

descongelação rápida utilizando-se DMSO a 1,5 M.

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Palavras-chave: criopreservação, folículos pré-antrais, canino, congelação lenta,

dimetilsulfóxido, propanodiol. Abstract

Cryopreservation of preantral follicles is potentially an important approach for

the conservation of endangered species in such way that techniques have been

developed in order to retrieve fertilizable oocytes from this large pool of gametes. The

aim of this study was to evaluate the effects of slow freezing-rapid thawing using

dimethyl sulfoxide (DMSO) or 1,3-propanediol (PROH) on morphology and viability of

canine preantral follicles. Slices of canine ovarian tissue were equilibrated for 20 min at

20ºC in minimum essential medium (MEM) containing either cryoprotectants at 1.5 M,

and then frozen-thawed. Morphology of follicles was analyzed through histology and

transmission electron microscopy (TEM), and viability assessed using trypan blue and

fluorescent probes. The results showed that exposure of canine ovarian tissue to both

cryoprotectants had no effect on follicular morphology and viability. After freezing-

thawing, follicles remained histologically normal, but TEM revealed damage to the

ultrastructure of those frozen in PROH. Viability was significantly reduced when either

compound was used. However, high percentages of viable follicles (65%) were

maintained with DMSO. In conclusion, this study demonstrated that cryopreservation of

viable canine preantral follicles with intact ultrastructure can be successfully

accomplished through slow freezing and rapid thawing using 1.5 M DMSO.

Additional keywords: cryopreservation, preantral follicles, canine, slow freezing,

dimethyl sulfoxide, propanediol.

Introduction

Along with the global warming, extinction of species is an increasing major

concern worldwide, since biodiversity is a central component of Earth’s life supporting

systems (Khuroo et al., 2007), and the removal of single species can affect the

functioning of global ecosystems (Myers et al., 2000). In this context, in addition to the

crucial protection of habitats, reproductive biotechnology has a great potential to

contribute for the conservation of endangered wild animal species.

Nevertheless, a limiting factor for the development of reproductive techniques,

and also for their efficiency, is the lack of abundant numbers of fertilizable oocytes.

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This problem could be addressed by using the large source of oocytes enclosed in

preantral follicles (Telfer, 2001), which exist in ovaries in number of thousands to

millions depending on the species. This category comprises around 90 to 95% of entire

follicular population, storing a vast majority of the oocytes in mammalian ovaries

(Figueiredo et al., 2006).

In vitro culture systems capable to promote growth and maturation of preantral

follicles have been developed for several species. Currently, birth of healthy offspring

after embryo transfer of in vitro fertilized oocytes derived from primordial follicles has

been accomplished only in mice (Eppig and Schroeder, 1989). The oocyte growth

period in this species is about 3 weeks, whereas, in non-rodents, the time required to

reach the mature stage is thought to be up to several months (Jewgenow and Paris,

2006). Therefore, the creation of such systems is a challenging task still at an early

stage of development, and long-term preservation of preantral follicles until the

establishment of routine culture is an important issue. It could permit to lengthen the

genetic lifespan of each rare female of an endangered species after death through the

storage of its large pool of gametes.

Cryopreservation of isolated primordial follicles through slow freezing was

successfully achieved in the mouse (Carroll and Gosden, 1993). Follicles were

transplanted to sterilized hosts which could afterwards produce normal offspring after

mating. Live young were also obtained after in vitro culture, maturation and fertilization

of mouse oocytes within frozen-thawed secondary follicles (Smitz and Cortvrindt,

1998). More recently, live births after orthotopic autotransplantation of cryopreserved

ovarian tissue were described in a woman (Donnez et al., 2004), as well as in ewes

(Bordes et al., 2005).

Although cryopreservation of preantral follicles from carnivores would be

specially important since many wild species of these are currently threatened by

extinction, few studies have been published on this topic. Jewgenow et al. (1998)

demonstrated that small preantral follicles isolated from cat ovaries survive

cryopreservation, remaining structurally intact and physiologically active after thawing.

In addition, Bosch et al. (2004) reported the survival of cat ovarian tissue subjected to

slow freezing-thawing and transplantation to an immunocompromised host, which was

followed by development of follicles from early up to the antral stage. With respect to

canids, to our knowledge, only one attempt to cryopreserve preantral follicles was

reported. Ishijima et al. (2006) vitrified canine ovarian cortex and xenotransplanted to

immunodeficient mice. Although antral follicle formation did not occur, grafted tissue

was morphological normal and proliferating cell nuclear antigen immunoreactivity was

detected in primary follicles. Nonetheless, detailed morphological and quantitative

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analyses on cryopreservation of canine preantral follicles are still to be performed, and

knowledge gathered in this species could be applied to endangered canids.

The aim of this study was to evaluate the cryopreservation of canine preantral

follicles through slow freezing of ovarian tissue using 1.5 M dimethyl sulfoxide (DMSO)

or 1.5 M 1,3-propanediol (PROH). For this purpose, morphology of follicles after

exposure to the cryoprotectants and freezing-thawing was analyzed through histology

and transmission electron microscopy (TEM), and viability was assessed through the

trypan blue dye exclusion test as well as using the fluorescent probes calcein-AM and

ethidium homodimer.

Materials and methods Collection and preservation of canine ovaries

Ovarian pairs were collected during ovariectomy of healthy 12-24 months old

bitches (Canis lupus familiaris) at local veterinary clinics. After removal from the bursa

ovarica and excision of eventual corpora lutea using a scalpel, ovaries were placed into

50 ml tubes (Corning Glass Works, Corning, NY, USA) containing 15 ml of HEPES-

buffered Minimum Essential Medium (H-MEM, osmolarity 280 mOsmol/l, pH7.2; Sigma,

St. Louis, MO, USA). Then, transportation to the laboratory was carried out at 4°C

within one hour in thermoflasks filled with water. All procedures were performed

aseptically.

Experiment 1: Morphological evaluation of follicles after exposure to cryoprotectants


At the laboratory, seven fragments of cortex (approximately 3 x 3 x 1 mm) were

cut from each pair of ovaries (n=5), and one of these pieces was randomly selected as

fresh control and fixed for histology and ultrastructural analysis. The remaining samples

were placed into cryovials containing 1.8 ml of freezing medium which consisted of

Minimum Essential Medium (MEM) supplemented with 10% fetal bovine serum (FBS)

and 1.5 M DMSO or 1.5 M PROH or without cryoprotectant (freezing control), prepared

immediately prior to use under sterile conditions. After incubation at 20ºC for 20 min

(equilibration period), half of the fragments was subjected to cryoprotectant removal

according to the method described by Candy et al. (1997). Briefly, three washes of 5

min each in MEM with 10% FBS were performed at room temperature (RT,

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approximately 25°C). Samples were subsequently fixed for histology and TEM in order

to evaluate effects of exposure to cryoprotectants on preantral follicles.

The cryovials with the remaining fragments were transferred to a biological

programmable freezer (Freeze Control, CryoLogic Pty Ltd.,Waverley, Australia) pre-set

at 20 °C. The following program was used to freeze the tissue samples: cooling from

20 to -7°C at a rate of 2°C/min; holding at -7°C for 15 min (after 5 min, ice crystal

formation - seeding - was induced manually by touching the vials with a forceps pre-

chilled in liquid nitrogen); temperature was then lowered 0.3°C/min to -30°C, and

further reduced by 0.15°C/min to -33°C; finally, the vials were plunged into liquid

nitrogen (-196°C) and stored for 7 days. Thereafter, ovarian tissue pieces were thawed

through rapid warming by exposing the cryovials to air at RT for 1 min, followed by

immersion in a water bath at 38ºC for 3 min. Cryoprotectant removal was carried out as

describe for the exposure test, and pieces of ovarian cortex were fixed for histological

and ultrastructural analyses. This experiment was repeated five times.

Experiment II: Viability assessment of follicles exposed to cryoprotectants and frozen-

thawed

A second study using a similar experimental protocol as described above in the

morphological investigation was performed with the objective of analyzing effects of

exposure to cryoprotectants and freezing-thawing on viability of preantral follicles. Five

ovaries were used, and fragments from the fresh control and testing groups undergone

follicle isolation followed by trypan blue dye exclusion test as described later. Based on

the results of this assay and experiment I, viability of follicles cryopreserved with the

substance which provided the best outcome was further analyzed using a more

accurate method of assessment based on fluorescent probes. Additional pairs of

ovaries (n=5) were cut into fragments, from which one was immediately processed for

follicle isolation and analyzed using trypan blue as well as calcein-AM and ethidium

homodimer. Remaining fractions were subjected to exposure to the selected

cryoprotectant, frozen-thawed and analyzed after each procedure similarly as for the

control. All samples were evaluated in duplicate.

Histological analysis




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wax and serially sectioned at 7 μm. Every fifth section was mounted on glass slides,








of granulosa cells, or (ii) degenerated, when the oocyte exhibited pycnotic nucleus

and/or ooplasma shrinkage, and occasionally granulosa cells layers became

disorganized, detached from the basement membrane and/or included enlarged cells.

To avoid evaluating and counting a follicle more than once, preantral follicles were

analyzed only in the sections where oocyte nucleus was observed.


In order to better examine follicular morphology, TEM was performed to analyze

ultrastructure of preantral follicles from the control as well as from treatments that did

not differ statistically from control in histology. A portion with a maximum dimension of

1 mm3 was cut from each fragment of ovarian tissue and fixed in modified Karnovsky

solution (2% paraformaldeyde and 2% glutaraldeyde in 0.1 M sodium cacodylate buffer

pH 7.2) for 3 h at room temperature (RT, approximately 25°C). After three washes in

sodium cacodylate buffer, specimens were post-fixed in 1% osmium tetroxide, 0.8%

potassium ferricyanide and 5 mM calcium chloride in 0.1 M sodium cacodylate buffer

for 1 h at RT. The samples were then dehydrated through a gradient of acetone

solutions and thereafter embedded in Spurr’s epoxy resin. Afterwards, semi-thin

sections (3 μm) were cut, stained with toluidine blue and analyzed by light microscopy

at a 400x magnification. Ultra-thin sections (60–70 nm) were obtained from preantral

follicles classified as morphologically normal in semi-thin sections, according to the

criteria adopted in histology. Subsequently, ultra-thin sections were contrasted with

uranyl acetate and lead citrate, and examined under a Jeol 1011 (Jeol, Tokyo)

transmission electron microscope operating at 80 kV.

Assessment of preantral follicles viability

Canine preantral follicles were isolated from ovarian fragments using the

mechanical method described by Figueiredo et al. (1993). Briefly, using a tissue

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chopper (The Mickle Laboratory Engineering Co., Gomshal, Surrey, UK) adjusted to a

sectioning interval of 87.5 μm, samples were cut into small pieces, which were placed

in MEM and suspended 40 times using a large Pasteur pipette (diameter of about 1600

μm) and subsequently 40 times with a smaller Pasteur pipette (diameter of

approximately 600 μm) to dissociate preantral follicles from stroma. The obtained

material was passed through 500- and 100-μm nylon mesh filters, resulting in a

suspension containing preantral follicles smaller than 100 μm in diameter. This

procedure was carried out within 10 min at RT.

Thereafter, viability of preantral follicles was assessed through trypan blue dye

exclusion test (Jewgenow et al., 1998). Briefly, 10 μl of 0.4% trypan blue (Sigma,

Deisenhofen, Germany) were added to 90 μl of the suspension of isolated preantral

follicles, which were examined using an inverted microscope after incubation for 5 min

at RT. Follicles were classified as viable if the oocyte and <10% granulosa cells

remained unstained, or as non-viable if uptake of the dye by the oocyte and/or ≥10%

granulosa cells occurred.

Preantral follicles were also analyzed using a two-color fluorescence cell

viability assay based on the simultaneous determination of live and dead cells by

calcein-AM and ethidium homodimer-1, respectively. Whilst the first probe detected

intracellular esterase activity of viable cells, the later labeled nucleic acids of non-viable

cells with plasma membrane disruption. The test was performed by adding 4 μM

calcein-AM and 2 μM ethidium homodimer-1 (Molecular Probes, Invitrogen, Karlsruhe,

Germany) to the suspension of isolated follicles, followed by incubation at 37°C for 10

min. After being labeled, follicles were washed three times in MEM and mounted on a

glass microscope slide in 5 μl antifading medium (DABCO, Sigma, Deisenhofen,

Germany) to prevent photobleaching, and finally examined using an a DMLB

fluorescence microscope (Leica, Germany). The emitted fluorescent signals of calcein-

AM and ethidium homodimer were collected at 488 and 568 nm respectively. Oocytes

and granulosa cells were considered live if the cytoplasm was stained positively with

calcein-AM (green) and chromatin was not labelled with ethidium homodimer (red).


All experiments were replicated five times. Kolmogorov-Smirnov and Bartlett’s

tests were applied to verify normal distribution of data and homogeneity of variances

respectively. Afterwards, analysis of variance (ANOVA) was performed using the GLM

procedure of the software SAS (SAS Institute Inc., Cary, NC, USA). Differences of

percentages of morphologically normal preantral follicles (MNPF) and viable follicles

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between control and treatments were determined by Dunnett’s test. Student Newman

Keuls’ (SNK) test was used to compare results among testing groups. Data were

expressed as means ± standard error of means (SEM). Differences were considered

statistically significant when P <0.05.

Results

Experiment 1: Morphological evaluation of follicles after exposure to cryoprotectants


A total of 1079 preantral follicles were examined in histology, with a range of

145 to 171 in each treatment. Morphologically normal preantral follicles (MNPF) in

control as well as after exposure to cryoprotectants and freezing-thawing exhibited a

spherical or elliptical oocyte with a large central or eccentric nucleus and uniform

cytoplasm. Granulosa cells without pycnotic nuclei were well-organized in layers

surrounding the oocyte and a distinguishable intact basement membrane could be

observed (Fig. 1a-c). Degenerated follicles showed a retraced oocyte with or without a

pycnotic nucleus, and occasionally a strongly eosinophilic cytoplasm (Fig. 1d). Layers

of granulosa cells remained unaltered or became disorganized into a low density mass

of cells which were many times swollen and/or detached from basement membrane.

Occurrence of pycnotic bodies in granulosa cells or rupture of the basement membrane

was not observed.

The percentages of MNPF observed in the control and after the exposure and

freezing-thawing tests are shown in figure 2. Immersion of ovarian fragments into the

cryopreservation medium, which contained either DMSO or PROH at 1.5 M had no

effect on the percentages of normal follicles as compared with the fresh control

(P>0.05). Similarly, freezing-thawing with either cryoprotectant maintained the

percentages of MNPF (P >0.05). Nevertheless, the absence of cryoprotectants in the

freezing medium (freezing control) led to a severe decrease of the percentages of

normal follicles (22%) as compared with the fresh control (90%) and freezing with

DMSO (84%) or PROH (80%) (P <0.05). For both cryoprotectants, it was observed that

percentages of MNPF after freezing-thawing were similar to the values obtained after

the exposure test (P <0.05).

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Fig. 1. Histological features of canine ovarian fragments before (a) and after freezing-

thawing with 1.5 M DMSO (b) or PROH (c, d). Morphologically normal preantral follicles

comprised an oocyte displaying a large nucleus (nu) and homogenous cytoplasm

surrounded by one or more layers of flattened or cuboidal granulosa cells (gc) and

without antrum (a, b and c). Degenerated preantral follicles often displayed oocyte

retraction (d, arrow) and disorganization of granulosa cell layers. Scale bars represent

10 µm (a, b and c) or 20 µm (d). PAS-hematoxilin.

109

Fig. 2. Percentages of morphologically normal preantral follicles in ovarian tissue

before (fresh control) and after exposure and freezing-thawing tests in medium

containing 1.5 M DMSO or PROH or without cryoprotectants (exposure and freezing

controls). (*) Differs significantly from the control (P <0.05); (a, b) different letters for the

same test denote significant differences between media (P <0.05); (A, B) different

letters for a given medium indicates significant differences among exposure and post-

thawing values (P <0.05).


TEM of frozen-thawed preantral follicles was performed in order to assess the

ultrastructure of follicles classified as normal at the histological level. At least five

follicles per group were analyzed. Normal follicles contained an oocyte displaying a

very homogenous cytoplasm plenty of round shaped mitochondria with continuous

membranes, few peripheral cristae and electron-dense granules. Some elongated

forms with parallel cristae could also be seen. Small Golgi apparatus cisternae were

rarely observed. Smooth and rough endoplasmic reticulum were present, either as

isolated aggregations or as complex associations with mitochondria. The nuclei of

oocytes were large and usually round, well delimited by the nuclear envelope. The

chromatin was uncondensed and a nucleolus could often be identified. A few vacuoles

were also observed. Granulosa cells were small and presented a high nucleus-to-

cytoplasm ratio. Their irregularly shaped nuclei contained loose chromatin in the inner

part and small peripheral aggregates of condensed chromatin. The cytoplasm exhibited

a great number of mitochondria and well-developed smooth and rough endoplasmic

110

reticulum. The cellular membranes of oocyte and surrounding granulosa cells were

closely juxtaposed, and sometimes few short microvilli could be observed. A distinct


ovarian stroma (Fig. 3a-c).


from control, after exposure to either cryoprotectants and freezing-thawing with DMSO.

When PROH was used for freezing, despite some follicles displayed normal

morphology, changes could be detected in follicles evaluated as normal in semi-thin

sections by light microscopy. The ooplasm presented a reduced electron density and

lower numbers of organelles, heterogeneously distributed in small clusters. Some large

vacuoles could also be seen. In granulosa cells, loss of cytoplasm content was noticed,

leading to the existence of very large empty spaces in some cases (Fig 3c).

Experiment II: Viability assessment of follicles exposed to cryoprotectants and frozen-

thawed

Preantral follicles from ovarian fragments of the control and testing groups were

mechanically isolated and viability was assessed by trypan blue dye exclusion test. A

total of 1,289 follicles were examined, with a range of 152 to 200 in each treatment.

The percentages of viable follicles observed in each treatment are presented in table 1.

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Fig. 3. Electron micrographs of canine preantral follicles from control (a) and frozen-

thawed using 1.5 M DMSO (b, d) or PROH (c). In Fig. 3a (2500x, scale bar= 10 µm)

and 3b (6000x, scale bar= 5 µm), normal follicles containing an oocyte with

homogenous cytoplasm and a large nucleus well delimited by the nuclear envelope are

displayed. A nucleolus could often be identified (nu), as well as a continuous basement

membrane (bm). In Fig. 3c (2500x, scale bar= 10 µm), observe the large vacuoles

(arrowheads) and a large empty space (asterisk) after freezing-thawing with PROH. In

Fig. 3d (6000x, scale bar= 5 µm), note the great number of intact mitochondria (m) and

smooth endoplasmic reticulum (ser) in the cytoplasm of an oocyte and surrounding

granulosa cells cryopreserved using DMSO. O, oocyte; GC, granulosa cells; v,

vacuole.

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Table 1. Percentages of viable follicles in the fresh control and after exposure to

cryoprotectants and slow freezing-thawing as assessed using trypan blue.

Treatments Exposure Freezing-thawing

Fresh control 77.68±2.89

No cryoprotectant 75.82±2.62aA 5.94±2.82*cB

1.5 M PROH 73.24±3.35aA 48.74±4.55*bB

1.5 M DMSO 73.76±2.63aA 65.68±1.66*aB

(*) Differs significantly from control (P <0.05); (a, b, c) different letters denotes

significant difference between values within a column (P <0.05); (A, B) different letters

indicate significant difference between values of a row (P <0,05).

Immersion of ovarian fragments into 1.5 M DMSO or 1.5 M PROH had no effect

on the percentages of viable follicles as compared with the fresh and test control

(medium without cryoprotectant) (P >0.05). Nevertheless, freezing in either 1.5 M

DMSO or 1.5 M PROH and rapid thawing resulted in a significant reduction of the

percentages of viable follicles in relation to the fresh control (P <0.05), which were

higher on the use of DMSO than when PROH was employed (P <0.05). The absence

of cryoprotectants in the freezing medium (cryopreservation control) led to a severe

and significant decrease of the percentages of viable follicles in comparison with the

fresh control and with freezing with DMSO or PROH. For both cryoprotectants, it was

observed that percentages of viable follicles after freezing-thawing were significantly

decreased as compared with the values obtained after the exposure test.

Based on the results of morphological evaluation (experiment I) and viability

assessment with trypan blue, which evidenced that freezing-thawing using 1.5 M

DMSO provides preservation of ultrastructure and higher percentages of viable follicles

than PROH, a second viability trial using the former cryoprotectant was performed. In

addition to trypan blue testing, a fluorescence cell viability assay based on labelling of

live and dead cells by calcein-AM and ethidium homodimer-1, respectively, was

employed (Fig. 4).

113

Fig. 4. Viability assessment of canine preantral follicles using fluorescent probes. (a)

An isolated preantral follicle classified as viable after slow freezing- rapid thawing with

1.5M DMSO, since cells were labeled by calcein-AM (b) (green fluorescence). (c)

Another follicle from the same treatment considered non-viable as cells were marked

with ethidium homodimer-1 (d) (red fluorescence). Scale bars represent 10 µm.

Percentages of viable follicles remained unaltered after exposure of ovarian

cortex fragments to 1.5 M DMSO in comparison to the control (P >0.05) as assessed

by trypan blue and calcein-AM/ethidium homodimer assays, whose values did not differ

from each other (P >0.05). However, a significant reduction in the percentages of

viable follicles relative to the fresh control and exposed fragments was observed after

freezing-thawing according to the two viability tests, which once more retrieved similar

results (Fig.5).

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Fig. 5. Percentages of viable canine preantral follicles in fresh ovaries and after

exposure and freezing-thawing using 1.5 M DMSO as assessed by trypan blue dye

exclusion test and labeling with calcein-AM and ethidium homodimer. (*) Differs

significantly from control (P <0.05); (a, b) different letters within the same test

(exposure or freezing-thawing) denote significant differences between values of the

viability assays (P <0.05); (A, B) different letters for a same viability assay indicates

significant differences between tests (P <0.05).

Discussion

The present study evaluated for the first time the cryopreservation of canine

preantral follicles through slow freezing, employing DMSO and PROH as

cryoprotectants. It was demonstrated that viability and ultrastructural integrity of such

follicles can be successfully maintained with the use of 1.5 M DMSO.

Exposure of ovarian fragments to either DMSO or PROH at 1.5 M did not cause

reduction of the percentages of MNPF follicles as evaluated in histology, and

ultrastructural analysis through TEM confirmed the integrity of such follicles.

Accordingly, Lucci et al. (2004) also observed no effect of such cryoprotectants at the

same concentrations on bovine preantral follicles. On other hand, caprine (Rodrigues

et al., 2004) and ovine (Santos et al., 2006) ovarian tissue exposed to 1.5 M DMSO or

PROH at similar conditions (temperature and time) showed a significant reduction of

the percentage of normal preantral follicles. Indeed, an osmotic stress due to the

presence of cryoprotectants in the freezing medium is one of the primary theories of

115

injury during cryopreservation, whilst the inherent toxicity of these compounds is also

an important consideration (Mullen et al., 2008). We hypothesize that these differences

reflect a species-specific resistance of preantral follicles enclosed in ovarian cortex to

exposure to DMSO and PROH at 1.5 M.

After freezing and thawing with either cryoprotectants, percentages of MNPF

also remained unchanged as evaluated by histology. However, TEM revealed

alterations in the ultrastructure of follicles frozen with PROH which were previously

evaluated as normal in histology. Therefore, TEM proved to be an essential tool to

detect damage due to the freezing-thawing process which can be observed only at the

ultrastructural level but not through light microscopy. The most severe damage

observed was made up of large empty spaces not surrounded by membranes. This

feature suggests the occurence of crystallization, and was also observed in glycerol-

frozen ovine embryos (Cocero et al., 2002). This relative inefficiency of PROH to

protect canine preantral follicles during freezing-thawing may be due to the fact that its

ability to prevent ice-crystal formation is limited at 1.5 M (Jain and Paulson, 2006).

Similarly, integrity of caprine (Rodrigues et al., 2004) and bovine (Lucci et al., 2004)

preantral follicles cryopreserved in 1.5 M DMSO was confirmed, whilst similar

modifications were found in follicles frozen-thawed with 1.5 M PROH.

Notwithstanding previous studies on cryopreservation of preantral follicles

provided important knowledge on this issue, most approaches were limited to analysis

of morphology, which is not always correlated with the viability of follicles (Santos et al.,

2007). Therefore, in the present work, we further analyzed the quality of follicles

through assessment of viability using the trypan blue dye exclusion test. After exposure

to either DMSO or PROH at 1.5 M, percentages of viable follicles remained similar to

those of the controls. Accordingly, Jewgenow et al. (1998) demonstrated that adding

these cryoprotectants at the same concentrations to isolated feline small preantral

follicles did not result in decrease of viability after in vitro culture in comparison to the

control. Amorim et al. (2004) observed that only exposure to DMSO or PROH at

concentrations higher than 2 M and 1.5 M respectively reduced the numbers of viable

preantral follicles isolated from ovine ovaries. Thus, our results show that addition of

DMSO or PROH at up to 1.5 M to canine ovarian tissue affects neither the integrity nor

the viability of preantral follicles as a consequence of osmotic and toxic effects of the

freezing solution.

When post-thaw viability of follicles was examined, a significant decrease was

found with the use of both cryoprotectants in comparison to the fresh control. However,

a high percentage (65%) of viable follicles was maintained through the employment of

1.5 M DMSO, whilst PROH did not show a comparable efficiency for follicular

116

preservation, which might be due to the ultrastructural damages detected in the

morphological analysis. In accordance, Jewgenow et al. (1998) showed that viability of

isolated feline preantral follicles was also reduced after freezing-thawing with either 1.5

M DMSO or PROH. Conversely, Rodrigues et al. (2005) observed that

cryopreservation using 1.5 M DMSO or PROH had no effect on viable caprine preantral

follicles, and this outcome was also achieved with ovine follicles after using 1.5 M

DMSO (Amorim et al., 2007). Hence, we suggest that carnivore preantral follicles are

less resistant to freezing-thawing with these cryoprotectants at 1.5 M than their

counterparts from small ruminants.

Percentages of viable follicles were much lower than those of morphologically

normal follicles in every treatment. This proves that a more precise determination of the

proportions of follicles with adequate quality for subsequent applications such as in

vitro culture may rely on viability assessment. In this context, we have further analyzed

follicles cryopreserved in DMSO using a more accurate method. For this purpose,

labeling of non-viable cells with disruption of plasma membrane was performed again

by using ethidium homodimer-1, which enabled a better visualization of dead cells

through fluorescent staining of nuclei. Concomitantly, identification of viable cells was

performed through detection of esterase activity with calcein-AM. These fluorescent

probes have been successfully used to assess viability of bovine early-staged follicles

(Schotanus et al.,1997; Van den Hurk et al., 1998) and after cryopreservation of

caprine preantral follicles (Santos et al., 2006, 2007). In the present study, results of

this assay were similar to values observed with trypan blue testing, which thus proved

to be a reliable, practical and quick method for preliminary viability assessment of

preantral follicles. Accordingly, Poeschmann et al. (2008) observed a significant

correlation between both methods while analyzing viability of feline preantral follicles.

Despite a significant decrease of viable follicles was observed after freezing-

thawing in the present study, we consider that preservation rates with the use of DMSO

to freeze canine preantral follicles are by this time high and can be further improved.

Since these follicles proved to not be affected by exposure to DMSO at 1.5 M at the

described conditions, concentration of this cryoprotectant could be increased up to a

threshold on which cytotoxic and/or osmotic effects are still inexistent and protection

against cryoinjuries is maximum. It must also be considered for such adjustment that

during cell dehydration, a delicate balance must be maintained between the removal of

free water that can form ice crystals and the removal of bound water, because

excessive loss of the latter results in loss of structural support to proteins and lipids

(Wright et al., 2004). The use of isolated follicles could also enhance results, since

117

mammalian cells frozen in situ are more susceptible to damage caused by this process

than cells in suspension (Armitage et al., 1995; Armitage and Juss, 1996).

Once established, knowledge on cryopreservation of canine preantral follicles

could be applied to endangered canids in order to lengthen the genetic lifespan of each

rare female. To this end, development of in vitro culture systems capable to promote

growth and maturation of this vast pool of oocytes has to be accomplished. However,

at present, such systems still do not lead to normal full follicle development in non-

rodent species, for which oocyte growth is a long-term, coordinated and complex

process, and the time required to reach the mature stage is thought to be up to several

months. Currently, xenotransplantation into immunodeficient recipients seems to be

one the most promising ways for obtaining oocytes from ovaries for further exploration

of assisted reproduction techniques (Jewgenow and Paris, 2006). Ishijima et al. (2006)

transplanted canine ovarian tissue into non-obese diabetic severe combined

immunodeficient mice and observed a significant shift from primordial to primary

follicles, in which the proliferating cell nuclear antigen could be detected. In addition,

Fassbender et al. (2007) demonstrated the survival of cat ovarian cortex tissue

transplanted under the kidney capsule of athymic nude rats. Follicular development

was monitored through high-resolution ultrasonography, and cumulus oocytes

complexes could be collected and in vitro matured.

In conclusion, cryopreservation of canine preantral follicles can be

accomplished through slow freezing and rapid thawing using 1.5 M DMSO or PROH.

However, only DMSO provided maintenance of high percentages of viable follicles with

intact ultrastructure. Further studies are necessary to evaluate developmental capacity

of such follicles, which demands the establishment of in vitro culture systems or use of

transplantation approaches.

Acknowledgements

During the period of this work, Claudio A. P. Lopes received financial support

from the National Council for Scientific and Technological Development (CNPq;

doctoral scholarship) and the Coordination for Improvement of Graduate Personnel

(CAPES; fellowship for studies abroad, PDEE, grant 5212/06-5), both institutions of the

Brazilian Government. José Ricardo de Figueiredo was also recipient of a grant from

CNPq. We would like to express our appreciation to the Center for Zoonosis Control

(CCZ) of Fortaleza Municipality (Brazil) and to the veterinary clinics of the animal

shelter Tierheim-Berlin and Tierklinik-Biesdorf (Germany), which gently provided the

canine ovaries.

118

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10 CAPÍTULO V

Potencial de anticorpos anti-zona pelúcida para a imunoesterilização de cães:

efeitos sobre folículos pré-antrais in vitro

Potential of anti-zona pellucida antibodies for the immunosterilization of dogs: effects

on preantral follicles in vitro

Journal of Reproduction and Development

(Submetido, 2008)

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Potential of anti-zona pellucida antibodies for the immunosterilization of dogs: effects on preantral follicles in vitro

Lopes CAPABD, Niu YC, Alves AMCVA, Chaves RNA, Campello CCA, Jewgenow KB, Figueiredo JRA

A Faculty of Veterinary, PPGCV, LAMOFOPA, State University of Ceará, Fortaleza, Brazil.

Postal address: Av. Paranjana 1700, Campus do Itaperi, CEP 60.740-903, Fortaleza, CE, Brazil.

B Institute for Zoo and Wildlife Research, Berlin, Germany.

Postal address: Postfach 601103, D-10252, Berlin, Germany.

CKunming Primate Research Center and Kunming Institute of Zoology, Chinese Academy of Sciences,

China.

Postal address: 32 Jiao Chang Dong Lu, Kunming, Yunnan 650223, China.

DCorresponding author. Email: [email protected]

Resumo

A superpopulação de cães constitui um grave problema em diversas cidades do

mundo, que tem sido controlado através da eliminação sistemática de animais sem

responsáveis, que podem atuar como reservatórios de zoonoses. A esterilização de cadelas

através de imunização contra as proteínas da zona pelúcida (ZP), estrutura que reveste os

oócitos, é uma alternativa promissora para o controle reprodutivo desta espécie. O objetivo

deste estudo foi avaliar in vitro o potencial de um antisoro contra ZP suína (pZP) de induzir

degeneração de folículos pré-antrais caninos e/ou de bloquear a ligação espermática à ZP.

Folículos pré-antrais foram isolados de ovários caninos e cultivados in vitro com soro anti-

pZP. Oócitos obtidos de folículos antrais foram tratados com o mesmo soro e co-incubados

com sêmen canino de modo a se analisar a ligação espermática. A avaliação da viabilidade

após o cultivo revelou que o soro anti-pZP foi efetivo para induzir degeneração de folículos

pré-antrais maiores que 100 µm, conforme comparação com o controle (soro pré-imune),

mas não teve efeito sobre folículos menores. Este processo parece envolver a ativação do

sistema complemento sérico. Anticorpos anti-pZP também se mostraram capazes de

bloquear a ligação espermática. Diante do exposto, concluímos que anticorpos anti-pZP

podem promover a eliminação de folículos pré-antrais caninos e inibir a ligação espermática,

constituindo-se, assim, uma importante perspectiva para o controle efetivo da reprodução

em cães.

Palavras-chave: Imunocontracepção, anticorpos, canino, zona pelúcida, folículos pré-antrais.

123

Abstract

Overpopulation of dogs in urban areas is a critical issue worldwide, which has been

controlled by means of systematic euthanasia of unowned animals that can act as zoonoses

reservoirs. Sterilization of bitches through immunization against proteins of the zona

pellucida (ZP), the egg coat, is a promising alternative for the reproduction control in this

species. The aim of this work was to evaluate in vitro the potential of an antiserum against

porcine ZP (pZP) to cause degeneration of canine preantral follicles and/or blocking of sperm

binding. Preantral follicles were isolated from bitch ovaries and in vitro cultured with anti-pZP

serum. Oocytes from antral follicles were also obtained, treated with the same serum and co-

incubated with dog semen in order to analyze sperm binding. Viability assessment after

culture showed that anti-pZP serum was effective to induce degeneration of preantral follicles

larger than 100 µm as compared to the control (pre-immune serum), but had no effect on

smaller follicles. This process might involve activation of the serum complement system.

Anti-pZP antibodies also proved capable of blocking sperm binding. In conclusion, anti-pZP

antibodies can promote the elimination of canine preantral follicle and inhibit sperm binding,

which is an important prospect for effective control of reproduction in dogs.

Keywords: Immunocontraception, antibodies, canine, zona pellucida, preantral follicles.

Introduction

Overpopulation of dogs is a critical problem is many cities around the world, which is

characterized by the occurrence of large groups of surplus animals without a responsible

owner. Since these individuals can act as zoonoses reservoirs, health authorities perform

systematic euthanasia in order to control this subpopulation. Each year, this situation results

in millions of deaths at shelters and spending in the billions of dollars (Frank e Carlisle-Frank,

2007).

Despite efforts in recent years to identify reliable methods of pharmacologic and

chemical sterilization for dogs, surgical methods have remained the mainstay. Although

these procedures are often viewed as ‘‘routine’’ surgeries, complications may result from

inappropriate techniques, and efforts should be made to follow good surgical and aseptic

standards (Howe, 2006). In addition, such methods can be too time consuming and

expensive to be performed on a large-scale (Kutzler e Wood, 2006), and hence are not

logistically and economically feasible to be applied to large canine populations, as is the case

in metropolis.

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In this context, immunocontraception arises as a promising perspective for the control

of reproduction in dogs. This approach consists of directing the body’s immune response

towards functionally and structurally important molecules that are involved in mammalian

reproduction. Sperm surface antigens, the zona pellucida (ZP), gonadotropins (LH and FSH)

or their receptors, and the gonadotropin releasing hormone (GnRH) have been identified and

are used as targets for immunocontraceptive vaccines in carnivores. At present, the best

contraceptive vaccines are zona pellucida based, which has been shown to cause long-

lasting contraception in several wildlife species (Jewgenow et al., 2006).

The ZP consists of an extracellular glycoprotein matrix which surrounds the

mammalian oocytes and plays fundamental roles in the fertilization process through the

regulation of sperm binding, induction of the acrossome reaction and of the block to

polyspermy (Wassarman, 2008). Active immunization against ZP proteins inhibits fertility in

many species by interfering with sperm–egg interaction during fertilization, and affecting the

growing pool of ovarian follicles. The synthesis of ZP takes place during oocyte growth.

Therefore, a immune reaction against ZP can induce destruction of early ovarian oocytes,

leading sometimes to irreversible infertility (Ringleb et al., 2004). Loss of primordial follicles is

a common outcome of the immunization against one ZP glycoprotein (ZP3), which might

occur due to the elimination of growing follicles by anti-ZP antibodies followed by accelerated

recruitment of the quiescent follicles (Aitken, 2002).

The aim of the present study was to evaluate in vitro the potential of serum containing

anti-pZP antibodies to cause degeneration of canine preantral follicles. It was also tested the

capacity of these antibodies to block canine sperm binding to homologous ZP.

Material e Methods

All chemicals were obtained from Sigma-Aldrich (Sigma Chemie GmbH, Deisenhofen,

Germany) unless stated otherwise and were of the highest purity available.

Animals and gonad collection

Ovaries and testes were obtained from dogs at the veterinary clinic of the animal

shelter of the City of Berlin (Tierheim Berlin - Tierschutzverein für Berlin und Umgebung

Corporation e.V.). Following excision, ovaries were placed into 15 mL of HEPES-buffered

Minimum Essential Medium (MEM, osmolarity 280 mOsmol/l) at 4ºC. Testes were stored at

125

the same temperature without any medium. Gonads were transported to the laboratory in

thermoflasks within 4 h and then immediately processed.

Collection of oocytes, preantral follicles and spermatozoa

Ovaries were dissected in M199 containing Earle’s salts, supplemented with 3 mg/ml

BSA, 0.6 mg/ml sodium lactate, 1.4 mg/ml Hepes, 0.25 mg/ml sodium pyruvate, 0.1 mg/ml

cysteine and 0.055 mg/ml gentamicin. Grade 1 oocytes displaying an even dark ooplasm

surrounded by a multilayered cumulus cell mass were selected, vortexed for 3 min to detach

cumulus cells and then washed three times.

Canine preantral follicles were isolated using the mechanical method described by

Figueiredo et al. (1993). Briefly, using a tissue chopper (The Mickle Laboratory Engineering

Co., Gomshal, Surrey, UK) adjusted to a sectioning interval of 100 µm, ovarian cortex was

cut into small pieces, which were placed in MEM and suspended 40 times using a large

Pasteur pipette (diameter of about 1600 µm) and subsequently 40 times with a smaller

Pasteur pipette (diameter of approximately 600 µm) to dissociate preantral follicles from the

stroma. The obtained material was passed through 500- and 100-μm nylon mesh filters,

resulting in a suspension containing preantral follicles smaller than 100 μm in diameter. This

procedure was carried out within 10 min at room temperature (RT, approximately 25°C).

Preantral follicles with a diameter of 100 to 200 μm were isolated from canine ovaries

as described by Mao et al. (2004). Briefly, ovarian cortical tissue (<1 mm thickness) was

sliced from the ovarian surface, placed in HEPES-buffered α-MEM and visualized under a

dissecting microscope (SMZ 645 Nikon, Tokyo, Japan) equipped with an ocular micrometer.

Follicles were then manually isolated with a 28-gauge needle and surgical blade. Those

displaying normal morphology, no sign of oocyte retraction and well-organized layers

granulosa cells adhered to an intact basement membrane were selected.

Canine spermatozoa were obtained from caudae epididymides by mincing them in

1ml TALP medium (Tyrode’s salt solution containing 6 mg/ml BSA, 1.2 mg/ml Hepes, 1.1

mg/ml sodium lactate, 0.11 mg/ml sodium pyruvate). Sperm cells were centrifuged at 500 g

for 7 min and resuspended in 100–200ml TALP medium. After estimation of sperm

concentration and motility, a final concentration of 1 x 105 motile sperm cells/ml was

established by addition of TALP medium.

126

In vitro culture of canine preantral follicles with anti-pZP serum

Serum containing anti-pZP polyclonal antibodies was produced through immunization

of rabbits as previously described by (Jewgenow et al. 2000). Canine preantral follicles

(n=2,560) were isolated as described previously and divided into two groups according to the

diameter (<100 µm or ≥100 µm) and in vitro cultured with 10% pre-immune serum (control)

or anti-pZP serum. The used medium consisted of α-MEM (osmolarity: 300 mOsm/L, pH:

7.2) supplemented with 1% ITS (6.25 µg/ml insulin, 6.25 µg/ml transferrin and 6.25 ng/ml

selenium), glutamine (2 mM), hypoxanthine (2 mM) and bovine serum albumin (BSA,1.25

mg/ml).

Assessment of preantral follicles viability

Viability of canine preantral follicles was assessed before and after in vitro cultured

through a test based on the simultaneous determination of live and dead cells by calcein-AM

and ethidium homodimer-1, respectively. Whilst the former compound detected intracellular

esterase activity of viable cells, the latter marked nucleic acids in non-viable cells with

damaged plasma membrane. The test was performed by adding by adding 4 μM calcein-AM

and 2 μM ethidium homodimer-1 (Molecular Probes, Invitrogen, Karlsruhe, Germany) to

isolated follicles, followed by incubation at 37°C for 10 min. After being labeled, follicles were

washed three times in MEM, mounted on a glass microscope slide in 5 μl antifading medium

(DABCO, Sigma, Deisenhofen, Germany) to prevent photobleaching, and finally examined

using an a DMLB fluorescence microscope (Leica, Germany). The emitted fluorescent

signals calcein-AM, and ethidium homodimer were collected at 488 and 568 nm,

respectively. Oocytes and granulosa cells were considered live if the cytoplasm was stained

positively with calcein-AM (green) and chromatin was not labelled with ethidium homodimer

(red).

In vitro test for sperm binding test

Denuded oocytes were incubated for 1 h at 37.5°C with pre-immune serum (control),

anti-pZP serum (undiluted or diluted 1:10 or 1:20) or in HEPES-buffered MEM (positive

control). Afterwards, oocytes were transferred to separate insemination drops containing 400

ml of IVF medium (TALP solution containing 10mg/ml heparin) and 1 x 105 motile

spermatozoa/ml. After 18–20 h of co-incubation at 39°C in an atmosphere of 5% CO2,

oocytes were washed three times in DPBS with 3 mg/ml BSA to remove loosely attached

127

spermatozoa and finally placed into DPBS supplemented with 4% (w/v) paraformaldehyde

and 0.02% (v/v) Triton-X100 for 45–60 min at 39 8C. After washing twice again, oocytes

were stained with 10mg/ml Hoechst 33258 for 45 min, mounted on slides and the number of

attached sperm heads was counted under fluorescence excitation using a DMLB Leica

Microscope (Wetzlar, Germany).


All experiments were replicated five times. Kolmogorov-Smirnov and Bartlett’s tests

were applied to verify normal distribution of data and homogeneity of variances respectively.

Afterwards, analysis of variance (ANOVA) was performed using the GLM procedure of the

software SAS (SAS Institute Inc., Cary, NC, USA). Differences of percentages of viable

preantral follicles between treatments were determined by Student Newman Keuls’ (SNK)

test. For the sperm binding experiments, differences between groups were assessed by the

non-parametric Mann–Whitney test. Data were expressed as means ± standard error of the

mean (SEM) and differences were considered statistically significant when P <0.05.

Results

Effect of anti-pZP serum on viability of canine preantral follicles

Viability of canine preantral follicles was assessed before and after in vitro culture

through a test based on the simultaneous determination of live and dead cells by calcein-AM

and ethidium homodimer-1, respectively. Percentages of viable follicles observed in each

treatment are showed in Figure 1.

128

Figure 1. Percentages of viable canine preantral follicles before (fresh control time 0 h) and

after in vitro culture for 24 h with in medium containing no serum or 10% pre-immune serum

(control) or anti-pZP. (a, b) different letters indicate significant differences (P <0.05) between

values within a follicular category (<100 µm or ≥100 µm).

After in vitro culture for 24 h in medium without serum, with 10% pre-immune serum

(control) or anti-pZP serum, it was observed a significant reduction (P <0,05) of the

percentages of viable follicles with diameter smaller than 100 µm in comparison to the fresh

control (time 0h). No significant difference (P >0,05) was found among the different culture

groups (P >0,05).

With respect to the group of follicles with diameter of 100-200 µm, a significant

decrease (P <0,05) of the percentages of viable follicles was also seen after in vitro culture in

medium without serum or with either sera in comparison to the fresh control. Moreover, it

was noticed that the presence of anti-pZP serum led to a massive reduction of the

percentages of viable follicles, which were significantly lower (P <0,05) than those obtained

with the addition of pre-immune serum (control) or with culture medium alone. It is

noteworthy that along with the oocyte, no single follicular cell could survive, which is an

important outcome considering the potential of remaining granulosa cells to develop cysts

(Mahi-Brown et al., 1998). In figure 2, viable and non-viable follicles analyzed using

fluorescent labels are presented.

a

129

Fig. 2. Viability evaluation of canine preantral follicles using fluorescent probes. (A) A follicle

in vitro cultured for 24 h with pre-immune serum (control) and classified as viable since cells

were labeled by calcein-AM (green fluorescence). The arrow indicates a thick zona pellucida

surrounding the oocyte. (B) A follicle cultured with anti-pZP serum and considered non-viable

as cells were marked with ethidium homodimer-1 (red fluorescence). Scale bars represent 20

µm.

Effect of anti-pZP antibodies on canine sperm binding to homologous oocytes

The capacity of anti-pZP antibodies to block binding of dog spermatozoa to bitch

oocytes was assessed through an in vitro test which consisted of co-incubation of oocytes

treated with pre-immune (control) or hyperimmune serum with sperm followed by counting of

Hoechst 33258 stained sperm heads bound to oocytes under a fluorescence microscope (fig.

3).

A B

130

Fig. 3. Assessment of anti-pZP antibodies ability to inhibit canine oocyte-sperm interaction.

(A) Oocyte incubated in MEM (positive control) displaying several bound spermatozoa; (B)

oocyte treated with pre-immune serum (control); (C) oocyte treated with anti-pZP serum

showing no bound spermatozoon; (D) oocyte treated with anti-pZP diluted 1:10. ZP: zona

pellucida.

A B

C D

ZP

ZP

ZP ZP

131

The mean numbers of bound spermatozoa bound per oocyte for each treatment are

presented in figure 4. Incubation of oocytes with pre-immune serum led to a significant

decrease (P <0,05) of the sperm binding as compared to the positive control (incubation in

MEM). Treatment of oocytes with anti-pZP serum reduced significantly the numbers of bound

sperm (P <0,05) in comparison to the test control (pre-immune serum) and to the positive

control. Conversely, anti-pZP serum diluted at 1:10 lowered (P <0,05) sperm binding in

relation to the positive control, but no significant difference (P >0,05) was found in relation to

treatment with pre-immune serum. Dilution of anti-pZP at 1:20 had no effect on numbers of

bound sperm as compared to the controls.

Fig. 4. Assessment of the contraceptive efficiency of anti-porcine zona pellucida (pZP)

antibodies in dogs using an in vitro test for canine sperm binding to homologous ZP. Bitch

oocytes were incubated with pre-immune serum (control), anti-pZP serum (undiluted or

diluted 1:10 or 1:20) or in HEPES-buffered MEM (positive control). Subsequently, oocytes

were transferred to IVF drops and co-incubated with 1 x 105 motile spermatozoa/ml for 18-20

h. After staining with Hoechst 33258, the number of attached sperm heads on each oocyte

was counted under a fluorescence microscope. (a, b, c) Different letters indicate significant

differences (P <0.05).

a

b

c

b

ab

132

Discussion

The present study demonstrates the ability of anti-ZP antibodies to cause atresia of

ovarian preantral follicles, and thus their potential to be used for the immunosterilization of

dogs. In most of the works on immunological contraception targeting ZP, including this one,

pZP glycoproteins have been used due to its high availability in abattoirs and the high degree

of homology with the ZP proteins of many mammalian species, which ensures the cross-

reactivity of produced antibodies to native zonae (Niu et al., 2006). We have used a serum

produced against purified pZP, which contained antibodies with high specificity for native

zona proteins as shown by ELISA, immunoelectrophoresis and immunohistology (Jewgenow

et al., 2000).

In order to analyze the effects of anti-pZP serum on viability of canine preantral

follicles, we used as experimental model an efficient in vitro culture system which has been

developed by our group to promote growth of preantral follicles from some mammalian

species, but it had not been employed hitherto in canine. The maintenance of high

percentages of viable follicles after culture observed in this study indicates that this system is

also suitable for canine preantral follicles. Moreover, since it enabled observations on

isolated follicles, it was possible to assess directly and exclusively the action of anti-ZP

antibodies on these structures, contributing for the elucidation of the pathogenesis of follicle

damage showed previously in studies in vivo. It has been postulated by many authors that

cytotoxic T-cell responses due to ZP immunization would be a major mechanism (Millar et

al., 1989; Rhim et al.,1992; Lou and Tung, 1993; Jackson et al., 1998; Lou et al. 2000). The

absence of immunological cells in our system indicates that antibodies can act solely for

follicle elimination. Another important aspect of the model used in this work is the possibility

of experimentation without use of animals.

Addition of anti-pZP serum to the culture medium had no effect on viability of follicles

with diameter smaller than 100 µm as compared to the use of medium alone or with 10%

pre-immune serum. However, pZP antiserum caused extensive atresia of preantral follicles

measuring 100-200 µm. Since such effect was not observed when follicles of this category

were cultured in medium containing pre-immune serum (control), polyclonal anti-ZP

antibodies previously confirmed in the hyperimmune serum, and not other components, may

have accounted for this outcome. We suggest that massive binding of antibodies to the ZP

led to the activation of the cascade of serum complement proteins, which in turn resulted in

the formation of the membrane-attack complexes (Janeway et al., 2001) and subsequent

degeneration of follicular cells. This hypothesis is supported by the study of Gwatkin et al.

(1977), who localized antibodies and complement in the zonae of oocytes from infertile mice

133

immunized with hamster zonae. Jackson et al. (1998) postulated that in mice, antibodies

binding to the developing ZP of growing eggs may kill oocytes via antibody-dependent cell-

mediated cytotoxicity or complement lysis. Another possible mechanism for this effect could

be the interruption of communication between oocytes and granulosa via gap junctions by

large amounts of bound antibodies as postulated by Mahi-Brown et al. (1988). However,

despite these authors observed deposit of electron-dense material at the surface of the ZP in

bitches immunized with pZP, no alterations of cell junctions were found.

The absence of effect of the anti-pZP serum on follicles smaller than 100 µm might be

explained by a lower frequency of antibody binding due to lesser amounts of ZP

glycoproteins within these follicles. The zona proteins are the products of three gene families,

named ZPA, ZPB, and ZPC (Harris et al., 1994). A vast majority of follicles in this size

category comprised primordial follicles (data not presented), for which only expression of

ZPA could be detected in the cytoplasm of oocytes (Blackmore et al., 2004). Furthermore,

Barber et al. (2001) showed through ultrastructural immunohistochemistry that anti-pZP

antibodies react with zona proteins present in the golgi apparatus of canine of oocytes within

developing follicles but not when they are at the primordial stage. Conversely, follicles of the

other group (100-200 µm) corresponded to mid and late secondary follicles, which present

peak mRNA transcription for ZPC, in addition to ZPA and ZPB expression (Blackmore et al.,

2004). A comparable sequential ZP expression pattern was also described by Jewgenow

and Fickel (1999) in cats, for which ZPB gene transcripts were firstly present in primary

follicles, whilst ZPA and ZPC were detectable only from transition to secondary stage

onwards.

We also suggest as a reason for the inability of anti-pZP serum to cause

degeneration to smaller follicles that the affinity of the present antibodies may be higher or

even specific to the ZP glycoproteins synthesized by late secondary follicles. Blackmore et

al. (2004) showed through probing canine ovarian tissue with a panel of lectins that patterns

of glycosylation of zona proteins occurs in a developmentally specific manner. Residues of α-

mannose were weakly labeled in granulosa cells of primary follicles and strongly labeled in

the ZP and in surrounding granulosa cells of secondary follicles. On other hand, using the

lectin obtained from Erythrina cristagalli (ECL), which binds D-galactose and N-acetyl

glucosamine, labeling was identified in the ZP and adjacent granulosa cells of secondary

follicles only. Therefore, in order to achieve immunosterilization targeting directly the pool of

oocytes within the quiescent follicles, a promising strategy could be the characterization of

the ZPA proteins already expressed in canine primordial follicles to be subsequently used in

immunogens. Grootenhuis et al. (1996) observed the binding of anti-ZP antibodies to oocytes

and granulosa cells of primordial follicles in several species, suggesting that complement

134

fixation following antibody binding may be the mechanism of depletion of these follicles

observed in studies in vivo.

Despite primordial follicles were not affected by anti-pZP serum in the present study,

the observed degeneration of late secondary follicles is also an important prospect for the

immunosterilization of bitches. Skinner et al. (1984) demonstrated that immunization of

rabbits with pZP resulted in a complete disappearance of growing follicles within 30 weeks

with associated reduction of the number of primordial follicles. These authors postulated that

developing follicles provide some signal that prevents the activation of primordial follicles,

and since the former are destroyed by anti-ZP antibodies, the latter start a suicidal growth

until ZP proteins are secreted. These progressive changes persist whilst ovaries are under

influence of such antibodies. Thus, the anti-ZP antibodies studied here could promote a

similar process in vivo leading to an immunological castration.

Mahi-Brown et al. (1988) observed that immunization of bitches with pZP caused

extensive oocyte destruction. These authors suggested that this consequence could

probably lead to permanent sterility. In some animals, follicles failed to survive beyond the

primordial stage. However, it was also noticed the occurrence of follicular cysts and

abnormal estrous cycles in which prolonged elevated 17 β-estradiol was recorded in most of

the treated females. Despite bitches were rendered infertile, this side effect is undesirable,

since some sequela such as bone marrow hypoplasia and/or pyometra can develop (Meyers-

Wallen, 2007). In the present study, complete follicular degeneration was achieved, i.e. no

single granulosa cell could survive along with the dead oocyte. Therefore, potential of cyst

formation would be unlikely if in vitro conditions can be established in vivo. We hypothesize

that levels of antibodies that could interact with follicles in our work were higher than those

attained within ovaries in the study of Mahi-Brown et al. (1988). Anti-pZP titres of the sera

used in the current study ranged from 1:100 000 to 1:160 000, whilst in active immunized

animals in the mentioned previous work, titers ranged from 1:1000 to 1:20 000. Hence, levels

of anti-pZP antibodies in the former study could possibly had not reached a threshold to

cause massive follicle degeneration, and remaining live granulosa cells may have developed

to the observed cysts. In accordance to this theory, Srivastava et al. (2002) reported an

increase in the number of atretic follicles associated with the antibody titres in bitches

immunized with recombinant canine ZP3.

Capacity of the used anti-ZP antibodies to block binding of spermatozoa to the canine

ZP was also evaluated in this study. We could observe a significant inhibition of sperm

binding when oocytes were incubated for 1 h with undiluted serum, but dilution (1:10 and

1:20) suppressed this effect. Nevertheless, bitches do not need to have extremely high anti-

135

ZP titers in order for their eggs to be impenetrable. Since oocytes in vivo are exposed

continuously to circulating immunoglobulins, there may be more thorough coating of their

zonae pellucidae than after in vitro incubation (Mahi-Brown et al., 1985). Oocytes recovered

from the ovary of one bitch immunized with pZP were not penetrable by spermatozoa,

whereas antiserum from this same bitch did not prevent sperm penetration (Mahi-Brown et

al., 1982). Hence, the anti-ZP antibodies tested in the present work have also potential to be

used for the immunocontraception of dogs through fertilization blocking.

In conclusion, anti-pZP antibodies are capable to cause atresia of canine preantral

follicles as the secondary stage of development is reached. This process might involve the

serum complement system. Therefore, immunosterilization of bitches can be potentially

accomplished through vaccination against pZP proteins or passive immunization with anti-

pZP antibodies. Contraception through blocking of sperm binding can possibly be achieved

by using these antibodies as well. Once established, such techniques could enable an

effective control of dog populations in many urban areas.

Acknowledgements

During the period of this work, Claudio A. P. Lopes received financial support from the

National Council for Scientific and Technological Development (CNPq; doctoral scholarship)

and the Coordination for Improvement of Graduate Personnel (CAPES; fellowship for studies

abroad, PDEE, grant 5212/06-5), both institutions of the Brazilian Government. José Ricardo

de Figueiredo was also recipient of a grant from CNPq. We would like to express our

appreciation to the veterinary clinics of the animal shelter Tierheim-Berlin and Tierklinik-

Biesdorf (Germany), which gently provided the canine ovaries.

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11 CONCLUSÕES

A preservação de folículos pré-antrais caninos durante o armazenamento é realizada

com maior eficiência através de hipotermia em um meio nutritivo. A manutenção da

viabilidade e integridade ultra-estrutural pode ser obtida a 4ºC e 20ºC em solução salina por 6

h ou em MEM por 12 h, sendo esta última solução também adequada a 38ºC por 6 h. Sugere-

se o uso de MEM a 4ºC por até 12 horas para a preservação ótima da qualidade de folículos

pré-antrais caninos destinados a aplicações como o cultivo in vitro.

A criopreservação de folículos pré-antrais caninos pode ser realizada através de

congelação lenta e descongelação rápida utilizando-se DMSO a 1,5 M. Este crioprotetor

proporciona a manutenção de altas percentagens de folículos viáveis com ultra-estrutura

intacta. Índices elevados de preservação da ultra-estrutura folicular podem também ser

obtidos com EG a 1,5 M. Entretanto, faz-se necessário avaliar a viabilidade dos folículos

criopreservados utilizando-se este composto.

Folículos pré-antrais caninos frescos ou criopreservados podem ser cultivados in vitro

mantendo sua viabilidade. Assim, pode-se utilizar este sistema como modelo experimental

para o teste de agentes esterilizantes.

Anticorpos anti-ZP são capazes de se ligar à ZP de oócitos caninos, causando bloqueio

da ligação de espermatozóides ou atresia de folículos pré-antrais.

139

12 PERSPECTIVAS

Os conhecimentos acerca da preservação de folículos pré-antrais caninos obtidos neste

estudo poderão servir como base para o desenvolvimento pleno desta técnica, que, uma vez

estabelecida, poderá ser aplicada a canídeos em risco de extinção de forma a se preservar o

patrimônio genético de cada fêmea rara. Ainda, o uso do método de congelação lenta e

descongelação rápida utilizando-se DMSO a 1,5 M já possibilita a manutenção de elevados

percentuais de folículos viáveis com ultra-estrutura intacta. Entretanto, é preciso avaliar a

capacidade de desenvolvimento de tais folículos, sendo necessário o estabelecimento de

sistemas de cultivo in vitro ou in vivo, sendo as técnicas de xenotransplante ou alotransplantes

recursos promissores. O método de cultivo in vitro utilizado neste trabalho também

demonstrou potencial para este propósito.

A alta eficiência da congelação utilizando-se EG a 1,5 M para a manutenção da ultra-

estrutura folicular indica o potencial deste composto para a criopreservação de folículos pré-

antrais caninos. No entanto, a viabilidade, bem como a capacidade de desenvolvimento

folicular, devem ser avaliados, sendo importante também a comparação direta da eficiência

deste crioprotetor àquela observada para o DMSO, que proporcionou os melhores resultados.

A demonstração da capacidade de anticorpos anti-ZP de causar atresia em folículos

pré-antrais caninos consolida a importante perspectiva do desenvolvimento de um método de

imunoesterilização de cães, que poderá se estabelecer através da composição de uma vacina

ou de um soro hiperimune. Isto poderá permitir o controle reprodutivo de cães nas cidades e

estabelecer uma abordagem racional, efetiva e humanitária para o controle populacional

destes animais.

140

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