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.

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±1,09 unidade arbitrária/UA) e da combinação FGF16+BMP15 (11,34±0,61 UA) em relação ao grupo controle (8,73±0,44 UA) e ao suplementado com FGF16 (9,42±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±1,24 % e 3,81±2,76 %, respectivamente) em comparação ao grupo FGF16 (22,54±2,80 % e 31,13±7,81 %, respectivamente). Ainda, o FGF16 causou aumento na incidência de fragmentação de DNA em CC, quando relacionado ao controle (16,04±1,45 %). A taxa de maturação nuclear oocitária foi superior (p=0,014) no grupo suplementado com BMP15 (93,60±4,03 %) em comparação aos grupos controle (80,80±2,49 %) e FGF16 (76,75±2,28 %), aproximando-se da totalidade. De forma inédita, descrevemos ação da BMP15 (10,79±0,72 ng/ml) no incremento da produção de progesterona, sendo maior (p=0,0113) do que a produzida nos grupos controle (8,38±0,39 ng/ml) e FGF16 (8,84±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±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±1.09 arbitrary unit/AU) and FGF16+BMP15 (11.34±0,61 AU) when compared to control (8.73±0.44 AU) and FGF16 groups (9.42±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±1.24 % and 3.81&plusmn2.76 %, respectively) in comparison to FGF16 group (22.54±2.80 % and 31.13±7.81 %, respectively). Moreover, FGF16 caused an increase in CC DNA fragmentation, when related to control (16.04±1.45 %). Oocyte nuclear maturation rate was higher (p=0.014) in groups supplemented with BMP15 (93.60±4.03 %) compared to control (80.80±2.49 %) and FGF16 treatments (76.75±2.28 %), almost reaching the totality of COCs. In an unprecedented way, we described the BMP15 increasing action on progesterone production (10.79±0,72 ng/ml; p=0.0113) when compared to control (8.38±0.39 ng/ml) and FGF16 groups (8.84±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.

Retinoblastoma-binding protein 4 (RBBP4) is a subunit of chromatin remodeling factor 1 (CAF-1) and is essential for mammalian oocyte maturation, embryo survival, and embryo implantation. RBBP4 also localizes to the chromatin and is a ubiquitously expressed nuclear protein. Previous methods used to study this protein include short interfacing RNAs (siRNAs) and CRISPR/Cas9. These techniques have limitations such as determining an indirect depletion of proteins, may trigger compensatory mechanisms, and may not be useful in non-dividing primary cells. A new, acute, and rapid endogenous protein depletion technique called Trim-Away, can overcome these limitations. Trim-Away is also widely applicable since it can be used with many off-the-shelf reagents. Trim-Away utilizes the TRIM21-antibody interaction within the cytosol and the ubiquitin-proteasome pathway (UPP) to target and degrade a protein of interest. Studying RBBP4 using Trim-Away can offer insight into possible new functions of RBBP4 and its maternal effect, and increase the knowledge on a new, acute, and endogenous protein depletion technique. Here we report that, RBBP4 is required for proper blastocyst development and RBBP4 is more abundant in MII oocytes than GVBD oocytes. We also report that the loss of RBBP4 hinders RNA synthesis and causes cell death in later stages of embryo development. While our Trim-Away methodology can deplete RBBP4 as early as the 2-cell stage in embryos, our oocyte Trim-Away protocol needs to be optimized.

Orientador: José Buratini Junior / Banca: Gisele Zoccal Mingoti / Banca: Juliano da Silveira / Banca: Marcelo Marcondes Seneda / Banca: Marcelo Fábio Gouveia Nogueira / Resumo: O oócito é responsável por regular os mecanismos da maturação do complexo cumulus oócito (COC) principalmente via secreção de fatores. parácrinos.Dentre os fatores secretados pelo oócito (OSF) desta-se o fator de crescimento dos fibroblastos 10 (FGF10), e dois fatores TGFβ mais estudados a proteína morfogenética óssea 15 (BMP15) e fator de crescimento de diferenciação 9 (GDF9) e estão associados com o desenvolvimento da competência oocitária. O presente trabalho investigou a regulação do oócito sobre as células do cumulus em dois cenários atuais da maturação in vitro, na presença de hormônio folículo estimulante (FSH) ou ampirregulina (AREG). A ausência do oócito afeta a expansão das células do cumulus e também o consumo de glicose e a produção de lactato e a adição de oócitos desnudos reverte o efeito da oocectomia na expansão mas não no consumo de glicose nem na produção de lactato. A oocectomia afeta também a expressão gênica reduzindo a abundância de RNAm de hialurona sintetase 2 HAS2 e elevando a expressão de prostaglandina sintase 2 (PTGS2), pentraxina 3 (PTX3) e proteína indutora do fator de necrose tumoral 6 (TNFAIP6) na presença de FSH e AREG. A adição de oócitos desnudos reverte o efeito da oocectomia sobre a expressão de RNAm de AREG às 4 horas de cultivo, HAS2 e PTGS2 às 22 horas de cultivo apenas quando os complexos foram cultivados na presença de AREG. Adicionalmente foi investigado a ação isolada do FGF10 sobre a diferenciação das células do cumulus. O FGF10 re... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: The oocyte is responsible for regulating mechanisms of cumulus oocyte complex (COC) maturation mainly through secretion of paracrine factors. Including oocyte secreted factors (OSF) are the fibroblast growth factor 10 (FGF10), and two TGF beta factors most studied, bone morphogenetic protein 15 (BMP15) differentiation and growth factor 9 (GDF9), and they are associated with the oocyte development of competence. This study investigated the regulation of oocyte cumulus cells on two current scenarios in vitro maturation in the presence of follicle stimulating hormone (FSH) or ampirregulina (AREG). The absence of oocyte affects the expansion of the cumulus cells and also the glucose consumption and lactate production and addition of denuded oocytes reverses the effect of the expansion oocectomia but not glucose consumption or lactate production. The oocectomia also affects gene expression by reducing the abundance of mRNA hialurona synthetase 2 HAS2 and raising synthase prostaglandin expression 2 (PTGS2), pentraxin 3 (PTX3) and inducing protein of the tumor necrosis factor 6 (TNFAIP6) in the presence of FSH and AREG. The addition of denuded oocytes reverses the effect of AREG on oocectomia mRNA expression at 4 hours of culture, HAS2 and PTGS2 to 22 hours of cultivation only when complexes were grown in the presence of AREG. Additionally it investigated the isolated FGF10 action on the differentiation of cumulus cells. FGF10 reverses the effect of on the expansion of the cells and the expression of HAS2 and GFPT1apenas to 22 hours in cumulus but not glucose consumption or lactate production, which shows a delayed effect of FGF10 necessitating the presence of other OSF to soon effect as seen previously in the literature. Complementary to the results, it appears that the cumulus cells have an intracumulus control Smad2-activation by the presence of GDF9 and BMP15 in cumulus cells. In summary the oocyte... (Complete abstract click electronic access below) / Doutor

A new macroscopic ovarian reproductive maturity index for haddock, Melanogrammus aeglefinus L, was developed to improve field collection of reproductive stage data. The index was tested, validated and revised based on a comparison with a laboratory histological staging method. The comparison of field and histological observations helped to improve the field index and methodologies and provided useful insight into the reproductive biology of Haddock. Although laboratory staging based on histology is inherently more accurate than any macroscopic field staging method, field observations can reveal weaknesses in the laboratory approach due to sampling bias. The revised field index includes three new macroscopic stages that represent a progression in final oocyte maturation from early to late, which were found to be reliable for staging spawning readiness in the field. This index was then used to study a population of Haddock in the Gulf of Maine to determine if it exhibits diel spawning periodicity. Commercial fishing vessels were chartered for 25 dedicated longlining trips to collect sexually mature haddock in the Southwestern Gulf of Maine at locations identified by commercial fishers as having spawning aggregations. In order to examine diel effects on haddock reproduction, the change in catch per unit effort and percentage of male and female haddock of all reproductive maturity stages together with the gonadosomatic index were observed across a 24 hour diel cycle. Only females in hydration stage 3 (defined as late final oocyte maturation stage ovaries with 50-75% of oocytes hydrated) were significantly affected by time of day with significant increases in both catch per unit effort and percentage of hydration stage 3 haddock during the night. Because H3 is the most advanced reproductive stage observed prior to a spawning event and therefore the best indicator of imminent spawning these results demonstrate that female haddock in Southwestern Gulf of Maine primarily spawn during night hours with a peak between 2100 and 0100 hours. No diel trend was observed for any male reproductive stages. Additionally, no diel trend was observed in male or female reproductive stages unrelated to spawning including immature, spent and resting.

No description available.

In-vitro produced embryos (IVP) differ greatly from in-vivo derived embryos (IVD) in gene expression, metabolism, development, and cryotolerance which limit the widespread use of this technology. In-vitro maturation (IVM) is one of the most important components for successful in-vitro embryo development. Growth differentiation factor 9 (GDF9) and bone morphogenetic protein 15 (BMP15) have been found to be essential during oocyte maturation and thus female fertility. These proteins are oocyte secreted factors (OSFs) and are produced with pro- and mature protein regions where the pro-regions are thought to aid in protein folding and dimerization where heterodimerization the two proteins has been termed cumulin (Motterhead et al., 2015). Cumulin was found to have significant effects on oocyte maturation, blastocyst rate and hatching rates. However, only recombinant mature forms of these proteins are available commercially and making pro-mature GDF9 and BMP15 as well as pro- and mature cumulin is problematic. A few studies have evaluated the mature versions finding slight, although non-significant effects on oocyte maturation and blastocyst rates. However, none have studied the effects of using both mature GDF9 and BMP15 on bovine oocytes; thus, it was tested in this thesis. For the first experiment we hypothesized cumulus–oocyte complexes (COCs) matured for 23 h in maturation media supplemented with the mature proteins would increase blastocyst development, decreased lipid levels, and increased mitochondrial activity. Additionally, cryopreservation of embryos induces oxidative damage. However, studies have shown adding individual antioxidants to cryopreservation medium help alleviate post-thaw oxidative stress by reducing the accumulation of reactive oxygen species (ROS) and detoxifying lipid peroxidation (Tarin and Trounson, 1993; Lane et al., 2002; Takahashi et al., 2013). Few studies have evaluated effects using combinations of antioxidants supplemented in slow-freezing media. For the second experiment we further hypothesized blastocysts slow frozen with antioxidants would have increased cryotolerance compared to controls. For the first experiment, bovine embryos were IVP in three treatment groups, J: a commercial IVM media, T: control (TCM 199 supplemented with gonadotrophins), and TGB: control supplemented with GDF9 (200 ng/µL) and BMP15 (100 ng/µL). For experiment 2, only IVM groups T and TGB were used. Embryos were produced in five then four replicates, respectively, from abattoir ovaries, oocytes were matured, fertilized with frozen-thawed semen from one of three bulls, and presumptive zygotes were cultured for 7-8 days. For experiment 1, stage 6–9 blastocysts were stained with Nile Red or Mitotracker Red CMX-Rosamine to evaluate lipid content and mitochondrial polarity, respectively, utilizing confocal microscopy at ×40. Five slices per embryo were evaluated and averaged for fluorescence. Blastocyst rates, Nile Red (sqrt transformed, outliers removed), and Mitotracker data were analyzed by an ANOVA and means separated by Tukey HSD. For experiment 2 stage 6–9 blastocysts were slow frozen then thawed in a 2x2 factorial and evaluated for re-expansion 24 hours post-thaw. Results indicate that there was no difference for blastocyst rates for experiment 1 and 2 (J: 26.7 0.02%, T: 26.9 0.02%, TGB: 24.2 0.031%, P > 0.1; and T: 22.0 0.020%, TGB: 21.8 0.024%, P > 0.1; respectively). TGB’s Nile Red fluorescence intensity was significantly lower (5.09 2.16 AFU, P < 0.0001) than T (12.0 2.11) and J (11.05 2.18). MitoTracker fluorescence was similar among all treatments (P > 0.05). There was no significant interactions or main effects seen between cryopreservation groups; however, T/AO (52.9 0.05%, n = 37) and T/C (39.8 0.05%, n =38) having on average a 13.1% higher re-expansion rate and AO overall had on average 6.2% higher re-expansion rates. There was no difference seen between TGB/AO and TGB/C. These results suggest that the mature forms of GDF9 and BMP15 supplemented during oocyte maturation can lower lipid content of resulting embryos, however they do not increase blastocyst rates, mitochondrial activity, or re-expansion rates after cryopreservation.

The production of embryos has been described as a revolutionary process with the ability to make cattle systems more successful. However, despite constant research done in the field of embryology, there remains a discrepancy between the quality of in vitro produced (IVP) and in vivo derived (IVD) embryos. This difference is potentially associated with the lack of synchronization between nuclear and cytoplasmic maturation events within the oocyte, which is carefully mediated in the ovarian environment and the cumulus oocyte complex (COC). The purpose of this thesis was to utilize a pre-maturation culture system to keep oocytes in an arrested germinal vesicle (GV) state before subjecting them to maturation. In the first half of the experiment, oocytes were pre-matured in C-type natriuretic peptide (CNP) supplemented medium for 12 hours. Following pre-maturation, oocytes were transferred to amphiregulin (AREG) for another 12 hours to develop. This procedure is known as CAPA-AREG. After fertilization and a 7- day culture, embryos were assessed for cleavage rate and blastocyst rate. Embryos were also subjected to staining in order to evaluate lipid content and mitochondrial activity via confocal microscopy and ImageJ software. Hurdles in acquiring data also encouraged an assessment on the IVF laboratory and how procedures can be optimized. Overall, embryology lab procedures must be strictly followed, and the laboratory environment must be maintained to the best of the staff’s ability to increase success rates. Due to mishaps in the laboratory, the effectiveness of CNP and AREG on improving bovine oocyte maturation and embryo development is still inconclusive. Further research is required to determine if CNP and AREG can be utilized in future bovine IVF procedures.

O objetivo deste estudo foi avaliar a eficiência da baixa tensão de oxigênio (5% de CO2; 5% de O2 e 90% de N2) na maturação oocitária in vitro em meio quimicamente definido (0,1% de PVA) ou suplementado com 10 % de PFF. Foram avaliados a migração dos grânulos corticais, a quantificação e distribuição da proteína HSP70, a maturação nuclear, as concentrações intracelulares da glutationa reduzida, a avaliação dos índices de penetração espermática e o desenvolvimento e qualidade de embriões fecundados in vitro em oócitos antes da maturação (0 hora) e após a maturação in vitro nos diferentes tratamentos (atmosfera = 5 ou 20% de O2 e suplementação do meio de maturação = 0,1% de PVA ou 10% de PFF). Para verificar a influência da atmosfera e da suplementação do meio no sistema de maturação foram avaliados o ciclo celular das células do cumulus, o número de células do cumulus por oócitos, as concentrações intracelulares de glutationa reduzida (GSH) das células do cumulus, as concentrações de TBARS no meio de maturação e a resistência das células do cumulus ao estresse oxidativo. Avaliações quanto às concentrações de GSH e HSP70 foram realizadas em oócitos in vivo, visando comparar a eficiência do sistema in vitro. Para a maioria dos parâmetros avaliados não houve interação entre a atmosfera e a suplementação do meio de maturação, indicando que o efeito da atmosfera independe do meio utilizado. Foi observado que em atmosfera com baixa tensão de O2 houve diminuição da concentração de glutationa nas células do cumulus, do índice de clivagem e do número total de blastômeros dos embriões e houve aumento do número de células por oócito após o período de maturação in vitro. A suplementação do meio de maturação com 0,1% de PVA diminuiu os índices de migração dos grânulos corticais, contudo não alterou os índices de penetração espermática. Os oócitos do grupo 0 hora apresentaram índices maiores de HSP70 do que cada um dos demais grupos, indicando provável diminuição dos estoques de HSP70 durante o período de maturação em todos os grupos de maturação in vitro. As células do cumulus provenientes de oócitos maturados em meio suplementado com 0,1% de PVA apresentaram maior número de células na fase S e G2/M do que o grupo 0 hora, entretanto isso não refletiu em aumento no número de células por oócito após a maturação. Este trabalho permite concluir que o uso de baixa tensão de oxigênio não melhora as condições do sistema de maturação oocitária in vitro em suínos, sendo que o efeito do uso desta atmosfera é, na maioria das variáveis avaliadas, independente do tipo de suplementação que se utiliza no meio de maturação in vitro. / The aim of this study was to evaluate the efficiency of low oxygen tension (5% CO2, 5% O2 and 90% N2) on in vitro oocyte maturation using defined media (0.1% PVA) or supplemented with 10% PFF. To achieve this goal, oocytes were evaluated prior to in vitro maturation (0 hour) and after in vitro maturation on different treatments (atmosphere = 5 or 20% O2 and media supplementation = 0.1% PVA or 10% PFF) for cortical granules migration, quantification and distribution of HSP70 protein, nuclear maturation, intracellular concentrations of reduced glutathione, evaluation of sperm penetration and development and quality of in vitro-produced embryos. Furthermore, to evaluate the effects on maturation system, several factors were analyzed, such as: the cell cycle phase of cumulus cells, the number of cumulus cells per oocyte, the intracellular concentrations of reduced glutathione in cumulus cells, the concentrations of TBARS in maturation media and the resistance of cumulus cells to the oxidative stress. Some of these evaluations were performed on in vivo oocytes, aiming to analyze oocyte competence after in vitro maturation. Concerning the majority of the parameters analyzed, there were no interaction between atmosphere and the supplementation of maturation media, indicating that the effect of atmosphere is independent of the media used. It was possible to observe that low tension atmosphere of O2 decreased glutathione concentrations in cumulus cells, cleavage rate and total number of embryo blastomeres and increased the number of cells per oocyte after the period of in vitro maturation. The maturation media supplementation with 0.1% PVA decreased the migration of cortical granules; however this fact did not have effect on in vitro sperm penetration levels. The 0 hour oocytes showed higher levels of HSP70 than each one of the in vitro maturated groups, indicating decrease of this protein stock during the maturation period in all of the in vitro maturated groups. The cumulus cells originated from maturated oocytes in supplemented media with 0.1% PVA showed higher number of cells in S and G2/M phase than 0 hour group; however this fact did not reflect in increased cell numbers per oocyte after maturation. Therefore, the use of low oxygen tension does not improve the conditions of the in vitro porcine oocyte maturation system and their effect in most of the factors analyzed is not dependent on the supplementation of maturation media.

O objetivo deste estudo foi avaliar a eficiência da baixa tensão de oxigênio (5% de CO2; 5% de O2 e 90% de N2) na maturação oocitária in vitro em meio quimicamente definido (0,1% de PVA) ou suplementado com 10 % de PFF. Foram avaliados a migração dos grânulos corticais, a quantificação e distribuição da proteína HSP70, a maturação nuclear, as concentrações intracelulares da glutationa reduzida, a avaliação dos índices de penetração espermática e o desenvolvimento e qualidade de embriões fecundados in vitro em oócitos antes da maturação (0 hora) e após a maturação in vitro nos diferentes tratamentos (atmosfera = 5 ou 20% de O2 e suplementação do meio de maturação = 0,1% de PVA ou 10% de PFF). Para verificar a influência da atmosfera e da suplementação do meio no sistema de maturação foram avaliados o ciclo celular das células do cumulus, o número de células do cumulus por oócitos, as concentrações intracelulares de glutationa reduzida (GSH) das células do cumulus, as concentrações de TBARS no meio de maturação e a resistência das células do cumulus ao estresse oxidativo. Avaliações quanto às concentrações de GSH e HSP70 foram realizadas em oócitos in vivo, visando comparar a eficiência do sistema in vitro. Para a maioria dos parâmetros avaliados não houve interação entre a atmosfera e a suplementação do meio de maturação, indicando que o efeito da atmosfera independe do meio utilizado. Foi observado que em atmosfera com baixa tensão de O2 houve diminuição da concentração de glutationa nas células do cumulus, do índice de clivagem e do número total de blastômeros dos embriões e houve aumento do número de células por oócito após o período de maturação in vitro. A suplementação do meio de maturação com 0,1% de PVA diminuiu os índices de migração dos grânulos corticais, contudo não alterou os índices de penetração espermática. Os oócitos do grupo 0 hora apresentaram índices maiores de HSP70 do que cada um dos demais grupos, indicando provável diminuição dos estoques de HSP70 durante o período de maturação em todos os grupos de maturação in vitro. As células do cumulus provenientes de oócitos maturados em meio suplementado com 0,1% de PVA apresentaram maior número de células na fase S e G2/M do que o grupo 0 hora, entretanto isso não refletiu em aumento no número de células por oócito após a maturação. Este trabalho permite concluir que o uso de baixa tensão de oxigênio não melhora as condições do sistema de maturação oocitária in vitro em suínos, sendo que o efeito do uso desta atmosfera é, na maioria das variáveis avaliadas, independente do tipo de suplementação que se utiliza no meio de maturação in vitro. / The aim of this study was to evaluate the efficiency of low oxygen tension (5% CO2, 5% O2 and 90% N2) on in vitro oocyte maturation using defined media (0.1% PVA) or supplemented with 10% PFF. To achieve this goal, oocytes were evaluated prior to in vitro maturation (0 hour) and after in vitro maturation on different treatments (atmosphere = 5 or 20% O2 and media supplementation = 0.1% PVA or 10% PFF) for cortical granules migration, quantification and distribution of HSP70 protein, nuclear maturation, intracellular concentrations of reduced glutathione, evaluation of sperm penetration and development and quality of in vitro-produced embryos. Furthermore, to evaluate the effects on maturation system, several factors were analyzed, such as: the cell cycle phase of cumulus cells, the number of cumulus cells per oocyte, the intracellular concentrations of reduced glutathione in cumulus cells, the concentrations of TBARS in maturation media and the resistance of cumulus cells to the oxidative stress. Some of these evaluations were performed on in vivo oocytes, aiming to analyze oocyte competence after in vitro maturation. Concerning the majority of the parameters analyzed, there were no interaction between atmosphere and the supplementation of maturation media, indicating that the effect of atmosphere is independent of the media used. It was possible to observe that low tension atmosphere of O2 decreased glutathione concentrations in cumulus cells, cleavage rate and total number of embryo blastomeres and increased the number of cells per oocyte after the period of in vitro maturation. The maturation media supplementation with 0.1% PVA decreased the migration of cortical granules; however this fact did not have effect on in vitro sperm penetration levels. The 0 hour oocytes showed higher levels of HSP70 than each one of the in vitro maturated groups, indicating decrease of this protein stock during the maturation period in all of the in vitro maturated groups. The cumulus cells originated from maturated oocytes in supplemented media with 0.1% PVA showed higher number of cells in S and G2/M phase than 0 hour group; however this fact did not reflect in increased cell numbers per oocyte after maturation. Therefore, the use of low oxygen tension does not improve the conditions of the in vitro porcine oocyte maturation system and their effect in most of the factors analyzed is not dependent on the supplementation of maturation media.