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.

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.

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.

Questiona-se um possível papel deletério da endometriose sobre a qualidade oocitária (QO), que é, em grande parte, condicionada pelo papel das células do cumulus. A função dessas células envolve a expressão de diversas moléculas codificadas por genes específicos, regulada a nível de transcrição, pós-transcrição e tradução. Os microRNAs atuam como reguladores póstranscricionais da expressão gênica, de modo que qualquer alteração nesse mecanismo pode levar a anormalidades no desenvolvimento oocitário. Desta forma, propusemos um estudo inédito da análise de microRNAs em células do cumulus (CC) de mulheres inférteis com e sem endometriose com o objetivo de evidenciar processos biológicos e vias de atuação correlacionados com o papel da endometriose na infertilidade e seu possível impacto na aquisição da competência oocitária. Foram incluídas no estudo 15 pacientes inférteis (5 controles com fator tubário e/ou masculino, 5 com endometriose estágios I/II e 5 com endometriose estágios III/IV) submetidas à estimulação ovariana controlada (EOC) para injeção intracitoplasmática de espermatozoide (ICSI). Imediatamente após a captação oocitária, as CCs foram isoladas e armazenadas para extração dos miRNAs. O perfil de 754 miRNAs foi analisado por meio da técnica de TaqMan®Array Human MicroRNA Cards. Considerou-se significativo p<0,05. Os miRNAs hsa-let-7f-1#, hsa-miR-1291, hsa-miR-140-5p, hsa-miR-218, hsa-miR-30b e hsa-miR-629-5p foram identificados menos expressos nas pacientes com endometriose I/II comparadas às controles. Os miRNAs hsa-miR-1291, hsa-miR-187-3p, hsamiR-30b, hsa-miR-532-3p e hsa-miR-629-5p foram identificados menos expressos nas pacientes com endometriose III/IV em relação às controles. Ao comparar-se os grupos endometriose I/II e endometriose III/IV entre si, os miRNAs hsa-miR-187-3p e hsa-miR-532- 3p foram menos expressos e os miRNAs hsa-let-7f-1# e hsa-miR-362-3p mais expressos nas pacientes com endometriose III/IV. A análise de enriquecimento identificou os genes regulados pelos miRNAs e as respectivas vias metabólicas em que estão envolvidos, sugerindo aumento de apoptose, diminuição de proliferação celular, alterações no controle do ciclo celular e no metabolismo energético das CCs de mulheres com a doença inicial e avançada. Os dados apontam para alterações na regulação pós-transcricional em CCs de mulheres inférteis com endometriose inicial e avançada, o que pode afetar processos e vias essenciais à aquisição de competência oocitária e estar envolvido na infertilidade associada à doença. Este estudo contribui para o entendimento da etiopatogênese da infertilidade relacionada à endometriose identificando mecanismos pós-transcricionais relacionados à piora da qualidade gamética nestas mulheres. / It is questioned a possible deleterious role of endometriosis on oocyte quality (OQ), which is largely conditioned by the function of cumulus cells. The role of these cells involve the expression of several molecules encoded by specific genes, regulated at transcription level, post-transcription and translation. The microRNAs act as transcriptional regulators of gene expression, thus any alteration in this mechanism can lead to abnormalities in oocyte development. In this way, we proposed an unpublished study of the microRNAs analysis in cumulus cells (CC) of infertile women with and without endometriosis, aiming to evidence the biological processes and pathways of action correlated with the presence of endometriosis in infertility and its possible impact on the acquisition of oocyte competence. Fifteen infertile patients (5 controls with tubal factor and/or male, 5 with stage I/II of endometriosis and 5 with stages III/IV of endometriosis) submitted to the controlled ovarian stimulation (EOC) for intracytoplasmic sperm injection (ICSI) were included in the study. Immediately after oocyte uptake, CCs were isolated and stored for miRNA extraction. The 754 miRNAs profile was analyzed using the TaqMan®Array Human MicroRNA Cards technique. Significant values were considered when p <0.05. The hsa-miR-218, hsa-miR-301 and hsa-miR-629-5p miRNAs were identified as less expressed in the patients with endometriosis I/II compared to controls. The miRNAs hsa-miR-1291, hsa-miR-187-3p, hsa-miR-30b, hsa-miR-532-3p and hsa-miR- 629-5p were identified as less expressed in patients with endometriosis III/IV compared to controls. Comparing the groups with endometriosis I/II and endometriosis III/IV, hsa-miR-187- 3p and hsa-miR-532-3p miRNAs were less expressed and hsa-let-7f-1# and hsa-miR-362-3p more expressed in patients with endometriosis III/IV. The enrichment analysis identified the genes regulated by the miRNAs and the respective metabolic pathways in which they are involved, suggesting apoptosis increase, decreased cell proliferation, alterations in the cell cycle control and energetic metabolism of the CCs of women with initial and advanced disease. The data point to changes in post-transcriptional regulation in CCs of infertile women with early and advanced endometriosis, which may affect processes and pathways essential for acquiring oocyte competence and resulting in infertility associated with the disease. This study contributes to the understanding of the etiopathogenesis of infertility related to endometriosis by identifying post-transcriptional mechanisms related to the deterioration of quality of gametes in these women.

Les cellules du cumulus (CCs) forment avec l'ovocyte le complexe ovocyte-cumulus (COC). Au cours de la folliculogénèse, un dialogue interdépendant régi par des jonctions communicantes se crée entre l'ovocyte et les CCs adjacentes. L'ovocyte en sécrétant certains facteurs, permettrait la différentiation et la prolifération des CCs qui, parallèlement, fournissent à l'ovocyte les nutriments nécessaires à sa maturation et son développement. La maturation nucléaire de l'ovocyte est définie par l'expulsion du 1er globule polaire au stade métaphase II (MII). Notre équipe a préalablement démontré que certains gènes exprimés dans les CCs chez l'humain, pouvaient prédire indirectement le potentiel implantatoire embryonnaire et la survenue d'une grossesse. Dans l'objectif d'identifier des marqueurs fiables de la maturité des ovocytes, nous avons analysé le profil transcriptomique des CCs en fonction du stade de maturité des ovocytes (VG, MI et MII). Dans un deuxième temps, nous avons étudié l'impact des conditions de maturation in vivo versus in vitro, sur le profil d'expression de gènes des CCs en fonction du stade de maturation ovocytaire. Nous avons mis en évidence, pour la première fois, une signature spécifique dans les CCs associée à la maturité nucléaire des ovocytes in vivo et in vitro. Nous avons également observé une sous-expression des gènes impliqués dans la maturation ovocytaire et l'expansion des CCs, et une sur-expression des gènes associés au cycle cellulaire dans les CCs dérivées d'ovocytes maturés in vitro, comparées aux CCs issus d'ovocytes in vivo. En comparant l'expression des gènes dans les CCs selon la condition de maturation et le stade de maturité de l'ovocyte, nous avons identifié deux voies de signalisation dominantes : la voie des lipides (transport du cholestérol et des triglycérides) fortement activée en condition in vivo, et le processus de réplication, recombinaison et réparation d'ADN, spécifique aux CCs in vitro. Nos résultats montrent que les conditions de maturation des COCs ont un impact sur la signature moléculaire des CCs. De plus, La signature moléculaire identifiée dans les CCs à différents stades de maturation ovocytaire nous a permis de définir la compétence des CCs. En considérant ce critère, nous avons observé que les ovocytes matures associés à des cellules du cumulus compétents (sur-expression de la signature des CCs au stade MII) présentent un potentiel de formation de blastocyste supérieur aux ovocytes MII entourés des cellules du cumulus incompétents (sur-expression de la signature des CCs au stade VG et/ou MI). Ces résultats ouvrent ainsi de nouvelles perspectives en application clinique. Des études supplémentaires sont toutefois nécessaires afin d'identifier, dans un premier temps, les facteurs qui influencent l'expression des gènes au cours de la maturation ovocytaire in vivo et comprendre ainsi les voies de signalisation altérées par les conditions de culture in vitro. / Cumulus cells (CCs) associated with the oocyte form the cumulus-oocyte complex (COC). During folliculogenesis, interdependent dialogue governed by gap junctions is created between the oocyte and adjacent CCs. The oocyte, by secreting certain factors allows the differentiation and proliferation of CCs which, at the same time, provide nutrients to the oocyte for its maturation and development. Nuclear maturation of oocytes is defined by its transition from germinal vesicle (GV) to metaphase I (MI) up to metaphase II (MII) phase. Our team previously shown that certain genes expressed in the human CCs could predict embryo and the pregnancy outcomes. We analyzed the transcriptomic profile of CCs according to oocyte nuclear maturation stages (GV, MI and MII). The aim of this study was to identify the CCs molecular signature according to nuclear maturation oocyte under in vivo and in vitro conditions. In addition, we studied the impact of culture conditions of the COCs under in vivo and in vitro on the gene expression profile of CCs. We have demonstrated that there is a specific signature in the human CCs associated with the nuclear maturity of human oocytes whatever the culture condition. We have also observed the under-expression of genes involved in oocyte maturation and CCs expansion, and the over-expression of genes associated with cell cycle function in the CCS derived from in vitro versus in vivo oocytes. By comparing gene expression in the CCs according to oocyte nuclear maturation stages, we have identified two dominant signaling pathways: the lipids pathway (cholesterol transport and triglyceride) strongly activated in in vivo conditions, and the process of replication, recombination and DNA repair, which appear to be specific to in vitro CCs. Our results suggest that the maturation conditions of COCs have an impact on the molecular signature of CCs.Moreover, our data showed that matures oocytes can be surrounded by competent (sur-expression of the identified molecular signature of CCs derived from oocyte at MII stage) or incompetent CCs (sur-expression of the identified molecular signature of CCs derived from oocyte at GV or/and MI stages). These results open new perspectives in clinical application.Further studies are needed to identify factors influencing gene expression during oocyte maturation in vivo. These data should help to better understand how/why signaling pathways are altered by culture conditions in vitro.

Ovulatory follicle development and associated oocyte maturation involve complex coordinated molecular and cellular mechanisms not fully understood. This study addresses, for the first time in any species, the relationships among diameter, vascularization, follicular-fluid factors, and gene expression for follicle growth, steroidogenesis, angiogenesis and apoptosis in granulosa/cumulus cells and oocytes during different stages from the beginning of largest/ovulatory follicle to impending ovulation. Follicle dynamics was tracked and follicle-wall blood flow evaluated daily through transrectal ultrasonography in mares. The largest follicle of the ovulatory wave was distributed in six diameter groups (predeviation, deviation, postdeviation, early preovulatory, preovulatory, and impending ovulation) according to the targeted diameter for complete aspiration and cell harvesting. The most remarkable findings were (i) positive association between follicle development, blood flow, intrafollicular FSH, LH, estradiol, progesterone, and mRNA expression for FSHR and LHCGR in granulosa cells of largest/ovulatory follicle; (ii) plateau/decrease in follicle diameter, blood flow, and granulosa cell mRNA for FSHR, LHCGR, IGF1R, VEGFR2, CYP19A1, and CASP3 at the preovulatory stage; (iii) higher StAR and BCL2 and lower CASP3 mRNA in granulosa cells at impending ovulation; (iv) greater IGF1R mRNA in the granulosa cells at the predeviation stage; and (v) lower FSHR, LHCGR, IGF1R, and VEGFR2 mRNA in cumulus cells, and greater LHCGR and IGF1R mRNA in oocytes at the ovulatory stage. This study is a critical advance in the understanding of molecular mechanisms of follicle development and oocyte maturation and is expected to be vital for future studies targeting potential markers for follicle selection, development, and ovulation.

Oocyte quality plays a critical role in establishment of pregnancies, embryo development, implantation and the health of offspring. The oocyte provides maternal factors necessary for the initial development of its embryo during the period of transcriptional silence. Despite the consistent increase in number of couples seeking assisted reproductive treatments, oocyte quality still remains as an obstacle in successful fertility treatments and the mechanisms governing the quality of oocyte are poorly understood. Among various factors that may potentially affect the quality of oocyte, the acquisition of oocyte developmental competence seems to mainly occur during the final stage of oocyte maturation. The correct temporal regulation of series of molecular events and the proper exchange of signals with surrounding follicular environment during this critical period will ensure the developmental competence of oocyte and its subsequent embryo. In order to identify molecular factors affecting oocyte quality, I have compared oocytes and cumulus cells of different qualities at a molecular level. I present in this thesis the pathways and molecules that may determine the developmental competence of oocyte as well as candidate molecular markers of oocyte and embryo quality. A cDNA microarray analysis was performed, comparing the transcriptomes of rhesus monkey MII oocytes of different qualities, high quality VVM oocytes and poor quality IVM oocytes. A small set of 59 Oocyte quality plays a critical role in establishment of pregnancies, embryo development, implantation and the health of offspring. The oocyte provides maternal factors necessary for the initial development of its embryo during the period of transcriptional silence. Despite the consistent increase in number of couples seeking assisted reproductive treatments, oocyte quality still remains as an obstacle in successful fertility treatments and the mechanisms governing the quality of oocyte are poorly understood. Among various factors that may potentially affect the quality of oocyte, the acquisition of oocyte developmental competence seems to mainly occur during the final stage of oocyte maturation. The correct temporal regulation of series of molecular events and the proper exchange of signals with surrounding follicular environment during this critical period will ensure the developmental competence of oocyte and its subsequent embryo. In order to identify molecular factors affecting oo was identified as differentially expressed between the two types of oocytes. These mRNAs are involved in steroid metabolism, cell-cell interactions, cellular homeostasis, cell adhesion, mRNA stability and translation. In addition, the overexpression of several imprinted genes in IVM oocytes were detected, indicating a possible loss of correct epigenetic programming during IVM. These results indicate that normal oocyte-somatic cell interactions may be disrupted during IVM and the interruptions of these interactions during the final phase of oocyte maturation may be the prime cause of reduced developmental competence of IVM oocytes. To elucidate oocyte quality factors linked to the cumulus cell phenotype, the transcriptomes of two types of rhesus monkey cumulus cells, IVM and VVM, were compared using a cDNA microarray analysis. In contrast to a relatively small difference between IVM and VVM oocytes, a large number of genes were differentially expressed between IVM and VVM rhesus cumulus cells. Moreover, a much larger number of differential mRNA expressions were observed comparing the transitions from pre-maturation cumulus cells to the IVM and VVM cumulus cells. The results from these array comparisons indicated that the cumulus cells may fail to achieve successfully normal gene regulation during IVM and thus make a remarkable amount of changes in gene expression to compensate for the loss. Numerous genes involved in lipid metabolism are incorrectly regulated during IVM, and the synthesis of sex hormones appears not suppressed during IVM. In addition, a panel of 24 cumulus cell markers of oocyte quality was identified. Genetic effects on oocyte quality were explored by comparing transcriptomes of oocytes obtained from two different inbred mouse strains, B6 and D2, and F1 hybrid. A clustering analysis and statistical tests showed that the transcriptome of F1 oocytes is more similar to the B6 transcriptome than to the D2 at both GV and MII stages. Also, comparison analyses of GV stage oocyte transcriptomes with MII oocyte transcriptomes from three different mouse strains indicated that the number of overdominance genes at the MII stage is bigger than the number of overdominance genes at the GV stage. In order to investigate how the genes gain the overdominance during the GV to MII transition, overdominance genes were categorized according to their mRNA expression patterns at GV and MII stages. The results showed that more than 80% of overdominance genes belong to one of the four major transition groups. The further evaluation of changes in array intensities from GV to MII stage transition revealed that F1 oocytes and inbred strain oocytes differentially regulate the mRNA abundance during oocyte maturation and that the differential regulation of mRNA abundance by the F1 genotype is responsible for the increase of the number of overdominance genes during maturation from GV stage to MII stage. A mRNA sequence analysis indicated that the gain of overdominant low in F1 mRNA expression pattern during maturation may be regulated by 3'UTR motif elements. The number of dominance genes also increase during GV to MII transition. At both GV and MII stages, there are more genes with B6 dominant mRNA expression pattern than those with D2 dominance pattern. Lipid metabolism, small molecule biochemistry and cell death are biofunctions overrepresented in both dominance and overdominance genes. In addition, `blebbing' was identified as a biofunction significantly downregulated in the F1 and B6 MII eggs, indicating that the cellular function may be involved in oocyte maturation. / Molecular Biology and Genetics / Accompanied by one .pdf file: YLEE_SupplementalTables.pdf

Cerca de 40% dos oócitos bovinos fecundados não completam o desenvolvimento da fase de pré-implantação. A aquisição da competência para o desenvolvimento do oócito depende de alterações morfológicas, bioquímicas e moleculares. Essas alterações ocorrem tanto nos oócitos como nas células do cumulus. O presente trabalho estudou a interação cumulus e oócito no processo de morte celular programada nos complexos de cumulus oophoros (COCs) de diferentes classes morfológicas em bovinos. Os COCs foram puncionados de ovários de abatedouro, selecionados e classificados em 3 qualidades morfológicas: A: cumulus completo; B: cumulus parcial; C: cumulus expandido (todos com ooplasma homogêneo). Os COCs foram utilizados para avaliação da maturação nuclear em 0 e em 24h de cultivo do grau de fragmentação do DNA das células do cumulus (CC) antes e após a maturação in vitro, avaliação da competência do desenvolvimento embrionário partenogenético até o 9º dia pós-ativação, avaliação da qualidade dos blastocistos e estimativa dos transcritos e proteínas BCL-2 e BAX em CC maturados por 24h. Os resultados obtidos demonstraram que a maioria dos oócitos imaturos do grupo COC-A estavam em estádio de VGi e apresentavam dano mínimo ou inexistente no DNA. Ao contrário, os COCs B e C após a retirada do folículo apresentavam-se em estádio mais avançado de VG e fragmentação do DNA. Após a maturação dos COCs houve um aumento significativo dos núcleos fragmentados nos grupos COC-C e principalmente no COC-B. A morfologia dos COCs alterou a quantidade de oócitos que conseguiram ultrapassar o bloqueio embrionário e desenvolver à blastocisto e não a qualidade dos blastocistos. A expressão da proteína BCL-2 nos COCs não diferiu entre as diferentes morfologias de COCs. No entanto, a razão entre as proteínas BCL-2/BAX foi maior nos grupos com o cumulus completo e no grupo com o cumulus expandido. O grupo de COC-B foi o que apresentou maior quantidade da proteína BAX e menor relação entre as proteínas BCL-2/BAX. Com base nestes resultados, conclui-se que em COCs de diferentes morfologias existe uma correlação negativa entre fragmentação nuclear e potencial de desenvolvimento embrionário, e ainda que a baixa razão das proteínas BCL-2/BAX está relacionada com o aumento de fragmentação nuclear nas CC. No entanto, essa relação não ocorre com transcritos dos genes BCL-2 e BAX / About 40% of fertilized bovine oocytes do not complete development during the preimplantation period. It is known that the in vitro embryo production system is influenced by several factors, among them, the morphological quality of cumulus-oocyte complexes (COCs). The present work aimed to study the cumulus-oocyte interaction on the process of programmed cell death in bovine COCs of different morphological classes. The COCs were obtained from bovine ovaries and classified according to the morphology of their cumulus cell layers, as follows: class A, compact and with many layers; class B, compact with few layers; class C, expanded (all classes with homogeneous ooplasm). Before in vitro maturation (IVM), the nuclear maturation in COCs and DNA fragmentation in cumulus cells (CC) were evaluated. After IVM, oocytes and CC were analyzed for nuclear maturation, DNA fragmentation and BCL-2 and BAX transcripts and proteins. The developmental competence and quality of parthenogenetic embryos at the 9th day post-activation were also analyzed. The results showed that the majority of class A immature oocytes were at iGV stage, with minimal or inexistent DNA fragmantation, contrasting with the other classes of oocytes. In immature class B and C oocytes, the fGV stage of nuclear maturation was the most frequent, with increased DNA fragmentation. After IVM, an increase in DNA fragmentation was observed in B and C COCs, mainly in B group. The morphological type of COCs was not related with blastocyst quality, but affected the proportion of embryos capable of overcoming developmental block and reaching the blastocyst stage. BCL-2 protein in CC had the same expression level in all the COCs groups. However, the BCL-2/BAX proteins ratio was higher in A and C groups. COC-B had the highest BAX expression and lower ratio. These data demonstrate that there is a negative correlation between DNA fragmentation in CC and embryo developmental potential in different morphological types of COCs, and that the lower the BCL2/BAX protein ratio the greates the DNA fragmentation in CC, but this relation does not occur with transcripts

Cerca de 40% dos oócitos bovinos fecundados não completam o desenvolvimento da fase de pré-implantação. A aquisição da competência para o desenvolvimento do oócito depende de alterações morfológicas, bioquímicas e moleculares. Essas alterações ocorrem tanto nos oócitos como nas células do cumulus. O presente trabalho estudou a interação cumulus e oócito no processo de morte celular programada nos complexos de cumulus oophoros (COCs) de diferentes classes morfológicas em bovinos. Os COCs foram puncionados de ovários de abatedouro, selecionados e classificados em 3 qualidades morfológicas: A: cumulus completo; B: cumulus parcial; C: cumulus expandido (todos com ooplasma homogêneo). Os COCs foram utilizados para avaliação da maturação nuclear em 0 e em 24h de cultivo do grau de fragmentação do DNA das células do cumulus (CC) antes e após a maturação in vitro, avaliação da competência do desenvolvimento embrionário partenogenético até o 9º dia pós-ativação, avaliação da qualidade dos blastocistos e estimativa dos transcritos e proteínas BCL-2 e BAX em CC maturados por 24h. Os resultados obtidos demonstraram que a maioria dos oócitos imaturos do grupo COC-A estavam em estádio de VGi e apresentavam dano mínimo ou inexistente no DNA. Ao contrário, os COCs B e C após a retirada do folículo apresentavam-se em estádio mais avançado de VG e fragmentação do DNA. Após a maturação dos COCs houve um aumento significativo dos núcleos fragmentados nos grupos COC-C e principalmente no COC-B. A morfologia dos COCs alterou a quantidade de oócitos que conseguiram ultrapassar o bloqueio embrionário e desenvolver à blastocisto e não a qualidade dos blastocistos. A expressão da proteína BCL-2 nos COCs não diferiu entre as diferentes morfologias de COCs. No entanto, a razão entre as proteínas BCL-2/BAX foi maior nos grupos com o cumulus completo e no grupo com o cumulus expandido. O grupo de COC-B foi o que apresentou maior quantidade da proteína BAX e menor relação entre as proteínas BCL-2/BAX. Com base nestes resultados, conclui-se que em COCs de diferentes morfologias existe uma correlação negativa entre fragmentação nuclear e potencial de desenvolvimento embrionário, e ainda que a baixa razão das proteínas BCL-2/BAX está relacionada com o aumento de fragmentação nuclear nas CC. No entanto, essa relação não ocorre com transcritos dos genes BCL-2 e BAX / About 40% of fertilized bovine oocytes do not complete development during the preimplantation period. It is known that the in vitro embryo production system is influenced by several factors, among them, the morphological quality of cumulus-oocyte complexes (COCs). The present work aimed to study the cumulus-oocyte interaction on the process of programmed cell death in bovine COCs of different morphological classes. The COCs were obtained from bovine ovaries and classified according to the morphology of their cumulus cell layers, as follows: class A, compact and with many layers; class B, compact with few layers; class C, expanded (all classes with homogeneous ooplasm). Before in vitro maturation (IVM), the nuclear maturation in COCs and DNA fragmentation in cumulus cells (CC) were evaluated. After IVM, oocytes and CC were analyzed for nuclear maturation, DNA fragmentation and BCL-2 and BAX transcripts and proteins. The developmental competence and quality of parthenogenetic embryos at the 9th day post-activation were also analyzed. The results showed that the majority of class A immature oocytes were at iGV stage, with minimal or inexistent DNA fragmantation, contrasting with the other classes of oocytes. In immature class B and C oocytes, the fGV stage of nuclear maturation was the most frequent, with increased DNA fragmentation. After IVM, an increase in DNA fragmentation was observed in B and C COCs, mainly in B group. The morphological type of COCs was not related with blastocyst quality, but affected the proportion of embryos capable of overcoming developmental block and reaching the blastocyst stage. BCL-2 protein in CC had the same expression level in all the COCs groups. However, the BCL-2/BAX proteins ratio was higher in A and C groups. COC-B had the highest BAX expression and lower ratio. These data demonstrate that there is a negative correlation between DNA fragmentation in CC and embryo developmental potential in different morphological types of COCs, and that the lower the BCL2/BAX protein ratio the greates the DNA fragmentation in CC, but this relation does not occur with transcripts

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