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FORTALEZA-CEAR
Dezembro de 2007
Veterinrias
do
Programa
de
Ps-
FORTALEZA-CEAR
Dezembro de 2007
Banca Examinadora
________________________________________________
Prof. Dr. Jos Ricardo de Figueiredo (Orientador)
Universidade Estadual do Cear
_________________________________________
Prof. Dr. Marcelo Marcondes Seneda (Examinador)
Universidade Estadual de Londrina
_________________________________________
Prof. Dr. Claudio Cabral Campello (Examinador)
Universidade Estadual do Cear
_________________________________________
Profa. Dra. Lcia Daniel Machado da Silva (Examinadora)
Universidade Estadual do Cear
AGRADECIMENTOS
No, no simples agradecer o que quero. Quero trazer para dentro do texto
aqueles que j o percorrem nas entrelinhas. E no s aos que me ajudaram efetivamente
na construo dessa Dissertao, mas aos amigos e colegas que partilham comigo idias
e sentimentos. queles que me ajudaram de alguma forma, no meu percurso nesses
quase dois anos e, principalmente, a seguir adiante, sem perder o que pulsa, o que vibra,
agradeo imensamente.
Em especial, a Deus, pela proteo, fora e coragem para enfrentar as
dificuldades na vida pessoal e profissional. E por me inspirar a certeza que nada nesse
mundo ocorre por acaso.
Ao meu esposo, Kenio Patrcio Lima de Oliveira, pelo inestimvel apoio
familiar que preencheu as diversas falhas que fui tendo por fora das circunstncias. E
pela pacincia e compreenso reveladas ao longo desses meses, mas principalmente
pelo amor e exemplo de companheirismo no sucesso e no fracasso, na alegria e na
tristeza, na sade e na doena, nestes anos de nossa vidas.
Aos meus pais, Francisco Arajo Chaves e Eurinice Nogueira Chaves, por terem
sido contnuo apoio em todos estes anos, ensinando-me, principalmente, a importncia
da construo e coerncia de meus prprios valores. Pelo estmulo e apoio incondicional
desde a primeira hora, pela pacincia com que sempre me ouviram, e sensatez com que
sempre me ajudaram.
Aos meus familiares, em especial, aos meus irmos (Francisco Hernandes
Nogueira Chaves e Renata Maria Nogueira Chaves) pela oportunidade de
compartilharmos experincias. E meus sogros (Jos Patrcio de Oliveira e Iza Helena
Lima de Oliveira) por me receberem na famlia e pela compreenso nos momentos
difceis.
Universidade Estadual do Cear (UECE) e Faculdade de Veterinria
(FAVET) por todos os anos de ensino e aprendizagem.
Ao Dr. Jos Ricardo de Figueiredo, professor e orientador, que devido sua
excelncia profissional conferiu prestgio e valor ao meu trabalho. Agradeo pela
abertura de esprito revelada desde a primeira aula, que logo me abriu a porta que
rapidamente me encaminharia para o tema tratado nesta dissertao. Pela
disponibilidade revelada ao longo desses meses, pelas crticas e sugestes relevantes,
pelo apoio moral e amizade.
Ao meu co-orientador, Dr. Jos Roberto Viana Silva, pelo apoio e ensinamentos
dedicados durante o desenvolvimento desse trabalho.
RESUMO
10
ABSTRACT
The aim of this study was to evaluate the effect of storing goat ovarian fragments in
different temperatures and storage times on viability and growth of preantral follicles
cultured in vitro. Caprine ovaries (n=10) were collected at slaughterhouse and each
ovarian pair was divided into 19 fragments. One fragment was immediately fixed for
classical histology and transmission electron microscopy (fresh control day 0).
Remaining fragments were distributed in Minimal Essential Medium (MEM) and
maintained at 4, 20 or 35 C for 2 or 4 h. After each conservation period, fragments
(n=30) were fixed (non-cultured), while fragments (n=60) were cultured in vitro for 1 or
7 days at 39 C and 5% CO2 in supplemented MEM. Follicular morphology was
evaluated at the end of both conservation and culture periods, by classical histology and
transmission electron microscopy. After 7 days of culture, only ovarian fragments
stored at 4 C for 4 h maintained the percentage of morphologically normal follicles
similar to fresh control (P>0.05). In all treatments, a significant reduction (P>0.05) in
the number of primordial follicles and a concomitant increase in the developing follicles
was observed, indicating follicular activation. Additionally, there was an increase in
oocyte and follicular diameter after culture of ovarian cortex that had been previously
chilled at 4 C for 2 or 4 h. Transmission electron microscopy confirmed that ovarian
fragments conservation at 4 C for 4 h, 20 and 35 C for 2 h helps to maintain
morphological integrity of caprine preantral follicles after 7 days of culture. In
conclusion, goat ovarian fragments stored at 4 C up to 4 h keep follicular viability
during transportation and increase follicular growth during in vitro culture.
11
: Antro
AMH
: Hormnio Anti-Mlleriano
APAF-1
ATP
: Adenosina Trifosfato
Bcl
: gene anti-apopttico
BMP
BSA
BrdU
: Bromodesoxiuridina
Ca++
: on Clcio
CAD
CE
: Cear
CG
: Clulas da Granulosa
CGP
CO2
: Dixodo de carbono
CT
: Clulas da Teca
DF
: Distrito Federal
DNA
: cido Desoxirribonuclico
EGF
FAVET
: Faculdade de Veterinria
FGF
FOPA
FSH
FUNCAP
GC
GDF-9
GH
: Hormnio do crescimentoo
GLM
12
: Horas
HC
: Histologia Clssica
ICAD
IGF-I
ITS
K+
: on Potssio
KGF
KL
: Kit ligand
: Hormnio Luteinizante
LIF
: Mitocndria
MEM
MEM+
MET
mL
: Mililitro
mM
: Milimolar
mm
: Milmetro
mm3
: Milmetro Cbico
MOIFOPA
: Nmero
Na+
: on Sdio
Nm
: Nanmetro
: Ncleo
ng
: Nanograma
NGF
Nu
: Nucleo do ocito
NUBIS
: Ocito
13
PAS
PBS
PCNA
pFSH
pH
: Potencial Hidrogeninico
P<0,05
PPGCV
RNAm
SAS
SEM
ser
SNK
TCM 199
TEM
TNF
TUNEL
UECE
UnB
: Universidade de Braslia
VEGF
ZP
: Zona Pelcida
: Microlitro
: Micrmetro
: Micrograma
: Graus Celsius
: Porcentagem
400x
4.000x
10.000x
14
LISTA DE FIGURAS
03
05
07
09
10
13
17
19
23
40
43
47
15
48
49
50
16
LISTA DE TABELAS
08
41
45
46
17
SUMRIO
1. INTRODUO
01
2. REVISO DE LITERATURA
02
02
2.2 Oognese
03
04
11
13
14
2.6.1 Apoptose
15
2.6.2 Necrose
17
19
21
22
24
25
26
3. JUSTIFICATIVA
28
4. HIPTESE CIENTFICA
29
5. OBJETIVOS
30
6. CAPTULO I
31
18
59
8. PERSPECTIVAS
60
REFERNCIAS BIBLIOGRFICAS
61
19
1. INTRODUO
20
2.
REVISO DE LITERATURA
21
2.2 Oognese
22
23
c
CG
CG
N
CG
CT
f
CT
ZP
A
O
CG O
CG
Figura 2. Folculos ovarianos aps colorao com cido Peridico de Schiff (PAS)Hematoxilina. Folculos pr-antrais: (a) primordial; (b) transio; (c) primrio e (d)
secundrio. Folculo antrais: (e) tercirio e (f) pr-ovulatrio. N: ncleo do ocito; O:
ocito; CG: clulas da granulosa; ZP: zona pelcida; A: antro; CT: clulas da teca.
24
25
transformao do formato destas clulas de pavimentoso para cbico. Durante esta fase,
os folculos que apresentam clulas da granulosa com ambos os formatos pavimentoso e
cbico so denominados intermedirios ou de transio (BARNETT et al., 2006).
Clulas epiteliais da
superfcie ovariana
Folculo primordial
Clulas
i ti
26
teca (SILVA et al., 2004b). Neste estdio, a zona pelcida claramente identificada ao
redor do ocito (PARROT & SKINNER, 2000). Alm disso, as clulas da granulosa
apresentam uma extensiva rede de junes do tipo gap, que so canais membranrios
que permitem a passagem de nutrientes, ons inorgnicos, mensageiros e pequenos
metablitos entre as clulas (KIDDER & MHAWI, 2002). O ncleo do ocito assume
uma posio exctrica e as organelas comeam a mover-se para a periferia (LUCCI et
al., 2001). Os folculos secundrios so observados em ovrios de fetos caprinos, ovinos
e bovinos aos 80, 120 e 210 dias de gestao, respectivamente (BEZERRA et al., 1998;
McNATTY et al., 1999; RSSE, 1983). Apesar de serem responsivos s
gonadotrofinas, os folculos secundrios podem desenvolver-se at o estdio antral com
uma quantidade mnima de FSH circulante (McGEE & HSUEH, 2000). Os dimetros
das categorias foliculares mencionadas anteriormente so mostrados na Tabela 1.
Tabela 1. Dimetro de folculos primordiais, primrios e secundrios e seus ocitos e
nmero de clulas da granulosa em seces histolgicas em caprinos.
Classe folicular
Dimetro (m)
N de CG
Folculo
Ocito
Primordial
20,0
15,9
1-14
Primrio
24,4
17,4
5-26
Secundrio
44,2
24,5
13-114
Com a intensa proliferao das clulas da granulosa, uma rea preenchida por
fluido folicular identificada na camada granulosa e, a partir de ento, os folculos
passam a ser classificados como antrais. O fluido antral pode servir como uma
importante fonte de substncias reguladoras ou moduladoras derivados do sangue ou
secrees de clulas foliculares como gonadotrofinas, esterides, fatores de
crescimento, enzimas, proteoglicanos e lipoproteas. Durante o desenvolvimento
folicular, a produo de fluido antral intensificada pelo aumento da vascularizao
folicular e permeabilidade dos vasos sanguneos, os quais esto fortemente relacionados
com o aumento do folculo antral (VAN DEN HURK & ZHAO, 2005). A formao dos
27
folculos antrais em caprinos, ovinos e bovinos observada aos 110, 135 e 230 dias,
respectivamente (BEZERRA et al., 1998; McNATTY et al., 1999; RSSE, 1983). A
partir deste estdio, o dimetro folicular aumenta acentuadamente devido ao
crescimento do ocito, multiplicao das clulas da granulosa, da teca e aumento da
cavidade antral.
O desenvolvimento dos folculos antrais caracterizado por uma fase de
crescimento, recrutamento, seleo e dominncia, sendo a formao de folculos provulatrios (ltimo estdio de desenvolvimento folicular) um pr-requisito para a
ovulao e formao do corpo lteo, bem como manuteno da fertilidade (Figura 4)
(DRUMMOND, 2006). O folculo pr-ovulatrio caracterizado por um ocito
circundado por clulas da granulosa especializadas que so denominadas de clulas do
cumulus. As clulas da granulosa de folculos pr-ovulatrios param de se multiplicar
em resposta ao hormnio luteinizante (LH) e iniciam o processo final de diferenciao.
A ovulao ocorre em resposta ao pico de LH. Em todas as espcies, a formao de
folculos pr-ovulatrios ocorre geralmente durante a puberdade (DRIANCOURT,
2001).
28
Lutelise
Recrutamento
Seleo
Dominncia
Atresia
29
30
31
32
33
2.6.1 Apoptose
A apoptose, tambm conhecida como morte celular programada, um evento
geneticamente determinado, ou seja, depende do balano de genes pr e antiapoptticos, e observada nos folculos ovarianos durante toda a vida, incluindo a fase
pr-natal (HUSSEIN, 2005). A apoptose geralmente mediada por mecanismos
intrnsecos, podendo tambm ser influenciada por fatores extrnsecos (JOHSTONE et
al., 2002).
34
da
apoptose
(HENGARTNER,
2000;
KAUFMANN
&
35
Normal
Condensao
Fragmentao
Corpos apoptticos
36
2.6.2 Necrose
A necrose, diferentemente da apoptose, considerada uma forma no
programada de morte celular e iniciada devido a algum estmulo circunstancial
resultando na rpida quebra da homeostasia celular (BRAS et al., 2005).
A necrose comumente ocorre devido a estmulos txicos, isqumicos,
degenerativos e imunolgicos, podendo tais fatores tambm induzir a apoptose. Alm
disso, alguns estmulos que levam a ocorrncia de apoptose podem em certas
circunstncias (como depleo de energia ou reduo da ativao das caspases) induzir
o fenmeno da necrose (ZEISS, 2003). Geralmente, a necrose iniciada por
mecanismos no celulares como isquemia, deficincia de nveis de ATP (BHATIA,
2004), trauma, levando a danos irreversveis na clula (McCULLY et al., 2004).
Recentes trabalhos tm sugerido que alm dessas causas, mecanismos ativos como
uma sobrecarga de Na+, acmulo de Ca2+ e mudanas na permeabilidade da mitocndria
podem tambm participar e levar ao processo necrtico (PADANILAM, 2003).
O padro bioqumico que leva a morte celular necrtica pouco conhecido. Em
injrias isqumicas ou hipxicas, a depleo de energia ocorre devido a uma deficiente
produo de ATP associada a um rpido consumo de ATP pelas bombas e devido
hidrlise e perda de ATP. O aumento do volume celular associado com a morte celular
necrtica iniciado devido ao influxo de Na+ e liberao de ATP devido ruptura da
membrana (PADANILAM, 2003). O aumento dos nveis de Na+ no citosol ativa a
bomba Na+-K+-Atpase, resultando na dissipao de ATP. Nos estdios iniciais da
injria, um simultneo efluxo de K+ mantm a homeostase inica. Severas deplees de
ATP levam a falhas no mecanismo de balano da bomba de ons, levando a um influxo
de Na+ e gu,a o qual resulta em inchao e ruptura da clula. Esse acmulo de Na+
concomitante com uma severa deficincia de ATP o maior responsvel pela necrose
(CARINI et al., 1995). O aumento dos nveis de Ca2+ ativa endonucleases para degradar
DNA, bem como proteases celulares ativas para degradar diversas estruturas e protenas
sinalizadoras (WANG, 2000).
A participao da mitocndria na necrose a na apoptose pode ocorrer pela
abertura de poros durante a permeabilidade mitocondrial transitria. Alguns segundos
mensageiros e protenas pr-apoptticas, incluindo membros da famlia Bcl-2, podem
induzir a abertura de poros na mitocndria (KROEMER & REED, 2000).
37
Normal
Inchao reversvel
Inchao irreversvel
Desintegrao
esteretipas
incluindo
aumento
de
volume
celular,
degenerao
38
39
40
41
In situ (fragmentos)
e
Isolado em suspenso
Figura 9. Modelos para cultivo in vitro de folculos pre-antrais. Folculos in situ (Fig. a,
b) e isolados (Fig. c, d, e, f). Adaptado de HARTSHORNE, 1997.
42
de folculos pr-antrais aps cultivo in vitro, porm sem a formao de antro. Nas
espcies bovinas (ITOH et al., 2002), ovinas (CECCONI et al., 1999), caprinas
(HUAMIN & YONG, 2000) e humana (ROY & TREACY, 1993), folculos pr-antrais
isolados foram cultivados in vitro e se desenvolveram at o estdio antral. Em sunos,
folculos secundrios crescidos in vitro chegaram at a ovulao, tiveram seus ocitos
fecundados in vitro, com desenvolvimento at estdio de blastocisto (WU et al., 2001).
Os maiores avanos do cultivo folicular foram obtidos em roedores, tendo sido
obtido o nascimento de crias viveis a partir do cultivo de ocitos provenientes de
folculos pr-antrais de camundongas, em que o ocito adquiriu competncia para
maturao, fertilizao e desenvolvimento embrionrio (O BRIEN et al., 2003). Tal
crescimento foi obtido atravs de dois sistemas de cultivo. O primeiro consistiu no
cultivo de ovrios inteiros para obteno da transio de primordial para primrio. O
segundo consistiu no isolamento e cultivo de folculos primrios e secundrios.
Provavelmente essa estratgia necessria para promover o crescimento de folculos
primordiais de espcies domsticas e primatas (SILVA et al., 2006). Embora a
utilizao de fragmentos ovarianos no cultivo permita a ativao folicular (HOVATTA
et al., 1999), a variedade de clulas presentes no cultivo in situ torna o sistema pouco
definido e impede estudos mais detalhados sobre a biologia dos ocitos e da
foliculognese nos estdios iniciais de desenvolvimento (MURUVI et al., 2005). O
presente desafio determinar condies de cultivo apropriadas para dar suporte
transio de folculos primrios para secundrios in vitro. Esta fase apresenta alta
sensibilidade degenerao em virtude da alta atividade biossinttica e consumo de
nutrientes, sendo a composio do meio um importante fator para a obteno de sucesso
durante o cultivo de folculos pr-antrais in vitro (WANDJI et al., 1997).
43
descreveram que a sobrevivncia dos folculos pr-antrais bovinos in vitro foi reduzida
na ausncia de hipoxantina e substratos energticos, tais como piruvato e glutamina. Foi
demonstrado tambm que uma mistura de piruvato (0,23 mM), glutamina (2 mM) e
hipoxantina ao meio de cultivo de base (MEM), suplementado com ITS (insulina 6,25
g/ml, transferrina - 6,25 g/ml e selnio - 6,25 g/ml) aumentou significativamente a
porcentagem de folculos morfologicamente normais durante o cultivo. JEWGENOW et
al. (1998) mostraram tambm que a adio de piruvato, glutamina e hipoxantina ao
meio de cultivo (MEM) so essenciais para o crescimento de folculos pr-antrais
felinos in vitro.
Insulina normalmente adicionada ao meio de cultivo como fator de
sobrevivncia, permitindo um melhor aproveitamento das fontes de energia do meio e
aumentando os precussores metablicos como aminocidos e glicose (LIU et al., 2002).
Os antioxidantes (selnio e transferrina) so relatados como importantes
substncias a serem adicionadas ao meio. Alguns autores sugerem que o processo de
maturao folicular est relacionado aos altos nveis de transferrina e seus receptores na
clula e que selnio pode ser adicionado ao meio de cultivo para ativar enzimas
envolvidas na detoxificao e eliminao de radicais livres (DEMEESTERE et al.,
2005). SILVA et al. (2004b) demonstraram a importncia da adio de tais substncias
ao MEM durante o cultivo de folculos pr-antrais caprinos inclusos no tecido ovariano.
O FSH usualmente adicionado ao meio de cultivo de folculos pr-antrais em
camundongas e grandes mamferos (MAO et al., 2002). Em caprinos, a adio de 50
ng/mL de FSH ao meio de cultivo de folculos pr-antrais inclusos em tecido ovariano
foi responsvel pela preservao da viabilidade folicular, aumento dos dimetros
folicular e oocitrio bem como pela manuteno da integridade ultra-estrutural dos
folculos (MATOS et al., 2007). MITCHELL et al. (2002) revelaram que na ausncia de
FSH somente 10% dos folculos sobreviveram durante crescimento in vitro.
44
45
detectar esses produtos de secreo folicular, podem ser utilizadas anlises de alta
sensibilidade, entretanto, anlises das concentraes de RNAm indicando a expresso
gnica ou imunohistoqumica utilizando anticorpos para protenas especficas so as
tcnicas mais indicadas (HARTSHORNE, 1997). Estes so alguns exemplos das
diferentes tcnicas que podem ser utilizadas para avaliar a qualidade do folculo aps o
cultivo in vitro (MATOS, 2007). Contudo, a melhor anlise da capacidade de preservar
folculos pr-antrais apropriadamente, consiste na obteno da sua ativao (i.e.,
transio de folculos primordiais para primrios) e crescimento in vitro (SANTOS et
al., 2006). Sendo assim, CECCONI et al. (2004a) confirmam a importncia do cultivo
in vitro como uma ferramenta de avaliao de folculos pr-antrais ovinos preservados a
baixas temperaturas.
46
3. JUSTIFICATIVA
A criao de caprinos no Brasil tem crescido bastante nos ltimos anos como um
ramo econmico rentvel. Dentre vrios aspectos, a explorao de caprinos no nordeste
brasileiro destaca-se pelo seu efetivo, uma vez que mais de 90% do rebanho nacional
explorado nessa regio e por apresentar material gentico de excelente qualidade para
produo de pele, carne e adequada produo de leite.
Desta forma, essencial o desenvolvimento de tcnicas que aumentem o
potencial reprodutivo de animais de alta produtividade. O desenvolvimento de um
sistema de cultivo eficiente poder fornecer subsdios para uma melhor compreenso
acerca dos fatores que regulam a foliculognese na fase pr-antral, fatores esses
necessrios para a sobrevivncia, a ativao e o incio do crescimento folicular. O
melhor conhecimento da foliculognese caprina uma condio necessria para
aumentar os ndices reprodutivos destes animais.
Mesmo com a possibilidade de recuperar milhares de folculos pr-antrais a
partir de um nico ovrio, um fator limitante da MOIFOPA consiste na manuteno da
qualidade folicular aps remoo e transporte dos ovrios, uma vez que na maioria das
vezes esses animais encontram-se distantes dos laboratrios de reproduo. Por esta
razo, torna-se fundamental a utilizao de protocolos eficientes de preservao.
Embora j tenha sido mostrado que possvel preservar ocitos imaturos inclusos em
folculos pr-antrais, ainda no conhecido se as condies de transporte (tempo e
temperatura) dos ovrios afetam a eficincia do cultivo in vitro. Apesar de vrios meios
para o transporte j terem sido testados, ainda no foi avaliada a eficincia do MEM. O
crescimento in vitro de folculos preservados in vitro poder viabilizar o aproveitamento
dos milhares de ocitos presentes em ovrios mamferos, revolucionando a produo in
vitro de embries.
47
4. HIPTESE CIENTFICA
48
5. OBJETIVOS
49
6. CAPTULO I
Resfriamento de fragmentos ovarianos durante o transporte melhoram
a viabilidade e crescimento de folculos pr-antrais caprinos cultivados
in vitro
RESUMO
O objetivo do presente estudo foi avaliar o efeito da conservao de fragmentos
ovarianos em diferentes temperaturas e tempos de conservao sobre a viabilidade e
crescimento in vitro de folculos pr-antrais caprinos aps cultivo in vitro. Os ovrios
caprinos (n=10) foram obtidos de animais de abatedouro. Cada par ovariano foi dividido
em 19 fragmentos. Um fragmento foi imediatamente fixado para histologia clssica e
microscopia eletrnica de transmisso (controle fresco dia 0). Os fragmentos restantes
foram distribudos em Meio Essencial Mnimo (MEM) e mantidos a 4, 20 ou 35C por
2 ou 4 h. Aps cada tempo de conservao, fragmentos (n=30) foram fixados (no
cultivados), enquanto fragmentos (n=60) foram cultivados in vitro por 1 ou 7 dias a
39C e 5% de CO2 em MEM suplementado. Aps cada perodo de conservao e/ou
cultivo, os fragmentos foram submetidos histologia clssica e microscopia eletrnica
de transmisso a fim de avaliar a morfologia folicular. Aps 7 dias de cultivo, somente
os fragmentos ovarianos armazenados a 4C por 4 h mantiveram o percentual de
folculos morfologicamente normais equivalentes ao controle fresco (P>0,05).
Observou-se que em todos os tratamentos ocorreu uma reduo (P>0,05) no nmero de
folculos primordiais e um concomitante aumento nos folculos em desenvolvimento,
indicando ativao. Alm disso, houve um aumento no dimetro oocitrio e folicular
aps o cultivo de crtex ovariano submetido conservao na temperatura de 4 C por 2
e 4 h. A microscopia eletrnica de transmisso confirmou a histologia clssica,
mostrando que a conservao na temperatura de 4 C por 4 h, 20 e 35 C por 2 h ajudou
a manter a integridade morfolgica dos folculos pr-antrais caprinos aps 7 dias de
cultivo. Em concluso, os fragmentos ovarainos caprinos armazenados a 4C por at 4 h
melhoram a viabilidade folicular durante transporte e aumenta o crescimento folicular
durante cultivo in vitro.
Palavras-chave: caprino, conservao, cultivo in vitro, folculo pr-antral.
50
CAPTULO I
Ovarian fragments chilling during transportation improves viability and growth of
goat preantral follicles cultured in vitro
R.N. Chavesa, F.S. Martinsa, M.V.A. Saraivaa, J.J.H. Celestinoa, C.A.P. Lopesa, J.C.
Correiaa, I.B. Lima Verdea, M.H.T. Matosa, S.N. Bob, K.P.O. Nameb, C.C. Campelloa,
J.R.V. Silvac, J.R. Figueiredoa
51
Abstract
The aim of this study was to evaluate the effect of storing goat ovarian fragments in
different temperatures and storage times on viability and growth of preantral follicles
cultured in vitro. Caprine ovaries (n=10) were collected at slaughterhouse and each
ovarian pair was divided into 19 fragments. One fragment was immediately fixed for
classical histology and transmission electron microscopy (fresh control day 0).
Remaining fragments were distributed in Minimal Essential Medium (MEM) and
maintained at 4, 20 or 35 C for 2 or 4 h. After each conservation period, fragments
(n=30) were fixed (non-cultured), while fragments (n=60) were cultured in vitro for 1 or
7 days at 39 C and 5% CO2 in supplemented MEM. Follicular morphology was
evaluated at the end of both conservation and culture periods, by classical histology and
transmission electron microscopy. After 7 days of culture, only ovarian fragments
stored at 4 C for 4 h maintained the percentage of morphologically normal follicles
similar to fresh control (P>0.05). In all treatments, a significant reduction (P>0.05) in
the number of primordial follicles and a concomitant increase in the developing follicles
was observed, indicating follicular activation. Additionally, there was an increase in
oocyte and follicular diameter after culture of ovarian cortex that had been previously
chilled at 4 C for 2 or 4 h. Transmission electron microscopy confirmed that ovarian
fragments conservation at 4 C for 4 h, 20 and 35 C for 2 h helps to maintain
morphological integrity of caprine preantral follicles after 7 days of culture. In
conclusion, goat ovarian fragments stored at 4 C up to 4 h keep follicular viability
during transportation and increase follicular growth during in vitro culture.
52
Introduction
53
2000; ovine: ANDRADE et al., 2002; bovine: LUCCI et al., 2004; swine:
WONGSRIKEAO et al., 2005; equine: PEDERSEN et al., 2004; canine: TAS et al.,
2006), but none of them evaluated the ability of chilled preantral follicles to develop in
vitro. Since follicular quality is essential for the success of follicle growth and oocyte
maturation, it is very important to evaluate if transportation conditions (time and
temperature) of caprine ovaries affect follicular viability during in vitro culture.
The present study aimed to evaluate the effect of the preservation of caprine
ovarian tissue in different temperatures and incubation times on the viability and growth
of preantral follicles cultured in vitro.
Ovaries (n=10) from five adult non-pregnant mixed breed goats were collected
at a local slaughterhouse. Immediately after collection, ovaries were stripped of
surrounding fat tissue and ligaments, washed one time in 70% alcohol and twice in
MEM. Still in the slaughterhouse, the ovarian pair from each animal was divided into
19 fragments of approximately 3 x 3 mm (1 mm thick) consisting of the ovarian cortex
of the two ovaries. An ovarian fragment was randomly taken and immediately fixed in
Carnoy and 2% paraformaldehyde and 2.5% glutaraldehyde in 0.1 M sodium cacodylate
buffer for histology and Transmission Electronic Microscopy (TEM), respectively
(fresh control day 0). The other 18 fragments were distributed into tubes containing 5
ml of MEM supplemented with 100 g/ml penicillin and 100 g/ml streptomycin and
54
After conservation period, the half of the ovarian fragments (n=30) were fixed
for histological and ultrastructural studies (non-cultured fragments). Other fragments
(n=60) were individually cultured in 24-well culture dishes (Corning Inc., Corning, NY)
containing 1 mL of culture medium at 39 C, in presence of 5% CO2 in air for 1 or 7
days. Fragments were cultured using a modification of the method described by
FIGUEIREDO et al. (1994) for bovine preantral follicles. The culture medium consisted
of MEM+, consisting of MEM with antibiotics (100 g/mL of penicillin, 100 g/mL of
streptomycin), 0.25 g/mL amphotericin B, 1.25 mg/mL BSA, ITS (insulin 6.25 g/mL,
transferrin 6.25 g/mL and selenium 6.25 ng/mL), 0.23 mM pyruvate, 2 mM glutamine,
2 mM hypoxanthine and pFSH (50 ng/mL - provided by Dr J.F. Beckers, Lige,
Belgium). Unless otherwise indicated, reagents were obtained from Sigma Chemical
Co. (St. Louis, MO). Fresh medium was prepared immediately before use. Every two
days, the total culture medium was replaced by fresh medium. Each treatment was
repeated 5 times.
55
section were stained using standard protocols with Periodic Acid Schiff and
hematoxilyn (PAS staining system, Sigma, Inc., St. Louis, MO, USA). Sections were
examined by light microscopy (Zeiss, Germany) at 400X magnification.
Preantral follicles were counted and evaluated only when oocyte nuclei were
visible. According to the developmental stage, preantral follicles were classified as
primordial (oocyte surrounded by one layer of flattened pre-granulosa cells) or
developing, which ones were divided into intermediate (oocyte surrounded by one layer
of flattened pre-granulosa and cuboidal granulosa cells), primary (oocyte surrounded by
one layer of cuboidal granulosa cells) or secondary (oocyte surrounded by two or more
layers of cuboidal granulosa cells) (Silva et al., 2000).
Each follicle was evaluated according to oocyte and granulosa cells integrity,
presence or absence of pyknotic bodies, ooplasmic retraction and organization of
granulosa cells. Acoording to those parameters, preantral follicles were classified as
normal, when a morphologically normal oocyte with a non-pyknotic nucleus was
surrounded by granulosa cells organized in discrete layers. Degenerated follicles
presented retracted oocyte with pyknotic nuclei, and/or surrounded by disorganized
granulosa cells detached from the basement membrane. From each treatment, 150
follicles were randomly evaluated.
56
oocyte and follicle diameters were measured with the aid of an ocular micrometer and
the averages of minor and major axes were reported as oocyte and follicle diameters,
respectively.
Statistical analysis
57
respectively. Analysis of variance was made using GLM procedure of SAS (1999) and
Dunnetts test was applied for comparison of control groups against each treatment
tested (STEEL et al., 1997) regarding to follicle and oocyte diameters before and after
culture. Student Newman Keuls (SNK) test was used to compare mean diameters
among days of culture and percentages of morphologically normal follicles in noncultured tissue or after 1 or 7 days culture. In order to avoid type II error because of the
higher coefficient of variation, Duncans test was applied to compare the percentage of
primordial or developing follicles obtained after 1 or 7 days among the various
treatments and Students t-test was used to compare means between days of culture.
Differences among groups were considered significant when p<0.05 and results were
expressed as means standard error of means (SEM).
Results
Effect of the storage conditions on the follicular morphology before and after in vitro
culture
A total of 2,850 preantral follicles were analyzed, being 1,532 primordial, 977 in
transition, 316 primary and 25 secondary follicles. Histological analysis showed that
normal (Figure 1A) and degenerated follicles (Figures 1B e C) were found in fragments
of ovarian cortex before and after in vitro culture. The main characteristics in
degenerated follicles were retracted oocyte, pyknotic nucleus and disorganized
granulosa cells, with low cellular density.
58
GC
Nu
O
O
GC
Nu
GC
O
Nu
Table 1 shows the effect of temperature and time of storage of ovarian fragments
on the percentage of normal preantral follicles before and after 1 or 7 days of culture.
Before culture, the percentage of morphologically normal follicles kept in MEM at 4, 20
or 35 C for 2 or 4 h were similar to the fresh control - day 0 (P<0.05). In contrast, after
the first day of culture, ovarian tissue stored at 35C for 4 h had a significant reduction
(P<0.05) in the percentage of normal follicles when compared to the fresh control - day
0. However, there was no significant difference in the percentage of normal follicles in
non-cultured tissues after preservation or in tissues cultured for 1 day.
After 7 days culture of fragments stored at 4 C for 2 and 4 h, 74.6% and 71.9%
of follicles were morphologically normal, respectively. Interestingly, this percentage did
not differ from those found in fresh control day 0. On the other hand, a significant
reduction (P<0.05) in the percentage of normal follicles after both storage at 20 C or 35
C and cultured for 7 days was observed when compared to fresh control (P<0.05).
59
However, after 7 days of culture, there was no difference in the percentage of normal
follicles among treatments, except when fragments were stored at 35 C for 4 h, which
showed significant reduction in the percentage of normal follicles when compared to
storage at 4 C for 2 h.
After preservation, a significant reduction in follicular viability at day 1 of
culture was observed in fragments previously preserved at 20 C for 2 or 4 h when
compared to non-cultured fragments. Acoording to the progression of culture period
from 1 to 7 days, no reduction (P>0.05) in follicular viability was observed.
81.3 1.99
Cultured fragments
Treatments
Non- cultured
fragments
Day 1
4 C 2h
80.6 1.57 Aa
77.3 1.90 Aa
4 C 4h
77.9 0.33 Aa
73.9 1.31
Aa
20 C 2h
82.6 0.99 Aa
69.3 1.31
Ab
20 C 4h
83.9 0.79 Aa
69.9 1.17 Ab
35 C 2h
78.6 1.95 Aa
35 C 4h
74.6 1.95 Aa
Day 7
74.6 0.33 Aa
Different letters in the same column denote significant differences among treatments in the
same period (P<0,05).
a, b
Different letters in the same row denote significant differences between culture periods within
the same treatment (P<0,05).
60
61
Figure 2. (A) Percentage (mean SEM) of primordial and (B) developing follicle
(transition, primary and secondary) in non-cultured (fresh control) and cultured tissue
for 1 or 7 days (P<0.05).
* Differs significantly from fresh control day 0 (P<0,05).
A, B
a, b
62
Follicular and oocyte diameters from fresh control - day 0 were 52.07 1.81 m
and 40.94 1.70 m, respectively (Tables 2 and 3). After 2 or 4 h of incubation,
preantral follicles enclosed in ovarian fragments stored at 4, 20 or 35 C did not show
changes in follicular and oocyte diameters when compared to the fresh control day 0.
As early as after 1 day of culture, in all treatments, except for ovarian fragments
stored at 35 C for 4 h, a significant increase in follicular diameter in monolaminar
follicles (primordial, transition and primary) was observed when compared to fresh
control day 0 (P<0.05). However, the oocyte diameter did not change in any of the
treatments. After 1 day of culture, only the tissues kept at 35 C for 2 h showed a
significant improviment on follicular diameter when compared to both preserved noncultured and cultured fragments. The culture of ovarian fragments for 7 days promoted a
significant increase in follicular diameter in all treatments when compared to both fresh
control and non-cultured fragments. Interestingly, only oocyte from follicles stored at 4
C had a significant increase in diameter when compared to non-cultured fragments.
With the progression of the culture period from 1 to 7 days, an increase in follicular
diameter was verified in all treatments (P<0.05). However, a significant increase in
oocyte diameter only occurred in follicles conserved at 4 C for 2 or 4 h.
63
Table 2. Follicular diameter (mean SEM; m) in fresh control day 0, preserved and
non-cultured and preserved and cultured tissues for 1 or 7 days.
Follicular diameter (mean SEM) m
Fresh control
day 0
52.07 0.40
Cultured fragments
Treatments
Non-cultured
fragments
Day 1
Day 7
4 C 2h
54.69 0.64 a
56.55 0.51 *a
66.43 1.02 * b
4 C 4h
54.23 0.35 a
56.39 0.95
*a
64.73 1.20* b
20 C 2h
54.38 0.76 a
55.77 0.94
*a
60.72 0.82 * b
20 C 4h
53.92 0.96 a
56.55 1.12 *a
60.56 1.01 *b
35 C 2h
52.68 0.65 a
55.31 0.89 * b
59.02 0.95 *c
35 C 4h
52.22 1.10 a
53.61 0.98 a
56.24 0.48 * b
Different letters in the same row denote significant differences between culture periods within
the same treatment (P<0,05).
64
Table 3. Oocyte diameter (mean SEM; m) of follicles from fresh control day 0,
preserved and non-cultured and preserved and cultured tissues for 1 or 7 days.
Oocyte diameter (mean SEM) m
Fresh control
day 0
40.94 0.38
Cultured fragments
Treatments
Non-cultured
fragments
Day 1
Day 7
4 C 2h
42.02 0.41 a
42.33 0.51 a
44.96 0.65 * b
4 C 4h
41.25 0.34 a
42.02 0.57
ab
43.41 0.85* b
20 C 2h
41.72 0.53 a
41.87 0.42
42.95 0.54 a
20 C 4h
40.79 0.48 a
41.56 0.42 a
42.18 0.56 a
35 C 2h
41.87 0.42 a
40.79 0.42 a
41.87 0.72 a
35 C 4h
40.63 0.46 a
39.71 0.51 a
41.10 0.32 a
Different letters in the same row denote significant differences between culture periods within
the same treatment (P<0,05).
65
smooth and rough endoplasmic reticulum. Circular mitochondria were the most
prevalent organelles, followed by smooth endoplasmic reticulum. Flattened granulosa
cells were well-organized around oocyte and showed a distinct elongated nucleus with
irregular membrane and high proportion nucleus-cytoplasm. Ultrastructural analysis
showed that fragments cultured for 7 days after conservation at 4 C for 4 h (Figure 4),
20 (Figure 5) and 35 C (Figure 6) for 2 h, preantral follicles were well-preserved and
similar to fresh control.
Nu
GC
m
O
ser
66
GC
Nu
ser
Figure 4. Electron micrograph of normal preantral follicle in ovarian tissue culture for 7
days (4 C 4 h). Note the great relation between the nucleus and the cytoplasm of
granulosa cells (arrow). O: oocyte; Nu: oocyte nucleus; GC: granulosa cells; m:
mitochondria; ser: smooth endoplasmic reticulum (x10000).
67
Nu
O
Figure 5. Electron micrograph of normal preantral follicle in ovarian tissue culture for 7
days (20 C 2 h). Note the presence of numerous elongated mitochondria in the
ooplasm. O: oocyte; Nu: oocyte nucleus; GC: granulosa cells; m: mitochondria (x4000).
68
Nu
Figure 6. Electron micrograph of normal preantral follicle in ovarian tissue culture for 7
days (35 C 2 h). O: oocyte; Nu: oocyte nucleus; GC: granulosa cells; m: mitochondria
(x4000).
69
Discussion
This study shows that goat preantral follicles can be successfully stored in
ovarian pieces at low temperatures maintaining the ability for in vitrodevelopment.
Ovaries collected in slaughterhouse are generally used as source of oocytes for
in vitro culture studies. The storage of ovaries without blood supply may affect oocyte
quality, influencing the extracellular environment around the oocyte (WONGSRIKEAO
et al., 2005). In general, hypothermia is globally applied to preserve organs to minimize
the rates of energy consuming processes and to minimize the rates of tissue autolysis.
Isquemy is present during long periods of storage causing rapid depletion of energetic
cellular stocks resulting in acidification of the medium (SALEHI et al., 2004).
After 2 or 4 h of incubation, caprine preantral follicle viability did not differ
either among temperatures nor in relation to fresh control day 0. Similar results were
observed after storage of ovarian fragments at 4 C for up to 24 (COSTA et al., 2002) or
12 h (CARVALHO et al., 2001), at 20 C for 4 h (SANTOS et al., 2002) and at 39 C
for 2 h (MATOS et al., 2004). All these works related the maintenance of the
percentage of morphologically normal preantral follicles similar to control.
It is well-known that there are a variable number of degenerated follicles during
in vitro culture (TELFER et al., 2000). In the present study, after 1 or 7 days of culture,
the percentage of morphologically normal preantral follicles was kept similar to fresh
control only in fragments maintained at 4 oC for 2 or 4 h. These results may be due to
hypothermia caused by low temperatures, which reduces the cellular metabolism and
increases follicle resistance to reduction of nutrients and oxygen during in vitro
preservation (SANTOS et al., 2002). However, with the increase of the temperature, the
medium composition becomes an important factor to maintain follicular viability. MEM
70
is a medium rich in nutrients and widely used for in vitro culture of bovine (SAHA et
al., 2000), ovine (CECCONI et al., 1999) and caprine (MARTINS et al., 2005)
preantral follicles. LUCCI et al. (2007) observed that after storing at 4 C for 18 h or at
20 C for 6 h, developing swine follicles were able to continue growth in a 3-day culture
system, showing a pattern similar to the observed in fresh follicles.
Preservation at 35 C for 4 h significantly reduced the percentages of normal
preantral follicles in comparison to controls in 1 and 7 days of culture. With similar
results, others authors showed a reduction in preantral follicle viability from caprine
ovaries kept at 32 C and further in vitro cultured for 5 days (SILVA et al., 2004;
MARTINS et al., 2005). In our study, at 20 C for 2 or 4 h, this reduction was only
observed after 7 days of culture. These results showed that storage of ovarian fragments
in association of low oxygen tension with temperatures close to the physiological limit
(35 C) or subnormal (20 C) may result in great rates of follicular degeneration
(SANTOS et al., 2002). A long term in vitro culture (7 days) caused probably a
reduction in the percentage of follicular viability mainteine at higher temperatures, since
the metabolism was accelerated and more nutrients are required (TAS et al., 2006).
After 1 day of culture, the percentages of primordial and developing follicles
were similar to control values. However, after 7 days of culture, high percentages of
primordial follicle activation were observed in ovarian cortex that had been preserved in
all temperatures and times of storage. Similar results were reported during short-term
culture of bovine ovarian tissue (BRAW-TAL and YOSSEFI, 1997), where activation
occurred from the second day of culture onward. Furthermore, Matos et al. (2007)
demonstrated that FSH, at 50 ng/mL, promoted caprine primordial follicle activation
and survival.
71
All cultured follicles had a bigger diameter than those from fresh control, except
from fragments preserved at 35 C for 4 h in the first day of culture. Nevertheless,
oocyte diameter, after 7 days of culture, was only significantly higher than control when
ovarian fragments were previously stored at the temperature of 4 C for 2 or 4 h,
suggesting that granulosa cells in cultured tissue were stimulated to proliferate quickly
and that, probably high temperatures during the transportation of ovaries are not benefic
to a further oocyte maturation. MIYANO and MANABE (2007) affirmed that oocytes
from primordial follicles when enter the growth phase take a long period to reach their
final size. Our results were similar to those found by SILVA et al. (2004) and MATOS
et al (2007), in which the use of FSH after culture for 5 or 7 days promoted an increase
in follicular diameter by the increase in oocyte diameter and proliferation of goat
granulosa cells.
Histological analysis of follicles both preserved at 4 C for up to 4 h and
cultured in vitro revealed their morphological integrity , and this result was confirmed
by ultrastructural analysis. MATOS et al. (2007) obtained satisfactory results in
ultrastructural studies of caprine preantral follicles cultured for 7 days after
transportation to the laboratory in saline solution at 32 oC.
In conclusion, our study showed that the storage of ovarian tissue at 4 C for up
to 4 h before culture was the best condition to the transportation of caprine preantral
follicles without affecting their morphology and the ability for in vitro development.
Preservation of ovarian fragments during the transportation in these conditions will be
useful in the future to optimize the use of oocytes enclosed in preantral follicles for the
reproductive prupose.
72
Acknowledgements
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7. CONCLUSO
78
8. PERSPECTIVAS
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