Este trabalho teve objetivou-se avaliar o efeito da melatonina adicionada ao meio de maturação (meio B199) sobre a maturação nuclear, a distribuição dos grânulos corticais e dos microtúbulos, a produção in vitro de embriões e sobre a apoptose das células do cumulus (CC) e dos embriões. Os complexos cumulus-ovócitos (COCs), aspirados de folículos ovarianos obtidos de ovários de vacas oriundas de abatedouros, foram cultivados por 24 h, conforme os tratamentos: 1) Grupo Controle: meio B199 acrescidos de 0,5 µg/mL de FSH e 5,0 µg/mL de LH; 2) Grupo MEL: meio B199 com 1 µg/mL de melatonina; 3) Grupo MEL/FSH/LH: meio B199 com 1 µg/mL de melatonina e 0,5 µg/mL de FSH e 5,0 µg/mL de LH. Após a maturação in vitro (MIV), os COCs de todos os grupos foram submetidos à fecundação in vitro (FIV) e desenvolvimento embrionário in vitro. A condição de cultivo em todas as etapas foi microgotas, sob óleo de silicone, com atmosfera de 5% de CO2 em ar, a 38,5ºC. Foram avaliadas as taxas de clivagem e de embriões produzidos no dia 7 (D-7) pós-inseminação in vitro. Após a MIV, os ovócitos de todos os grupos também foram corados com Hoechst 33342, com anticorpo monoclonal anti-tubulina conjugada com FITC ou cultivados com aglutinina Lens Culinaris conjugada a FITC para avaliação da taxa de maturação nuclear (progressão da meiose), distribuição dos microtúbulos e dos grânulos corticais, respectivamente. Para avaliação do grau de apoptose das CC e dos embriões, utilizou-se a técnica do cometa. Não houve diferença (P > 0,05) entre o grupo controle e os tratados com melatonina (MEL e MEL/FSH/LH) na taxa de ovócitos em metáfase II (66,18±4,04; 66,46±4,04 e 59,61±4,04), no percentual de blastocistos com baixo grau de apoptose (31,46±6,01; 38,36±6,01 e 32,92±6,01), na taxa de clivagem (85,70±2,47; 88,52±2,47 e 85,65±2,47) e nem na taxa de embriões no D-7 (54,47±3,67; 60,01±3,67 e 58,40±3,67). A distribuição dos microtúbulos e dos grânulos corticais também não foi alterada pelos grupos tratados com melatonina. O percentual de CC que não apresentaram dano no DNA no grupo MEL (37,64±2,41) foi superior (P<0,01) ao grupo MEL/FSH/LH (28,08±2,41) e ao grupo controle (17,83±2,41). Os resultados sugerem que a adição de melatonina durante o cultivo in vitro para maturação de ovócitos bovinos protegeu as CCs de danos no DNA promovendo a diminuição da incidência de apoptose e foi capaz de suportar o desenvolvimento embrionário produzindo taxas de embriões no D-7 similares as obtidas no sistema tradicional de produção in vitro de embriões. / The aim of this study was to examine the effect of addition of melatonin in maturation medium (B199) on the nuclear maturation, the cortical granules and microtubules distribution, the in vitro production of embryos and the DNA damage (apoptosis) of cumulus cells and embryos. The bovine cumulus-oocyte complexes (COCs) aspirated from follicles from abattoir ovaries were cultured for 24 h according to treatments: 1) Control group: B199 medium with 0.5 µg/ml FSH and 5.0 µg/ml LH; 2) MEL group: B199 medium with 1 µg/ml melatonin; 3) MEL/FSH/LH group: B199 medium with 1 µg/ml melatonin, 0.5 µg/ml FSH and 5.0 µg/ml LH. After in vitro maturation (IVM) the COCs of all groups were submitted to in vitro fertilization and in vitro embryo development. All cultures were in droplets under oil, at 38.5ºC in na atmosphere of 5% CO2 in air. The cleavage rate and the percentage of embryos produced on Day 7 (D-7) after in vitro insemination were evaluated. After IVM, the oocytes of all groups were stained with Hoechst 33342, or stained with FITCconjugated anti-?-tubulin antibody or cultured with FITC-conjugated Lens Culinaris agglutinin to evaluate the nuclear maturation rate, microtubules and cortical granules distribution, respectively. The incidence of apoptosis of the CC and embryos was also measured by Comet assay. There was no difference (P > 0.05) among groups on metaphase II oocyte rates (Control = 68.18±4.04; MEL = 66.46±4.04; MEL/FSH/LH = 59.61±4.04), on the percentage of blastocysts with low incidence of apoptosis (Control = 31,46±6,01; MEL = 38,36±6,01; MEL/FSH/LH = 2,92±6,01), on cleavage rate (Control = 85.70±2.47; MEL = 88.52±2.47; MEL/FSH/LH = 85.65±2.47) and on D-7 embryo rate (Control = 54.47± 3.67; MEL = 60.01±3.67; MEL/FSH/LH = 58.40±3.67). The distribution of microtubules and cortical granules was not affected by the groups treated with melatonin. The percentage of cumulus cells with no DNA damage in MEL group (37.64±2.41) was significantly superior to MEL/FSH/LH (28.08±2.41) and control groups (17.83±2.41). The results suggest that the addition of melatonin to the IVM medium protected the cumulus cells from DNA damage by diminishing the incidence of apoptosis and also supported the embryo development by producing D-7 embryo rates similar to those obtained in traditional system of bovine embryo in vitro production.

Pas de résumés / No abstract

O óxido nítrico (NO) é um mensageiro químico gerado pela atividade da enzima óxido nítrico sintase (NOS) a qual foi detectada em vários órgãos incluído o sistema reprodutor. O sistema NOS/NO parece desempenhar papel importante na maturação oocitária entre outras funções. No entanto, apesar das evidências, há poucos estudos sobre o papel desse sistema em oócitos da espécie bovina. Sabe-se que o NO atua pela via da guanilato ciclase (GC) estimulando a produção do nucleotídeo GMPc, que por sua vez é capaz de influenciar os níveis de outro nucleotídeo, o AMPc, que é um importante elemento da via de sinalização das gonadotrofinas nos oócitos e no controle da maturação oocitária. O objetivo do presente estudo foi de investigar o envolvimento da via do GMPc na ação do sistema NOS/NO na maturação in vitro (MIV) de oócitos bovinos e seu efeito sobre a via do AMPc. Com a maior concentração estudada do doador de NO (10-7M de SNAP), apenas 36% dos oócitos conseguiram alcançar o estágio de RVG (P< 0,05), após 9 horas de maturação. Esse atraso também foi observado com diferentes concentrações do estimulador de GC (5, 10 ou 50&mu;M de Proptoporfirina IX) e pelo análogo de GMPc (1, 2 e 4mM de 8-Br-GMPc ), que apresentaram uma taxa média de RVG de 50% para os tratamentos e 70% para os grupos controles sem as drogas (P<0,05). No início da maturação (0h), os níveis de GMPc foram de 5,29 pmol/oócito sofrendo uma queda logo na primeira hora de cultivo para 2,97 pmol nos oócitos do grupo controle e 1,54 pmol nos cultivados com associação de 10-7M de SNAP (doador de NO) e 100&mu;M de OQD (inibidor de GC (P<0,05). No grupo de oócitos cultivados apenas com SNAP, os níveis de GMPc se mantiveram em 4,51 pmol/oócito, semelhante ao grupo imaturo (0h de cultivo, P>0,05). O doador de NO manteve estável o nível de GMPc somente na primeira hora de maturação. Após 3 e 6 h de MIV, os níveis de GMPc permaneceram baixos e similares (0,07 a 2,46 pmol/oócito, P>0,05) nos grupos controle (sem drogas), tratado com doador de NO (10-7M de SNAP) associado ou não ao inibidor de guanilato ciclase (100&mu;M de OQD). Também foi observada uma queda nos níveis de AMPc em relação ao grupo imaturo (32,42 fmol de AMPc/oócito) para os demais grupos (P<0,05), que apresentaram aproximadamente, 12,0 a 16,0 fmol de AMPc/oócito durante a primeira hora, 3,3 a 8,0 fmol/oócito durante a terceira hora e 7,4 a 18,3 durante a sexta hora de maturação (P>0,05). O NO afetou os níveis de GMPc no início da maturação, mas não os níveis de AMPc. O NO e o GMPc podem atuar no controle da expressão gênica de uma série de proteínas envolvidas no controles dos níveis de AMPc e GMPc ou suas funções. Esse controle pode ser efeito direto do NO (PKG2, PDE3A, PDE4D e PDE8A), do GMPc (ADCY6) ou do NO via GMPc (PKA1) e varia com o compartimento considerado (oócito ou células do cumulus). Esses resultados demonstraram a inter-relação das vias NO/GMPc/AMPc e toda a sua complexidade dependendo do tipo celular e da fase da maturação de oócitos bovinos. / The NOS/NO system seems to play an important role in oocyte maturation besides other functions. However, despite the evidence, there are few studies on the possible role of this system in bovine oocytes. It is known that NO acts via guanylate cyclase (GC) by stimulating the production of the nucleotide cGMP, which in turn can influence the levels of another nucleotide, cAMP, which is an important element of the signaling pathway of gonadotropins in oocytes and in the control of oocyte maturation. The aim of the present study was to investigate the involvement of the cGMP pathway in the action of the NOS/NO system on the in vitro maturation (IVM) of bovine oocytes and its effect on the cAMP pathway. The highest studied concentration of the NO donor (10-7M SNAP), only 36% of oocytes were able to undergo GVBD (P<0.05) after 9 hours of maturation. This delay was also observed with different concentrations of the GC stimulator (5, 10 or 50&mu;M Proptoporfirin IX) and the cGMP analogue (1, 2 and 4 mM 8-Br-cGMP), which had an average of 50% GVBD for treatment groups and 70% for control groups without drugs (P<0.05). At the beginning of maturation (0 h) cGMP levels were 5.29 pmol/oocyte and decreased within the first hour of culture to 2.97 pmol and 1.54 pmol in the control group and in oocytes cultured in 10-7M SNAP (NO donor) associated with 100&mu;M OQD (GC inhibitor; P<0.05). In the group of oocytes cultured only with SNAP, cGMP levels remained at 4.51 pmol/oocyte similar to the immature group (0 h culture, P> 0.05). The increase of NO maintained cGMP levels stable only during the first hours of maturation. After 3 and 6 h IVM, cGMP levels remained low and similar (0.01 to 2.5 pmol/ ocyte, P>0.05) in control (without drugs), treated with NO donor (SNAP 10-7M) with or without the guanylate cyclase inhibitor (100&mu;M OQD). A decrease in cAMP levels was also observed when compared with the immature group (32.42 fmol cAMP/oocyte) for the other groups (P <0.05), which showed 12.0 to 16.0 fmol cAMP/oocyte after the first hour, 3.3 to 8.0 fmol/oocyte after the third hour and 7.4 to 18.3 after the sixth hour of IVM (P>0.05). NO and cGMP may act to control gene expression in a series of proteins involved in control of the levels of cAMP and cGMP or their functions. The control may be a direct effect of NO (PKG2, PDE3, PDE4D and PDE8A), cGMP (ADCY6) or NO via cGMP (PKA1) and varies with the compartment considered (oocyte or cumulus cells). The results showed the interrelationship of the NO/cGMP/cAMP pathway and all its complexity depending on the cell type and the stage of maturation in bovine oocytes.

Este estudo demonstrou que oócitos bovinos são capazes de progredir normalmente a maturação nuclear até a fase de M II quando cultivados in vitro na presença ou ausência de células da granulosa, na presença ou ausência de soro de vaca nas diversas fases do ciclo estral e/ou SFB e na presença de BSA. A progesterona promoveu um retardo na retomada da meiose, mas não impediu que os oócitos atingissem M II, exceto na concentração de 2,5 µg/ml. Além de promover um retardo inicial na retomada da meiose, a testosterona também prejudicou a progressão da meiose até M II. O estradiol também promoveu este retardo inicial, mas isto foi revertido no final da cultura, onde se verificou que quanto maior a concentração de estradiol até a dose de 5,0 µg/ml, maior a porcentagem de oócitos que atingiram M II. Porém, não se observou diferença significativa da porcentagem de oócitos que atingiram M II após maturação no grupo controle (TCM199 com BSA) e nos grupos onde se adicionou estradiol ao meio, em concentrações crescentes. Verificou-se que as células da granulosa e/ou células do cumulus foram capazes de secretar progesterona e estradiol no meio de cultura, quando estimuladas pelo soro bovino, que lhes forneceu precursores (testosterona). As células do cumulus dos COCs cultivados foram capazes de secretar progesterona quando estimuladas por BSA, SFB e estradiol e foram capazes de secretar estradiol quando estimuladas por BSA, progesterona e testosterona. O trabalho demonstrou que a MIV de oócitos bovinos ocorre normalmente na ausência de soro bovino e na ausência de células foliculares e demonstrou que a adição de estradiol não afeta a maturação e é desnecessária, uma vez que as células do cumulus o produzem no meio durante as 24 horas de cultura. / This study has demonstrated that culture medium supplementation with cycling cow serum and/or FCS, granulosa cells or BSA did not affect meiosis progression from GV to M II stage of bovine oocytes matured in vitro. Progesterone impaired meiosis resumption, but it was reverted after 24 hours of culture, except in the concentration of 2,5 µg/ml. Testosterone impaired meiosis resumption and meiosis progression to M II. Estradiol impaired meiosis progression, but it was reverted at the end of the culture, when it was observed that the higher the estradiol concentration in the medium, the higher the number of oocytes reaching M II. However, when IVM of bovine oocytes matured in medium supplemented with only BSA was compared to IVM of bovine oocytes matured in medium supplemented with BSA plus estradiol, no statistical difference was found. Data showed that granulosa cells and/or cumulus cells were able to produce progesterone and estradiol in the culture medium, when stimulated by bovine serum. Cumulus cells were able to produce progesterone when stimulated by BSA, FCS and estradiol and were still able to produce estradiol when stimulated by BSA, progesterone and testosterone. This study has demonstrated that IVM of bovine oocytes can proceed normally in the absence of bovine serum and granulosa cells, and has additionally demonstrated that the medium supplementation with estradiol did not affect nuclear maturation and it is still not necessary, once cumulus cells are able to produce it during the 24 hours of culture.

O interesse em criopreservar tecido ovariano, embriões e oócitos, principalmente quando se trata de pacientes oncológicas que irão ser submetidas a tratamentos potencialmente esterilizantes, vem crescendo nas últimas duas décadas. Uma das técnicas propostas para se preservar a fertilidade destas pacientes é o congelamento de oócitos, podendo estes ser obtidos já maturados in vivo após a hiperestimulação ovariana controlada ou na forma de oócitos imaturos na ausência de estimulação, nestes casos procede-se a maturação in vitro (MIV) de oócitos pré congelamento. No entanto sabe-se que a criopreservação causa danos de viabilidade e perda do potencial reprodutivo destes oócitos. Alguns autores têm demonstrado que esses danos podem ser reduzidos por meio de cultivos que modulam o perfil lipídico tanto de oócitos como embriões, fazendo com que estes sejam menos susceptíveis ao congelamento. Uma das técnicas que permite a verificação da composição lipídica de células e outras estruturas é a espectrometria de massas. Os objetivos deste estudo foram comparar o perfil lipídico de oócitos maturados in vitro na presença ou ausência de PL e correlacionar com o perfil lipídico e desenvolvimento embrionário dos embriões produzidos in vitro. Além disso, avaliamos o perfil lipídico dos meios de maturação usando oócitos bovinos como modelo experimental. CCOs foram maturados em meio TCM ou TCM + PL, contendo 10% de soro fetal bovino, 0,5 µg/ml de FSH, 5 ng/ml de LH e 1 mg/mL de 17?-estradiol em atmosfera úmida, com 5% de CO2 durante 24 horas. Após a MIV, os oócitos foram desnudados mecanicamente, lavados em PBS e armazenados a -80 ° C, até a análise de perfil lipídico. Oócitos, meios de maturação e blastocistos foram submetidos à técnica de MALDI-MS (ionização e dessorção a laser assistida por matriz /espectrometria de massas). Diferenças no perfil lipídico foram identificadas por PCA (análise de componentes principais). O perfil lipídico dos meios de maturação determinado por MALDI-MS permitiu a diferenciação entre TCM e TCM + PL. No entanto, a análise dos oócitos maturados in vitro demonstrou que o perfil lipídico dos grupos controle ou suplementado com PL não foram diferentes. Da mesma forma, não foram observadas diferenças no perfil lipídico e na embriogênese dos embriões resultantes. No entanto, diferenças no perfil lipídico entre COC e oócitos desnudos (ODs) maturados in vitro foram detectadas. Oócitos maturados com as células do cumulus contêm íons PC com maiores graus de insaturação dos resíduos de ácidos graxos, enquanto ODs contêm espécies de PC com ácidos graxos insaturados (18:0) ou monoinsaturados (18:1). O MALDI-MS permite a obtenção de perfis lipídicos informativos para meios de cultura e oócitos maturados in vitro. A identificação de mudanças no metabolismo lipídico de oócitos durante a MIV pode contribuir para determinar a suplementação lipídica adequada dos meios de MIV e soluções de vitrificação, contribuindo para otimizar os protocolos de criopreservação de oócitos humanos. / The interest in ovarian tissue, embryos and oocytes cryopreservation has been growing in the last two decades, especially in patients who are faced with potentially sterilizing treatments. One of the techniques proposed to preserve the fertility of these patients is the oocyte cryopreservation. The oocytes can be obtained matured in vivo after controlled ovarian hyperstimulation or as immature oocytes, in the absence of stimulation. In these cases, an option is to proceed the in vitro maturation (IVM) of oocytes before cryopreservation. However, it is known that damage induced by cryopreservation is associated with loss of viability and reproductive potential of oocytes. Some authors have demonstrated that such damage can be reduced by culture media that modulate lipid profile in both oocytes and embryos, making them less susceptible to freezing. One technique that enables the determination of the lipid composition of cells and other structures is mass spectrometry. The objectives of this study were to compare lipid profiles of oocytes matured in vitro in the presence or absence of PL and relate this information to lipid profile and preimplantational development of IVM-derived embryos. Also, we evaluated the lipid profiles of culture media using bovine oocytes as an experimental model. COCs were matured in TCM or TCM + PL, both containing 10% fetal calf serum, 0,5µg/ml FSH, 5 µg/mL LH, and 1 µg/mL 17?-estradiol, with 5% CO2 for 24 h. After IVM, oocytes were mechanically denuded, washed in PBS and stored at -80°C, until lipid analysis. Oocytes, maturation media and blastocysts were submitted to the MALDI-MS (matrix-assisted laser desorption/ionization mass spectrometry). Differences in lipid profile were addressed by principal component analysis. Maturation media lipid fingerprint by MALDI-MS allows differentiation among TCM and TCM+PL. However, the MALDI-MS of the in vitro matured oocytes demonstrated that lipid profile of control or PL-supplemented groups were not different. Similarly, no differences were observed in the lipid profile and embryogenesis of resulting embryos. Nevertheless, differences in lipid profiles between COCs and denuded oocytes (DOs) matured in vitro were indicated to occur. The former contain PC ions with higher degrees of unsaturation in the fatty acid residues, while DOs contain PC ions with unsaturated (18:0) or monoenoic (18:1) fatty acids. The MALDI-MS has allowed obtaining informative lipid profiles for culture media and IVM-oocytes. Identification of lipid changes during IVM may contribute to determine appropriate lipid supplementation of IVM/vitrification media and to improve cryopreservation of human oocytes.

<p><i>In vitro</i> production of embryos allows efficient management of herd genetics, reduction of disease impact, and if used in combination with other reproductive technologies it could aid in preserving the threatened genetic diversity of swine. <i>In vitro</i> maturation (IVM) is identified as a deficient step in porcine in vitro production (IVP) of embryo systems, which decreases the overall success of IVP. There are problems encountered in each step of IVP; chromosomal abnormalities and decreased cell numbers in blastocysts during <i>in vitro</i> culturing (IVC), and low monospermic fertilization rates during <i>in vitro</i> fertilization (IVF) may be a result of insufficient IVM. As an addition to maturation media, porcine follicular fluid (pFF) can affect IVM. Estrogen can be found in high concentrations in pFF; possibly contributing to the effects seen when pFF is added to IVM. The objective of this thesis was to investigate the effects of estrogen supplementation during IVM on IVP of porcine embryos.</p> <p>The first objective was to evaluate the <i>in vitro</i> maturation rates of porcine oocytes in two maturation media: protein-free and 10% pFF supplemented. Nuclear maturation of oocytes was evaluated using Lamin/Dapi staining of oocytes matured in protein-free and 10% pFF maturation media to ensure the efficiency of the protein-free media. Protein-free and 10% pFF media mature oocytes at similar rates (91% and 89% respectively).</p> <p>The transcripts within the oocyte can be altered based on the <i>in vitro</i> maturation environment, so the second objective was to observe the expression of four chosen maternal effect genes: Basonuclin-1 (<i>BNC1</i>), Nucleoplasmin 2 (<i>NPM2</i>), Zygote arrest 1 (<i>ZAR1</i>), and Tripartite-motif protein-24 (<i>TRIM24</i>), using oocytes matured in 50 ng/ml, 100 ng/ml, or 1000 ng/ml of estradiol 17-&beta; (E₂), 10% pFF, or protein-free maturation media. Expression of maternal effect genes, was shown by the &Delta;Ct (cycle threshold) values, obtained from the difference between the Ct values of the normalizing gene (<i>GAPDH</i>) and the genes of interest evaluated through QRT-PCR. Values of &Delta;Ct were analyzed in place of fold change to avoid data manipulation. The &Delta;Ct expression of <i>TRIM24</i> in 0 ng/ml E₂ maturation medium and the 10% pFF maturation medium were significantly different (p<0.05) from the non-matured control, the other maternal determinant genes did not differ in their expression under any treatment.</p> <p>We hypothesized that estradiol's effects on IVM would be evident when analyzing cleavage and blastocyst formation rates. Cleavage and blastocyst formation rates were examined following <i>in vitro</i> fertilization of oocytes matured in 100 ng/ml E₂, 10% pFF, or a protein-free maturation medium to investigate the effect of estradiol on IVP embryos. Cleavage rates for the E₂ (n= 252; 60.2%) or 10% pFF (n= 256; 55.7%) additions to the maturation media did not differ (p>0.05) when compared to the protein-free maturation media (n=264; 54.9%). Both 10% pFF and E<syb>2</sub> groups had significantly higher blastocyst formation rates (p&le;0.05) than the protein-free maturation media (n=264; 3.5%), although no statistical difference was observed between the blastocyst formation rates of the 10% pFF (n=256; 12.4%) and E2 (n=252; 14.6%) groups.</o> <p>As a final study, the global gene expression of oocytes matured in a control protein-free media and the protein-free media supplemented with 100 ng/ml E₂ or 10% pFF was investigated using microarray analysis. Genes were not differentially expressed among the matured groups with the outlined threshold values of -2 &ge; log2(fold change) &ge; 2, and adjusted p-value &le;0.05. A total of 16 differentially expressed genes between the non-matured and all matured groups exceeded this threshold. Of these genes, 6 are novel transcribed regions with evidence of being an embryonic EST, and 1 is a novel protein-coding gene. The other genes are FBJ murine osteosarcoma viral oncogene homolog (<i>FOS</i>), Vimentin (<i>VIM</i>), Capthesin C (<i>CTSC</i>), Selenium binding protein 1 (<i>SELENBP1</i>), Poly(A) binding protein cytoplasmic 1 (<i>PABPC1</i>), Tissue factor pathway inhibitor 2 (<i>TFPI2</i>), Cysteine-rich, angiogenic inducer 61 (<i>CYR61</i>), Acyl-CoA synthetase long-chain family member 6 (<i>ACSL6</i>), and Phospholipase A2 group VII (<i>PLA2G7</i>).</p> <p>In conclusion, successful nuclear maturation of oocytes derived of prepubertal gilt abbatoire derived ovaries can be achieved without pFF or hormone supplementation. The expression of maternal determinant genes is not affected in a dose dependant manner, and removal of E₂ or supplementation of pFF during maturation may alter the expression of <i>TRIM24</i> from the non-matured control; where no other maternal effect gene changes through maturation. Estradiol has a similar effect as pFF during <i>in vitro</i> maturation of porcine oocytes as seen by cleavage and blastocyst formation rates. And media does not affect the global gene expression of porcine oocytes, though there is a temporal control of gene expression through maturation.</p>

<p><i>In vitro</i> production of embryos allows efficient management of herd genetics, reduction of disease impact, and if used in combination with other reproductive technologies it could aid in preserving the threatened genetic diversity of swine. <i>In vitro</i> maturation (IVM) is identified as a deficient step in porcine in vitro production (IVP) of embryo systems, which decreases the overall success of IVP. There are problems encountered in each step of IVP; chromosomal abnormalities and decreased cell numbers in blastocysts during <i>in vitro</i> culturing (IVC), and low monospermic fertilization rates during <i>in vitro</i> fertilization (IVF) may be a result of insufficient IVM. As an addition to maturation media, porcine follicular fluid (pFF) can affect IVM. Estrogen can be found in high concentrations in pFF; possibly contributing to the effects seen when pFF is added to IVM. The objective of this thesis was to investigate the effects of estrogen supplementation during IVM on IVP of porcine embryos.</p> <p>The first objective was to evaluate the <i>in vitro</i> maturation rates of porcine oocytes in two maturation media: protein-free and 10% pFF supplemented. Nuclear maturation of oocytes was evaluated using Lamin/Dapi staining of oocytes matured in protein-free and 10% pFF maturation media to ensure the efficiency of the protein-free media. Protein-free and 10% pFF media mature oocytes at similar rates (91% and 89% respectively).</p> <p>The transcripts within the oocyte can be altered based on the <i>in vitro</i> maturation environment, so the second objective was to observe the expression of four chosen maternal effect genes: Basonuclin-1 (<i>BNC1</i>), Nucleoplasmin 2 (<i>NPM2</i>), Zygote arrest 1 (<i>ZAR1</i>), and Tripartite-motif protein-24 (<i>TRIM24</i>), using oocytes matured in 50 ng/ml, 100 ng/ml, or 1000 ng/ml of estradiol 17-&beta; (E₂), 10% pFF, or protein-free maturation media. Expression of maternal effect genes, was shown by the &Delta;Ct (cycle threshold) values, obtained from the difference between the Ct values of the normalizing gene (<i>GAPDH</i>) and the genes of interest evaluated through QRT-PCR. Values of &Delta;Ct were analyzed in place of fold change to avoid data manipulation. The &Delta;Ct expression of <i>TRIM24</i> in 0 ng/ml E₂ maturation medium and the 10% pFF maturation medium were significantly different (p<0.05) from the non-matured control, the other maternal determinant genes did not differ in their expression under any treatment.</p> <p>We hypothesized that estradiol's effects on IVM would be evident when analyzing cleavage and blastocyst formation rates. Cleavage and blastocyst formation rates were examined following <i>in vitro</i> fertilization of oocytes matured in 100 ng/ml E₂, 10% pFF, or a protein-free maturation medium to investigate the effect of estradiol on IVP embryos. Cleavage rates for the E₂ (n= 252; 60.2%) or 10% pFF (n= 256; 55.7%) additions to the maturation media did not differ (p>0.05) when compared to the protein-free maturation media (n=264; 54.9%). Both 10% pFF and E<syb>2</sub> groups had significantly higher blastocyst formation rates (p&le;0.05) than the protein-free maturation media (n=264; 3.5%), although no statistical difference was observed between the blastocyst formation rates of the 10% pFF (n=256; 12.4%) and E2 (n=252; 14.6%) groups.</o> <p>As a final study, the global gene expression of oocytes matured in a control protein-free media and the protein-free media supplemented with 100 ng/ml E₂ or 10% pFF was investigated using microarray analysis. Genes were not differentially expressed among the matured groups with the outlined threshold values of -2 &ge; log2(fold change) &ge; 2, and adjusted p-value &le;0.05. A total of 16 differentially expressed genes between the non-matured and all matured groups exceeded this threshold. Of these genes, 6 are novel transcribed regions with evidence of being an embryonic EST, and 1 is a novel protein-coding gene. The other genes are FBJ murine osteosarcoma viral oncogene homolog (<i>FOS</i>), Vimentin (<i>VIM</i>), Capthesin C (<i>CTSC</i>), Selenium binding protein 1 (<i>SELENBP1</i>), Poly(A) binding protein cytoplasmic 1 (<i>PABPC1</i>), Tissue factor pathway inhibitor 2 (<i>TFPI2</i>), Cysteine-rich, angiogenic inducer 61 (<i>CYR61</i>), Acyl-CoA synthetase long-chain family member 6 (<i>ACSL6</i>), and Phospholipase A2 group VII (<i>PLA2G7</i>).</p> <p>In conclusion, successful nuclear maturation of oocytes derived of prepubertal gilt abbatoire derived ovaries can be achieved without pFF or hormone supplementation. The expression of maternal determinant genes is not affected in a dose dependant manner, and removal of E₂ or supplementation of pFF during maturation may alter the expression of <i>TRIM24</i> from the non-matured control; where no other maternal effect gene changes through maturation. Estradiol has a similar effect as pFF during <i>in vitro</i> maturation of porcine oocytes as seen by cleavage and blastocyst formation rates. And media does not affect the global gene expression of porcine oocytes, though there is a temporal control of gene expression through maturation.</p>

Orientadora: Gisele Zoccal Mingoti / Banca: Joaquim Mansano Garcia / Banca: Flávio Vieira Meirelles / Resumo: Butirolactona I (Bl-I) pode ser utilizada em sistemas PIV de embriões para bloquear a meiose durante o transporte de oócitos obtidos de OPU. O objetivo deste estudo foi verificar a concentração de BL-I eficaz durante o transporte de oócito bovinos. Oócitos (n=4581) foram pré-maturados em criotubos contendo meio com 10 ou 100μM de Bl-I, acrescido ou não de HEPES (20mM), no período de 5 horas em estufa portátil Minitub e transferidos para incubadora com atmosfera e temperatura controlada, permanecendo por mais 19 horas. Em seguida foram maturados a 38,5ºC em atmosfera de 5% de CO2 durante 20 horas, fecundados e os zigotos cultivados. Foram avaliadas a maturação nuclear e a maturação citoplasmática após pré-MIV e MIV (24h.-controles e 20h.-tratados). A fertilização foi avaliada após 18 h. da inseminação. Os embriões foram analisados quanto ao seu desenvolvimento e qualidade. Os dados foram avaliados por ANOVA (p<o,o5). A maioria dos oócitos tratados permaneceu em GV e após MIV houve a reversão do bloqueio, mas a eficiência foi menor com 10 μM de Bl-I. A maturação citoplasmática foi beneficiada de acordo a distribuição das mitocôndrias, no entanto, quanto à distribuição dos grânulos corticais apenas o bloqueio realizado com 100 μM Bl-I permitiu uma maturação. A taxa de fecundação foi prejudicada pelo tempo de transporte, mesmo nos oócitos tratados. A ocorrência de polispermia foi correlacionada à porcentagem de oócitos com grânulos corticais imaturos. O desenvolvimento embrionário sofreu um atraso pela utilização de 100μM de Bl-I, porém boa porcentagem de blastocistos de qualidade foi atingida. Assim, o bloqueio com 100μM de Bl-I possui efeitos benéficos na PIV de embriões de oócitos transportados por longo tempo. / Abstract: Butirolactone-I (Bl-I), can be used in systems for in vitro production (IVP) of embryos to block meiosis during the transport of oocytes from OPU. The objective of this study is to assess the concentration of BL-I more efficient during the transport of bovine oocytes. Oocytes (n = 4581) were prematured in criotubes containing medium with 10 or 100 μM of Bl-I with or without HEPES (20mM) in a portable incubator at 38,5°C without CO2 equilibration in the first 5 h, being later transferred to a incubator at 38.5°C and 5% CO2 in humidified air during the 19 hours remaining. Following were matured, fertilized and the zygotes cultured at 38.5°C and 5% CO2. Were evaluated the nuclear maturation and the cytoplasmic maturation after pre-IVM and after IVM (24h. controls and 20 h. treatments). Fertilization was assessed 18 hours after insemination. The embryos development and quality were analyzed. The data were evaluated by ANOVA (P< 0, 05). Most remained in GV oocytes treated and after IVM was the reversal of the blockage, but the efficiency was lower with 10 μM of Bl-I. The cytoplasmic maturation during the time of meiosis blockage was benefited from the distribution agreement and potential of mitochondria, however, about the distribution of cortical granules only blocking conducted with 100μM Bl-I had a complete maturation. The rate of fertilization was damage by the time of transport, and the meiosis blockage with Bl-I did not improve the percentage of fertilized oocytes. The occurrence of polispermia was correlated with the percentage of oocytes with immature cortical granules. The embryo developmentb was delayed by the use of 100μM of Bl-I, but a good percentage of blastocysts of good quality reached. So, the use of 100μM of Bl-I has beneficial effects on the IPV of embryos from oocytes transported at a long time. / Mestre

Dentre os principais desafios que persistem no campo da biologia da reprodução está a compreensão da natureza dos processos celulares e moleculares que determinam a qualidade dos oócitos. Um dos fenômenos a serem melhor compreendidos é a aquisição da competência do oócito, e qual o papel desempenhado pelo ambiente folicular que circunda o gameta no seu potencial de desenvolvimento. As células foliculares, especialmente as células do cumulus, certamente desempenham um papel fundamental na aquisição da competência de oócitos in vivo. Durante a maturação in vitro (MIV) do oócito observa-se expansão e mucificação das células da granulosa que formam o complexo cumulus oophorus-oócito (CCO), em função da intensa síntese de componentes da matriz extracelular. Essas modificações no aspecto do cumulus são utilizadas como indicativo da ocorrência de maturação oocitária e contribuem para que ocorra a fecundação. A expressão de proteínas associadas à matriz extracelular das células do cumulus pode estar sob influência de fatores de origem oocitária, e também pode estar relacionada à composição do meio de MIV. Os objetivos deste trabalho foram: 1) avaliar a expressão dos transcritos dos genes que codificam para as proteínas ácido hialurônico sintase 2 (HAS2), link protein 1 (HAPLN1), conexina 43 (GJA1) e b-actina (ACTB) em complexos cumuli-oócitos (CCOs) bovinos não maturados, e submetidos à maturação in vitro em meios com diferentes suplementações protéicas; e 2) avaliar as taxas de maturação nuclear dos oócitos submetidos às diferentes condições de MIV. Os CCOs foram obtidos a partir de ovários coletados de fêmeas bovinas logo após o abate, selecionados morfologicamente e distribuídos em três grupos experimentais: G1: CCOs não maturados; G2: CCOs submetidos à MIV em meio TCM suplementado com soro fetal bovino (SFB); G3: CCOs submetidos à MIV em meio TCM suplementado com albumina sérica bovina (BSA). A MIV foi realizada em a 39oC, 5% de CO2 e máxima umidade relativa, por 22 a 24 horas. Para a extração do RNA total das amostras de CCOs foi utilizado o reagente TRIzol®. O RNA total foi submetido à captação específica do mRNA através de separação magnética (Dynabeads® mRNATM DIRECT Micro Kit). Os mRNAs foram transcritos reversamente em cDNA utilizando-se a técnica de RT-PCR, para avaliar os padrões de expressão dos transcritos. Parte dos oócitos dos grupos G2 e G3 foi desnudada das células do cumulus, e submetida à coloração com Hoechst 33342, para avaliação da maturação nuclear. A análise dos resultados de abundância relativa dos mRNAs de interesse mostrou diferença significativa entre os diferentes grupos testados para os transcritos de HAS2 (p=0,000), link protein 1 (p=0,001), conexina 43 (p=0,007) e b-actina (p=0,011), sendo maior nos grupos de CCOs submetidos à MIV em meio suplementado com SFB. A avaliação da morfologia nuclear não mostrou diferenças significativas entre as taxas de maturação nos grupos G2 e G3. Pode-se concluir que a exposição de CCOs bovinos à diferentes condições de MIV influenciou a expressão dos transcritos de HAS2, link protein 1, conexina 43 e b-actina. Entretanto, a MIV em presença de SFB ou BSA não mostrou diferença nas taxas de retomada da meiose e de maturação nuclear. / Understanding the cellular and molecular processes that determine the oocytes quality is one of the main challenges that persist in biology of reproduction. The acquisition of oocyte competence, and the role played by the follicular environment surrounding the gamete in its development potential are phenomena to be better understood. Follicular cells, especially the cumulus cells, certainly play a role in the oocyte competence acquisition in vivo. During in vitro maturation (IVM) of oocytes was observed expansion and mucification of granulosa cells forming the cumulus-oocyte complex (COC), due to the intense synthesis of extracellular matrix components. These changes in the appearance of cumulus are used as indicative of the oocyte maturation occurrence and contribute to fertilization to occur. The expression of proteins associated with the extracellular matrix of cumulus cells may be under the influence of oocyte origin, and may also be related to the composition of the IVM medium. The aim of this work were 1) to evaluate the expression of gene transcripts coding for proteins hyaluronic acid synthase 2 (HAS2), link protein 1 (HAPLN1), connexin 43 (GJA1) and actin-b (ACTB) in bovine cumulus-oocyte complexes (COCs) not matured or submitted to IVM in media with different proteic supplements, and 2) assess the rates of nuclear maturation of oocytes subjected to different conditions of IVM. The COCs were obtained from ovaries collected from cows immediately after slaughter, morphologically selected and divided into three groups: G1: not matured COCs; G2: COCs submitted to IVM in TCM supplemented with fetal calf serum (FCS); and G3: COCs submitted to IVM in TCM supplemented with bovine serum albumin (BSA). IVM was performed at 39°C in 5% CO2 and maximum relative humidity for 22 to 24 hours. For extraction of total RNA of COCs samples, TRIzol® reagent was used. Total RNA was subjected to capture of specific mRNA by magnetic separation (Dynabeads® mRNATM DIRECT Micro Kit). The mRNAs were reverse-transcribed into cDNA using the RT-PCR to evaluate the expression patterns of transcripts. Some of the oocytes from G2 and G3 was stripped of cumulus cells, and subjected to staining with Hoechst 33342 to assess nuclear maturation. The analysis of relative abundance of interest mRNAs showed a significant difference between the different groups tested for transcripts of HAS2 (p = 0.000), link protein 1 (p = 0.001), connexin 43 (p = 0.007) and actin-b (p = 0.011), being higher in the groups of COCs submitted to IVM in medium supplemented with FCS. The evaluation of nuclear morphology showed no significant differences between maturation rates of G2 and G3. In conclusion, the IVM in media supplemented with FCS or BSA showed differeces in HAS2, link protein 1, connexin 43 and actin-b transcripts expression of bovine COCs. However, the nuclear maturation rates of oocytes subjected to IVM in media with different proteic supplements was not affected.

O desenvolvimento de meios de cultivo eficazes para a maturação in vitro de oócitos caninos tem sido um desafio para os pesquisadores em reprodução animal. O estresse oxidativo desempenha papel fundamental na maturação oocitária, uma vez que altas concentrações de espécies reativas ao oxigênio levam ao dano celular e apoptose. O objetivo desta pesquisa foi determinar as taxas de maturação nuclear de oócitos caninos na presença de cisteína e α-tocoferol, substâncias conhecidas por suas propriedades antioxidantes. No experimento 1, avaliou-se a taxa de maturação nuclear de oócitos caninos mantidos em meio TCM-199 modificado (A) (controle) ou acrescido de 0,57mM de cisteína (B). No experimento 2, determinou-se as taxas de maturação nuclear na presença de diferentes concentrações de α-tocoferol em duas etapas independentes: experimento 1 (0, 10, 50μM) e experimento 2 (0, 100 e 500μM). Os dados foram analisados pelo teste do Qui-quadrado (P < 0,05). No experimento 1, não foi observada diferença estatística entre as taxas de Metáfase I/Anáfase I (MI/AI) e Metáfase II (MII) entre os oócitos do grupo controle (A) (7,5%, 16/213; 1,9%, 4/213) e os oócitos do grupo acrescido de cisteína (B) (6,3%, 14/223; 0,9%, 2/223). No experimento 2, não houve diferença significativa nas taxas de MI e MII dos oócitos entre os tratamentos da primeira etapa: controle (26,8%, 29/108), 10μM (22,6%, 26/115) e 50μM (29,1%, 30/103). Na segunda parte do experimento, a percentagem de oócitos que retomaram a meiose (VGBD à MII) foi de 31,9% (31/97), 34,9% (39/112) e 51,5% (54/105) para os grupos controle,100 e 500μM de α-tocoferol respectivamente (P < 0,05). Entretanto, nenhum aumento nas taxas de MII foi observado quando foram utilizadas as concentrações de 100μM (0,9%, 1/112) ou 500μM (1,0%, 1/105) de α- tocoferol no meio. Concluiu-se que, o TCM-199 modificado suplementado com cisteína não foi eficaz em elevar os índices de maturação in vitro de oócitos caninos. Ainda, a suplementação de α-tocoferol não afetou as taxas de MII de oócitos caninos. / The development of effective culture media for in vitro maturation of canine oocytes has been a challenge for researchers in animal reproduction. Oxidative stress plays a critical role in oocyte maturation since high concentrations of reactive oxygen species lead to cell damage and apoptosis. This research aimed to determine the rates of nuclear maturation of canine oocytes in the presence of cysteine and α-tocopherol, substances known for its antioxidant properties. In experiment 1, the rates of oocytes nuclear maturation exposed to modified TCM-199 (A) (control) or added with 0.57mM cysteine (B) were evaluated. In experiment 2, the rates of oocytes nuclear maturation in the presence of different concentrations of α-tocopherol were determined in two independent essays: experiment 1 (0, 10 and 50μM) and experiment 2 (0, 100 and 500μM). Data were analyzed by the Chi-square test (P < 0.05). The data obtained in experiment 1 allowed no difference among the oocytes in Metaphase I/Anaphase (MI/AI) (7.5%, 16/213; 1.9%, 4/213) and Metaphase II (MII) (6.3%, 14/223; 0.9%, 2/223) rates observed in the medium without or supplemented with cysteine respectively. In experiment 2, there was no significant difference in the rates of MI and MII oocytes among treatments designed for the first essay: control (26.8%, 29/108), 10μM (22.6%, 26/115) and 50μM (29.1%, 30/103). In the second part of the experiment, the percentage of oocytes that resumed meiosis (GVBD to MII) was 31.9% (31/97), 34.9% (39/112) and 51.5% (54/105) for control, 100 and 500μM groups respectively (P < 0.05). However, no improvement on rates of MII was observed when either 100 or 500μM α-tocopherol was added in medium. It was concluded that, modified TCM-199 supplemented with cysteine, was not effective in raising the rates of in vitro maturation of bitch oocytes. Moreover, α-tocopherol supplement did not affect the rates of MII in canine oocytes.