Oocyte maturation (OM) in teleosts is under precise hormonal control by estrogens and progestins. We show here that estrogens activate an epidermal growth factor receptor (EGFR) signaling pathway through the G protein-coupled estrogen receptor (GPER) to maintain meiotic arrest of full-grown zebrafish (Danio rerio) oocytes in an in vitro germinal vesicle breakdown (GVBD) bioassay. A GPER- specific agonist decreased OM and a GPER-specific antagonist increased spontaneous OM, whereas specific nuclear estrogen receptor (ERα and ERβ) agonists did not affect OM, which suggests the inhibitory action of estrogens on OM are solely mediated through GPER. Furthermore, a peptide-bound estrogen, which cannot enter the oocyte, decreased GVBD, showing that these estrogen actions are mediated through a membrane receptor. Treatment of oocytes with actinomycin D, a transcription inhibitor, did not block the inhibitory effects of estrogens on OM, indicating that estrogens act via a nongenomic mechanism to maintain oocyte meiotic arrest. EGFR mRNA was detected in denuded zebrafish oocytes by reverse transcription polymerase chain reaction (RT-PCR). Therefore, the potential role of transactivation of EGFR in estrogen inhibition of OM was investigated. The matrix metalloproteinase inhibitor, ilomastat, which prevents the release of heparin-bound epidermal growth factor (HB-EGF), increased spontaneous OM. Moreover, specific EGFR1 (ErbB1) inhibitors and inhibitors of extracellular-related kinase 1 and 2 (ERK1/2) increased spontaneous OM. Previously, estrogens have been shown to increase 3’-5’-cyclic adenosine mono phosphate (cAMP) levels through GPER in zebrafish oocytes during meiotic arrest. Taken together these present results suggest that estrogens also act through GPER to maintain meiotic arrest through a second signaling pathway involving transactivation of EGFR and activation of ERK 1 and 2. / text

In vitro maturation of dog oocytes has always been the main obstacle preventing reproductive biologist from producing canine in vitro cultured embryos. The unsuccessful oocyte maturation in canine species originates from their unique physiological and biological specifications. Ovulation of dominant follicles in bitch (6-12 in each oestrous cycle) occurs at prophase I stage of oocyte nucleus and meiotic resumption develops during 3-5 days of oviductal transition. During this PhD thesis, studies were designed in order to speculate characteristics of canine oocyte maturation in vitro in terms of maturation media components, gas composition of the incubator and hormonal requirements. Level of oxidative stress during 72h (culture period) of in vitro maturation showed that 5%O2, 5% CO2 and 90% N2 composition improves meiotic resumption and reduces degeneration rate significantly compared to 5% CO2 in air. Utilization of caffeine as a non specific phosphodiesterase inhibitor at 10mM for 12h at the beginning of the 72h culture (12+60) also improved MII maturation rate (16.9% ± 2.4; P < 0.05). Among several hormonal treatments recombinant porcine Growth Hormone (PGH) at 100ng/ml and Melatonin (MTN) at 100nM concentrations had outstanding improvement over meiotic resumption (28.9% ±10.0 and 56.2% ±8.6 respectively; P < 0.05). Attempts were made to study developmental potentials of optimally matured oocytes by parthenogenetic activation (PA) and in vitro fertilization (IVF) using chilled semen. Partial digestion of the zona pellucida prior to IVF improved the cleavage rate at 48h 6.4% ± 0.3 and resulted in production of a single 8 cell embryo. Moreover; canine follicular cells were culture in order to characterize their primary culture morphology and steroidogenic responsiveness to physiological and pharmaceutical substances. Immunolocalization of aromatase (CYP19) positive cell clumps, presumptive oestrogen producing colonies, was identified. This primary culture also maintained its steroidogenic machinery up to 96h (measured by radioimmunoassay) with a significant increase in production of estradiol and progesterone after 72h compare to the start of the culture.

Orientador: Gisele Zoccal Mingoti / Banca: Lindsay Unno Gimenes / Banca: Claudia Lima Verde Leal / Resumo: Este estudo foi desenvolvido com o objetivo de avaliar o efeito do precursor do peptídeo natriurético tipo C (NPPC) durante o cultivo de pré-maturação in vitro (pré-MIV) de oócitos bovinos sobre: 1) a progressão da maturação nuclear; 2) a expressão gênica das células do cumulus e 3) a aquisição da competência do oócito para o desenvolvimento embrionário in vitro. Os complexos cumulus-oócito (CCOs) foram pré-MIV com 100 nM NPPC por 8 horas (grupo NPPC) e, ao final do período, foram lavados para completa remoção do NPPC e submetidos à MIV por 22h. Após 8h de pré-MIV e após 8h de pré-MIV seguidas de 22h de MIV (duração total do cultivo = 30h) foram avaliadas a progressão da maturação nuclear e a expressão relativa de mRNA nas células do cumulus. O grupo controle (C) foi maturado na ausência de NPPC por até 30h, e as mesmas avaliações anteriores foram realizadas imediatamente após a remoção do ambiente folicular (C0), após 8h de cultivo (C8), após 22h de cultivo (C22) e após 30h de cultivo (C30). Em outro experimento, cujos tratamentos foram idênticos aos supramencionados, os oócitos foram fecundados ao término da MIV e foi avaliado o desenvolvimento embrionário até a fase de blastocistos. Após 8h de cultivo de pré-MIV, a análise da progressão da meiose demonstrou que o grupo C0 apresentou 58,9% de estruturas com configuração de GV1, enquanto que o grupo C8 apresentou apenas 13,9% de estruturas nesta configuração (P<0,05). A proporção de GV1 no grupo NPPC foi semelhante a ambos e... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: The aim of this study was to evaluate the effect of the C-type natriuretic peptide precursor (NPPC) during pre in vitro maturation culture (pre-IVM) of bovine oocytes on: 1) nuclear maturation progress; 2) gene expression in cumulus cells and 3) acquisition of competence for in vitro embryo development. Cumulus oocyte complexes (COCs) were pre-IVM with 100 nM NPPC for 8 hours (NPPC group) and then were washed for the complete removal of NPPC and submitted to IVM for 22h. After 8h pre-IVM followed by 22h IVM (total culture time = 30h) oocytes were evaluated for nuclear maturation progress and cumulus cells for relative mRNA expression. Control group (C) was IVM in the absence of NPPC for up to 30h and the same evaluations were made immediately after follicle removal (C0) and after 8h (C8), 22h (C22) and after 30h of culture (C30). In another experiment with the same treatment, the oocytes were fertilized at the end of IVM and embryo development to the blastocyst stage was evaluated. After 8 hours of pre-IVM culture, meiosis progression analysis showed 58.9% of oocytes in GV1 configuration in C0, while C8 had only 13.9% (P<0.05). The GV1 rates in NPPC did not differ from any group (22.8%; P>0.05). On the other hand, C8 showed higher rates of GV3 in comparison with C0 (53.1% vs. 3.62%; p<0.05) and no differences compared to NPPC (38.7%; P<0.05). At the end of IVM culture, no differences between groups (C22 vs. NPPC vs. C30) were observed in nuclear maturation, however, higher rates of degenerated oocytes were observed in C30 in comparison with C22 (11.4% vs. 2.8%; P<0.05). The relative gene expression analysis in cumulus cells after 8h pre-IVM with NPPC showed an up-regulation in genes related to cumulus cells expansion (GREM1, PTGS2/COX2, PTX3, TNFAIP6 and VCAN), oocyte maturation (BDNF, EGFR, NOS3, PDE5A, PRKCD e STAT3) a... (Complete abstract click electronic access below) / Mestre

Orientador: Sony Dimas Bicudo / Banca: Eunice Oba / Resumo: O presente estudo visou avaliar a resposta ovariana a superestimulação utilizando 80UI de FSH e 300UI de eCG após ablação por laparoscopic ovum pic-up (LOPU1) de todos os folículos visíveis presentes em ambos os ovários. Determinar o melhor momento para realização de uma nova LOPU (LOPU2), mediante a avaliação ultrassonográfica dos ovários a cada 12 horas após LOPU1. E comparar a respostas ovariana do tratamento Duplo LOPU (DLOPU) com o tratamento One Shot adaptado de Baldassarre (1996), quanto a quantidades dos Complexos Cumulos-Oócito (COCs), e qualidade das estruturas obtidas e após processo de maturação in vitro (MIV) por 24 horas. Foi determinado o momento para realização da LOPU2 em 36 horas após a superestimulação. A média de folículos presentes na avaliação ultrassonográfica imediatamente antes a LOPU em ambos os tratamentos foi cerca de 9 estruturas, não diferindo da media de folículos aspirados, a taxa de recuperação comum aos dois tratamentos foi de 62,5%. A avaliação imediata das estruturas obtidas bem como a após 24 de MIV demonstrou que não houve diferença qualitativa COCs entre os tratamentos / Abstract: The present study aimed to evaluate the ovarian response to superstimulation using 80UI FSH and eCG 300UI after ablation LOPU (LOPU1) of all follicles present in both ovaries. Determine mlehor time to make a new LOPU (LOPU2) by ultrasound evaluation of the ovaries every 12 hours LOPU1. And comparing the responses ovarian DLOPU treatment with the treatment Ono Shot adapted Baldassarre (1996) with the DLOPU described here, the amounts of COC and quality of the structures obtained after process and IVM for 24 hours. It was determined the time for completion of LOPU2 in 36 hours after the overstimulation. The mean number of follicles present in the ultrasonographic evaluation immediately before the LOPU in both treatments was about 9 structures did not differ from follicles aspirated media, the recovery rate common to the two treatments was 62.5%. Immediate assessment of the structures obtained as well as after 24 IVM demonstrated that COCs do not hear qualitative difference between treatments / Mestre

A resposta da produção in vitro de embriões (PIVE) é reduzida quando comparada à in vivo. O aprimoramento do conhecimento dos mecanismos de maturação de oócitos bovinos permite fornecer embasamento para incrementar os sistemas in vitro, aproximando-os do ideal fisiológico. O presente estudo visou investigar os efeitos da suplementação dos meios de maturação com FGF16 (10 ng/ml), BMP15 (100 ng/ml) e a interação de ambos sobre parâmetros relevantes ao desenvolvimento do complexo cumulus oócito (COC), tais como: expansão as células do cumulus (CC), fragmentação de DNA em CC e oócito, maturação nuclear oocitária, metabolismo energético e produção de progesterona. Os COC foram maturados em meios de tratamento (controle, FGF16, BMP15 e FGF16+BMP15) e avaliados em diferentes momentos da MIV (0 e 22 horas). A análise da expansão das CC demonstrou efeito positivo (p=0,0071) da BMP15 (11,34&plusmn;1,09 unidade arbitrária/UA) e da combinação FGF16&#43;BMP15 (11,34&plusmn;0,61 UA) em relação ao grupo controle (8,73&plusmn;0,44 UA) e ao suplementado com FGF16 (9,42&plusmn;0,65 UA). A presença de fragmentação de DNA em CC (p=0,0015) e oócitos (p=0,036) foi significativamente menor em COC tratados com BMP15 (11,73&plusmn;1,24 % e 3,81&plusmn;2,76 %, respectivamente) em comparação ao grupo FGF16 (22,54&plusmn;2,80 % e 31,13&plusmn;7,81 %, respectivamente). Ainda, o FGF16 causou aumento na incidência de fragmentação de DNA em CC, quando relacionado ao controle (16,04&plusmn;1,45 %). A taxa de maturação nuclear oocitária foi superior (p=0,014) no grupo suplementado com BMP15 (93,60&plusmn;4,03 %) em comparação aos grupos controle (80,80&plusmn;2,49 %) e FGF16 (76,75&plusmn;2,28 %), aproximando-se da totalidade. De forma inédita, descrevemos ação da BMP15 (10,79&plusmn;0,72 ng/ml) no incremento da produção de progesterona, sendo maior (p=0,0113) do que a produzida nos grupos controle (8,38&plusmn;0,39 ng/ml) e FGF16 (8,84&plusmn;0,45 ng/ml). Não foi evidenciado efeito dos tratamentos sobre o consumo de glicose e a produção de lactato. O presente estudo reforça o envolvimento da BMP15 na foliculogênese e na diferenciação do COC. Deste modo, a adição da BMP15 (100ng/ml) aos convencionais protocolos de PIVE pode ser de grande valia para elevar a efetividade desta biotecnologia. A suplementação de FGF16 (10ng/ml) se mostrou indiferente ao processo de maturação, permitindo inferir que o FGF16 não tenha envolvimento nas etapas da maturação compreendidas pelo presente estudo in vitro. Não foi observada ação sinérgica entre o FGF16 e a BMP15. / In vitro embryo production (IVEP) efficiency is reduced when compared to in vivo. Gaining knowledge of bovine oocyte maturation mechanisms will provide bases to improve in vitro systems. The present study assessed the in vitro effects of fibroblast growth factor 16 (FGF16), bone morphogenetic protein 15 (BMP15) and their interaction on relevant parameters to cumulus oocyte complex (COC) development, such as: cumulus cells (CC) expansion, oocyte and CC DNA fragmentation, nuclear maturation, energetic metabolism and progesterone production. COCs were matured in control or supplemented media containing, FGF16 (10ng/ml), BMP15 (100ng/ml), FGF16&plusmn;BMP15 and analyzed at different times of IVM (0 and 22 hours). CC expansion evaluation demonstrated a positive effect (p=0.0071) of BMP15 (11.34&plusmn;1.09 arbitrary unit/AU) and FGF16+BMP15 (11.34&plusmn;0,61 AU) when compared to control (8.73&plusmn;0.44 AU) and FGF16 groups (9.42&plusmn;0.65 UA). The presence of DNA fragmentation in CC (p=0.0015) and oocytes (p=0.036) were lower in COCs treated in media supplemented with BMP15 (11.73&plusmn;1.24 % and 3.81&plusmn2.76 %, respectively) in comparison to FGF16 group (22.54&plusmn;2.80 % and 31.13&plusmn;7.81 %, respectively). Moreover, FGF16 caused an increase in CC DNA fragmentation, when related to control (16.04&plusmn;1.45 %). Oocyte nuclear maturation rate was higher (p=0.014) in groups supplemented with BMP15 (93.60&plusmn;4.03 %) compared to control (80.80&plusmn;2.49 %) and FGF16 treatments (76.75&plusmn;2.28 %), almost reaching the totality of COCs. In an unprecedented way, we described the BMP15 increasing action on progesterone production (10.79&plusmn;0,72 ng/ml; p=0.0113) when compared to control (8.38&plusmn;0.39 ng/ml) and FGF16 groups (8.84&plusmn;0.45 ng/ml). There were no differences in glucose consumption and lactate production. The present study reinforces BMP15 involvement in folliculogenesis and COC differentiation. FGF16 (10 ng/ml) media supplementation did not improve any of the outcomes measured, suggesting that FGF16 is not involved in the maturation steps analyzed in the present in vitro study. Thus, the inclusion of BMP15 (100 ng/ml) to conventional IVEP protocols can be valuable to increase the effectiveness of this biotechnology. Synergistic action between FGF16 and BMP15 was not observed.

Un contrôle précis de la maturation ovocytaire et de la ponte sont essentiels au succès de la reproduction sexuée au sein le règne animal. Ces processus sont coordonnés précisément par des signaux endocriniens et/ou environnementaux, selon les espèces, mais beaucoup reste à apprendre sur leurs régulations. Chez les cnidaires, de nombreuses méduses du groupe des hydrozoaires sont connues pour produire des gamètes en réponse à la transition nuit/jour. Pour caractériser les machineries cellulaires et moléculaires liant la réception de la lumière à l'initiation de la maturation ovocytaire, j'ai étudié la méduse hydrozoaire Clytia hemisphaerica. Mon travail de thèse s’est découpé en trois parties, chacune impliquant l'identification d'un composant moléculaire clé de ce processus.Mon étude initiale faisait partie d'une collaboration avec N. Takeda (Asamushi) et R. Deguchi (Sendai), chercheurs qui avaient, avant le début de ma thèse, identifié chez Clytia les Hormones d'Incitation de Maturation ovocytaire endogènes (MIH) comme étant des tétrapeptides de type WPRPamide, produit par clivage de deux précurseurs à neuropeptides. J'ai montré par hybridation in situ et immunofluorescence que les deux gènes précurseurs du MIH sont exprimés par un type de cellules neurosécrétrices localisées au niveau de l’ectoderme de la gonade, et que les peptides MIH sont sécrétés par ces mêmes cellules suite à une stimulation lumineuse. Cette étude a posé les bases permettant l'identification des régulateurs agissant en amont et en aval du MIH, et plus spécifiquement ceux impliqués dans la photoréception de l’ectoderme de la gonade et la réception du MIH par les ovocytes.Pour identifier le récepteur du MIH de Clytia (CheMIHR) dans les ovocytes, j'ai compilé à partir de données transcriptomiques issues de tissus de gonades, une liste de 16 protéines candidates de la famille des Récepteurs Couplés aux Protéines G (GPCR). J'ai cloné les 16 cDNAs et, utilisant une méthode de « deorphelinisation » de GPCR basée sur de la culture cellulaire (collaboration avec P. Bauknecht et G. Jékély; MPI, Tübingen), j’ai pu identifier un GPCR activée par des peptides MIH synthétiques. Sa fonction in vivo comme récepteur essentiel du MIH a été confirmée par la méthode d'édition génétique CRISPR/CAS9. La délétion ainsi produite, entraînant un déplacement du cadre de lecture au sein du gène CheMIHR, a détérioré la croissance des colonies de polypes et le comportement de ponte des méduses matures. Confirmant la fonction de CheMIHR, la maturation ovocytaire chez des mutants CheMIHR ne pouvait pas être déclenchée par la lumière ou par addition de MIH synthétiques, mais pouvait être rétablie en utilisant des analogues au cAMP, molécule connue pour agir en aval de la réception du MIH dans les ovocytes d’hydrozoaires. Des analyses phylogénétiques ont montré que Clytia MIHR est affilié à un sous-ensemble de familles de neuropeptides de bilaterians impliqués dans divers processus physiologiques, notamment la régulation de la reproduction. Des hybridations in situ sur les méduses Clytia, ont en outre montré l'expression des précurseurs de CheMIH et de CheMIHR dans des cellules neurales hors de la gonade, suggérant un rôle plus large du couple CheMIH-MIHR que la seule initiation de la maturation ovocytaire.Pour mieux comprendre la photoréception des gonades chez Clyita, j'ai montré que la ponte est sélectivement incitée par la lumière bleu-cyan, et mis en évidence, grâce à l’analyse de données de transcriptome de gonade, qu’un photopigment de la famille des Opsin (Opsin9) est hautement exprimé dans l'ectoderme. De façon saisissante, les hybridations in situ ont montré que le gène Opsin9 est exprimé dans les mêmes cellules sécrétant le MIH. L'introduction d'une mutation de changement de cadre de lecture dans le gène Opsin9 via la technologie CRISPR/Cas9 a empêché la maturation ovocytaire et la ponte des méduses mutantes en réponse à la lumière... / Tight control of oocyte maturation and of gamete release is essential for successful sexual reproduction in the animal kingdom. These processes are precisely coordinated by endocrine and/or environmental cues, depending on the species, but much remains to be learned about their regulation. Within the Cnidaria, many hydrozoan jellyfish are known to spawn mature gametes following dark/light transitions. To characterise the cellular and molecular machinery linking light reception and oocyte maturation initiation, I have studied the hydrozoan jellyfish Clytia hemisphaerica. My thesis work had three parts, each involving the identification of a key molecular component of this process.My initial study was part of a collaboration with N. Takeda (Asamushi) and R. Deguchi (Sendai), who identified the endogenous oocyte Maturation-Inducing Hormones (MIH) in Clytia as WPRPamide-related tetrapeptides, generated by cleavage of two neuropeptide precursors. I showed by in situ hybridization and immunofluorescence that Clytia MIH is produced by neurosecretory cells of the gonad ectoderm that co-express the two precursor genes, and that it is secreted upon light stimulation. This study paved the way for identification of regulators acting upstream and downstream of MIH release in the gonads, specifically the ones involved in photoreception in the gonad ectoderm, and in MIH reception by the oocytes. To identify the Clytia MIH receptor (CheMIHR) in the oocytes, I compiled a shortlist of 16 candidate G protein-coupled receptors (GPCRs) from gonad transcriptome data. I cloned all 16 cDNAs and, using a cell culture-based "GPCR deorphanization" assay (collaboration with P. Bauknecht and G. Jékély; MPI, Tübingen), identified one GPCR that was activated by synthetic MIH peptides. Its in vivo function as the essential MIH receptor was confirmed by CRISPR/Cas9 gene editing. Introduction of a frame-shift mutation in the CheMIHR gene impaired growth of Clytia polyp colonies and also the spawning behaviour of mature medusae. Confirming the function of CheMIHR, oocyte maturation in CheMIHR mutants could not be triggered by light or by synthetic MIH, but could be restored using cell-permeable analogues of cAMP, known to act downstream of MIH reception in hydrozoan oocytes. Phylogenetic analyses showed that Clytia MIHR is related to a subset of bilaterian neuropeptide hormone receptor families involved in diverse physiological processes, including regulation of reproduction. Accordingly, in situ hybridization showed the expression of Clytia MIH precursors and MIHR in non-gonadal neural cells, suggesting a wider role of Clytia MIH-MIHR besides oocyte maturation initiation.To address gonad photoreception, I showed that Clytia spawning is selectively induced by blue-cyan light, and then identified using gonad transcriptome data an opsin photopigment (Opsin9) highly expressed in the ectoderm. Strikingly, in situ hybridization showed that Opsin9 is expressed in the MIH-secreting cells. Introduction of a frame-shift mutation into the Opsin9 gene via CRISPR/Cas9 prevented oocyte maturation and spawning of mutant jellyfish in response to light. Anti-MIH immunofluorescence and rescue experiments with synthetic MIH showed that the essential function of Opsin9 is upstream of MIH release. Spawning in Clytia thus appears to be regulated by a dual function photosensory-neurosecretory cell type, perhaps retained from a distant metazoan ancestor...

Orientador: Fernanda da Cruz Landim / Banca: João Carlos Pinheiro Ferreira / Banca: Maria Denise Lopes / Banca: Alício martins Júnior / Banca: Edson Guimarães Lo Turco / Resumo: Este trabalho teve como objetivo geral averiguar o efeito da BL-I e da ROS em bloquear temporariamente a retomada da meiose, atuando sobre os fatores que controlam o ciclo celular meiótico de oócitos bovinos e a expansão das células do cumulus oophoros. No Experimento I, os oócitos permaneceram em meio MIV durante 6, 12 e 24 h na presença de duas concentrações de ROS (12,5 μM e 25 μM) ou na presença de BL-I (50 μ e 100 μM) ou na presença da associação de ROS (6,25 μM) + BL-I (25 μM) e em seguida foram cultivados em meio MIV livre de fármacos por 18 h, 12 h ou 24 h. Após esse tempo, os oócitos inibidos por 6 ou 12 h foram fertilizados e cultivados in vitro. O tratamento com ROS e BL-I não resultou em atraso na quebra da vesícula germinativa em relação ao controle. Entretanto, quando utilizados por 24 h, elevadas taxas de degeneração oocitária foram encontradas. Quando estes fármacos foram utilizados por 12 h com reversão por mais 12 h, verificamos uma menor taxa produção de blastocistos em relação ao grupo controle. No entanto, após a retirada do bloqueio por 6 h e maturação por 18 h um maior número de oócitos tratados tanto com ROS, como BL-I encontraram-se em MII. Da mesma forma, ao reduzirmos a dose e o tempo de inibição, os grupos tratados por 6 h de inibição meiótica e reversão por 18 h apresentaram taxas de produção embrionária similares ao grupo controle. No Experimento II, os oócitos permaneceram em meio MIV durante 6h na presença de 12,5 μM de ROS ou na presença de 50 μM de BL-I ou com a associação de ROS (6,25 μM) + BL-I (25 μM) sendo em seguida cultivados em meio MIV livre de fármacos por 18 h. Após esse tempo, os oócitos inibidos foram fertilizados, cultivados in vitro e os embriões produzidos foram submetidos a vitrificação. Os grupos BI-I e ROS + BL-I apresentaram superioridade de produção embrionária, tanto em relação ao grupo ROS como o controle. O grupo ... / Abstract: This work had as main objective to investigate the effect of BL-I and ROS in temporarily blocking the resumption of meiosis, acting on the factors that control the meiotic cell cycle of bovine oocytes and expansion of cumulus oophoros cells. In Experiment I, the oocytes remained in IVM mediafor 6, 12 and 24h in the presence of two concentrations of ROS (12.5 μM and 25 μM) or in the presence of BL-I (50 μ to 100 mM) or the association of ROS (6.25 μM) + BL-I (25 μM) and then cultured in a drug-free IVM media for 18, 12 or 24 h. After this time, the inhibited oocytes by 6 or 12 h were fertilized and cultured in vitro. Treatment with ROS and BL-I did not resulted in delay in germinal vesicle breakdown in relation to control. However, when used for 24 h, higher rates of oocyte degeneration were found. When these drugs were used for 12 h with reversion for more than 12 h, a lower blastocyst production rate compared to the control group was verified. However, after removal of the blockage for 6 h and maturation for 18 h, a higher number of oocytes treated with both ROS and BL-I were found in MII. Similarly, when the dose and duration of inhibition were reduced, the groups treated for 6 h of meiotic inhibition and reversion for 18 h showed similar embryo production rates to the control group. In Experiment II, oocytes remained in IVM mediafor 6 h in the presence of 12.5 μM of ROS or the presence of 50 μM of BL-I or the association of ROS (6.25 μM) + BL-I (25 μM) and then cultured in drug-free IVM media for 18 h. After this time, the inhibited oocytes were fertilized in vitro and the produced embryos were submitted to vitrification. The BI-I and ROS + BL-I groups showed superiority in embryo production, both in relation to the control as ROS group. The ROS + BL-I group showed higher rate of re-expansion after vitrification. Additionally, embryos from BL-I and BL-I + ROS showed lower number of apoptotic cells when compared to ... / Doutor

Primordial follicles are the first small follicles to appear in the mammalian ovary. Women are born with a fixed number of primordial follicles in the ovaries. Once formed, the pool of primordial follicles serves as a source of developing follicles and oocytes. The first aim of this thesis was to investigate the functional role of the intra-oocyte signaling pathways, especially the phosphatidylinositol-3 kinase (PI3K) and mammalian target of rapamycin complex 1 (mTORC1) pathways in the regulation of primordial follicle activation and survival. We found that a primordial follicle remains dormant when the PI3K and mTORC1 signaling in its oocyte is activated to an appropriate level, which is just sufficient to maintain its survival, but not sufficient for its growth initiation. Hyperactivation of either of these signaling pathways causes global activation of the entire pool of primordial follicles leading to the exhaustion of all the follicles in young adulthood in mice. Mammalian oocytes, while growing within the follicles, remain arrested at prophase I of meiosis. Oocytes within the fully-grown antral follicles resume meiosis upon a preovulatory surge of leutinizing hormone (LH), which indicates that LH mediates the resumption of meiosis. The prophase I arrest in the follicle-enclosed oocyte is the result of low maturation promoting factor (MPF) activity, and resumption of meiosis upon the arrival of hormonal signals is mediated by activation of MPF. MPF is a complex of cyclin dependent kinase 1 (Cdk1) and cyclin B1, which is essential and sufficient for entry into mitosis. Although much of the mitotic cell cycle machinery is shared during meiosis, lack of Cdk2  in mice leads to a postnatal loss of all oocytes, indicating that Cdk2 is important for oocyte survival, and probably oocyte meiosis also. There have been conflicting results earlier about the role of Cdk2 in metaphase II arrest of Xenopus  oocytes. Thus the second aim of the thesis was to identify the specific Cdk that is essential for mouse oocyte meiotic maturation. We generated mouse models with oocytespecific deletion of Cdk1  or Cdk2  and studied the specific requirements of Cdk1 and Cdk2 during resumption of oocyte meiosis. We found that only Cdk1 is essential and sufficient for the oocyte meiotic maturation. Cdk1 does not only phosphorylate the meiotic phosphoproteins during meiosis resumption but also phosphorylates and suppresses the downstream protein phosphatase 1, which is essential for protecting the Cdk1 substrates from dephosphorylation.

Female gametes, or oocytes grow and mature in a niche environment maintained by the somatic cells of the ovarian follicle. At ovulation ovarian follicle cells respond to the luteinising hormone (LH) surge coordinating the final maturation, meiotic resumption and release of oocytes. Simultaneously, production of a unique “mucified” extracellular matrix surrounding the oocyte through synthesis of Hyaluronan (HA) and HA cross-linking proteins produces an “expanded” and stabilised cumulus oocyte matrix with a specific composition, structure and function. In vitro maturation (IVM) of oocytes is a procedure by which cumulus oocyte complexes (COCs) are stimulated to produce cumulus matrix and undergo oocyte maturation ex vivo. In vitro maturation is a useful procedure for studying oocyte competence as well as offering health benefits for patients undergoing assisted reproduction. Oocytes derived from IVM have much lower developmental competence than in vivo matured oocytes, likely as a result of altered environmental conditions and gene expression leading to suboptimal maturation and/or inappropriate metabolic control in oocytes. Cumulus matrix expansion is widely used as an indicator of good oocyte developmental potential, however, the mechanism(s) that endow oocyte quality and how these may be influenced by the cumulus matrix are poorly understood. To better understand the process by which cumulus matrix is linked to the final stages of oocyte maturation, I undertook investigation of mouse COC matrix composition and function after in vivo maturation in comparison to IVM. The gene responsible for Hyaluronan synthesis, Has2, was not impaired under IVM conditions. In contrast, two key extracellular matrix proteins; Versican and Adamts1, which are normally selectively incorporated into periovulatory COCs in vivo, were greater than 10-fold reduced in IVM whether stimulated with Egf and/or FSH. This work is the first to show that commonly used IVM conditions result in altered gene expression in cumulus cells. Furthermore, the absence of Adamts1 and Versican suggest that COC matrix may be functionally insufficient. Although associated with good developmental potential, the function of the COC matrix in oocyte maturation is unknown. I assessed the properties of COC matrix that control metabolite supply to oocytes by examining transport of fluorescently labelled glucose and cholesterol across mouse COCs. Profound differences in the control of metabolite supply to oocytes in IVM were observed. In vivo matured complexes were capable of excluding glucose from the entire COC and cholesterol was excluded from oocytes. Conversely IVM COCs were more permissive to rapid equilibration of glucose and cholesterol concentrations across the complex and in oocytes. In fact both metabolites accumulated rapidly in IVM oocytes resulting in inverse gradient patterns of glucose and cholesterol abundance with highest concentrations accumulating in the oocyte after IVM vs highest concentrations surrounding the COC after in vivo maturation conditions. As oocytes are highly sensitive to high glucose my results indicate that metabolic balance in IVM may be disrupted due to impaired molecular filtration properties of the mucified COC matrix that controls supply of hydrophilic and lipophylic substrates. Importantly these novel findings can explain the glucose sensitivity of IVM oocytes and identifies a mechanism by which IVM may lead to poorer oocyte developmental competence. To translate these findings into the improvement of IVM I generated recombinant expression plasmid constructs for several Adamts1 and Versican functional domains. The efficacy of Versican as an IVM supplement that activates cumulus cell signal transduction was proved in principle, by showing enhanced COC matrix expansion when added to mouse IVM cultures. Similar mechanisms are likely to be functional in human COCs since I demonstrated VERSICAN and ADAMTS1 expression in human in vivo matured cumulus and granulosa cells. This work has advanced our understanding of oocyte maturation and will lead to improvements in IVM and healthier outcomes from reproductive therapies. / http://proxy.library.adelaide.edu.au/login?url= http://library.adelaide.edu.au/cgi-bin/Pwebrecon.cgi?BBID=1342419 / Thesis (Ph.D.) -- University of Adelaide, School of Paediatrics and Reproductive Health, 2008

Female gametes, or oocytes grow and mature in a niche environment maintained by the somatic cells of the ovarian follicle. At ovulation ovarian follicle cells respond to the luteinising hormone (LH) surge coordinating the final maturation, meiotic resumption and release of oocytes. Simultaneously, production of a unique “mucified” extracellular matrix surrounding the oocyte through synthesis of Hyaluronan (HA) and HA cross-linking proteins produces an “expanded” and stabilised cumulus oocyte matrix with a specific composition, structure and function. In vitro maturation (IVM) of oocytes is a procedure by which cumulus oocyte complexes (COCs) are stimulated to produce cumulus matrix and undergo oocyte maturation ex vivo. In vitro maturation is a useful procedure for studying oocyte competence as well as offering health benefits for patients undergoing assisted reproduction. Oocytes derived from IVM have much lower developmental competence than in vivo matured oocytes, likely as a result of altered environmental conditions and gene expression leading to suboptimal maturation and/or inappropriate metabolic control in oocytes. Cumulus matrix expansion is widely used as an indicator of good oocyte developmental potential, however, the mechanism(s) that endow oocyte quality and how these may be influenced by the cumulus matrix are poorly understood. To better understand the process by which cumulus matrix is linked to the final stages of oocyte maturation, I undertook investigation of mouse COC matrix composition and function after in vivo maturation in comparison to IVM. The gene responsible for Hyaluronan synthesis, Has2, was not impaired under IVM conditions. In contrast, two key extracellular matrix proteins; Versican and Adamts1, which are normally selectively incorporated into periovulatory COCs in vivo, were greater than 10-fold reduced in IVM whether stimulated with Egf and/or FSH. This work is the first to show that commonly used IVM conditions result in altered gene expression in cumulus cells. Furthermore, the absence of Adamts1 and Versican suggest that COC matrix may be functionally insufficient. Although associated with good developmental potential, the function of the COC matrix in oocyte maturation is unknown. I assessed the properties of COC matrix that control metabolite supply to oocytes by examining transport of fluorescently labelled glucose and cholesterol across mouse COCs. Profound differences in the control of metabolite supply to oocytes in IVM were observed. In vivo matured complexes were capable of excluding glucose from the entire COC and cholesterol was excluded from oocytes. Conversely IVM COCs were more permissive to rapid equilibration of glucose and cholesterol concentrations across the complex and in oocytes. In fact both metabolites accumulated rapidly in IVM oocytes resulting in inverse gradient patterns of glucose and cholesterol abundance with highest concentrations accumulating in the oocyte after IVM vs highest concentrations surrounding the COC after in vivo maturation conditions. As oocytes are highly sensitive to high glucose my results indicate that metabolic balance in IVM may be disrupted due to impaired molecular filtration properties of the mucified COC matrix that controls supply of hydrophilic and lipophylic substrates. Importantly these novel findings can explain the glucose sensitivity of IVM oocytes and identifies a mechanism by which IVM may lead to poorer oocyte developmental competence. To translate these findings into the improvement of IVM I generated recombinant expression plasmid constructs for several Adamts1 and Versican functional domains. The efficacy of Versican as an IVM supplement that activates cumulus cell signal transduction was proved in principle, by showing enhanced COC matrix expansion when added to mouse IVM cultures. Similar mechanisms are likely to be functional in human COCs since I demonstrated VERSICAN and ADAMTS1 expression in human in vivo matured cumulus and granulosa cells. This work has advanced our understanding of oocyte maturation and will lead to improvements in IVM and healthier outcomes from reproductive therapies. / http://proxy.library.adelaide.edu.au/login?url= http://library.adelaide.edu.au/cgi-bin/Pwebrecon.cgi?BBID=1342419 / Thesis (Ph.D.) -- University of Adelaide, School of Paediatrics and Reproductive Health, 2008