RESUMO
Oocyte meiotic maturation failure and chromosome abnormality is one of the main causes of infertility, abortion, and diseases. The mono-orientation of sister chromatids during the first meiosis is important for ensuring accurate chromosome segregation in oocytes. MEIKIN is a germ cell-specific protein that can regulate the mono-orientation of sister chromatids and the protection of the centromeric cohesin complex during meiosis I. Here we found that MEIKIN is a maternal protein that was highly expressed in mouse oocytes before the metaphase I (MI) stage, but became degraded by the MII stage and dramatically reduced after fertilization. Strikingly, MEIKIN underwent phosphorylation modification after germinal vesicle breakdown (GVBD), indicating its possible function in subsequent cellular event regulation. We further showed that MEIKIN phosphorylation was mediated by PLK1 at its carboxyl terminal region and its C-terminus was its key functional domain. To clarify the biological significance of meikin degradation during later stages of oocyte maturation, exogenous expression of MEIKIN was employed, which showed that suppression of MEIKIN degradation resulted in chromosome misalignment, cyclin B1 and Securin degradation failure, and MI arrest through a spindle assembly checkpoint (SAC)-independent mechanism. Exogenous expression of MEIKIN also inhibited metaphase II (MII) exit and early embryo development. These results indicate that proper MEIKIN expression level and its C-terminal phosphorylation by PLK1 are critical for regulating the metaphase-anaphase transition in meiotic oocyte. The findings of this study are important for understanding the regulation of chromosome segregation and the prevention meiotic abnormality.
RESUMO
As a member of Ubiquitin-specific protease subfamily, ubiquitin specific protease 7 (USP7) has been reported to participate in a variety of cellular processes, including cell cycle, apoptosis, DNA damage response, and epigenetic modification. However, its function in preimplantation embryos is still obscure. To investigate the functions of USP7 during preimplantation embryo development, we used siRNA to degrade endogenous USP7 messenger RNA. We found that USP7 knockdown significantly decreased the development rate of mouse early embryos. Moreover, depletion of USP7 induced the accumulation of the DNA lesions and apoptotic blastomeres in early embryos. In addition, USP7 knockdown caused an abnormal H3K27me3 modification in 2-cell embryos. Overall, our results indicate that USP7 maintains genome stability perhaps via regulating H3K27me3 and DNA damage, consequently controlling the embryo quality.
Assuntos
Histonas , Ubiquitina Tiolesterase , Animais , Camundongos , Peptidase 7 Específica de Ubiquitina/genética , Peptidase 7 Específica de Ubiquitina/metabolismo , Histonas/genética , Ubiquitina Tiolesterase/genética , Ubiquitina Tiolesterase/metabolismo , Dano ao DNA/genética , Proteases Específicas de Ubiquitina/genéticaRESUMO
BACKGROUND: As women with low ovarian reserve embark on the challenging journey of in-vitro fertilisation (IVF) treatment, the choice between natural and mildly stimulated cycles becomes a pivotal consideration. It is unclear which of these two regimens is superior for women with low ovarian reserve. Our study aims to assess the impact of natural cycles on embryo quality and pregnancy outcomes in women with low ovarian reserve undergoing IVF treatment compared to mildly stimulated cycles. METHODS: This retrospective study enrolled consecutive patients with low ovarian reserve who underwent IVF/intracytoplasmic sperm injection (ICSI) at Guangdong Second Provincial General Hospital between January 2017 and April 2021. The primary outcome for pregnancy rate of 478 natural cycles and 448 mild stimulated cycles was compared. Secondary outcomes included embryo quality and oocyte retrieval time of natural cycles. RESULTS: The pregnancy rate in the natural cycle group was significantly higher than that in the mildly stimulated cycle group (51.8% vs. 40.1%, p = 0.046). Moreover, natural cycles exhibited higher rates of available embryos (84.1% vs. 78.6%, p = 0.040), high-quality embryos (61.8% vs. 53.2%, p = 0.008), and utilisation of oocytes (73% vs. 65%, p = 0.001) compared to mildly stimulated cycles. Oocyte retrievals in natural cycles were predominantly performed between 7:00 and 19:00, with 94.9% occurring during this time frame. In natural cycles with high-quality embryos, 96.4% of oocyte retrievals were also conducted between 7:00 and 19:00. CONCLUSION: Natural cycles with appropriately timed oocyte retrieval may present a valuable option for patients with low ovarian reserve.
In the realm of in-vitro fertilisation (IVF) treatment, women with low ovarian reserve often face the crucial decision of opting for natural or mildly stimulated cycles. This retrospective study, conducted between January 2017 and April 2021 at Guangdong Second Provincial General Hospital, delves into the impact of these cycles on pregnancy outcomes. Examining 478 natural cycles and 448 mildly stimulated cycles, the study reveals a notably higher pregnancy rate in the natural cycle group (51.8% vs. 40.1%). Additionally, natural cycles demonstrated higher rates of available embryos, high-quality embryos, and oocyte utilisation compared to their mildly stimulated counterparts. The findings suggest that natural cycles, with proper oocyte retrieval timing, could be a favourable choice for those with low ovarian reserve seeking IVF treatment.
Assuntos
Reserva Ovariana , Resultado da Gravidez , Feminino , Humanos , Masculino , Gravidez , Estudos de Coortes , Estudos Retrospectivos , Sêmen , Recuperação de Oócitos , Taxa de GravidezRESUMO
Strategies to maximize individual fertility chances are constant requirements of ART. In vitro folliculogenesis may represent a valid option to create a large source of immature ovarian follicles in ART. Efforts are being made to set up mammalian follicle culture protocols with suitable FSH stimuli. In this study, a new type of recombinant FSH (KN015) with a prolonged half-life is proposed as an alternative to canonical FSH. KN015 supports the in vitro development of mouse follicles from primary to preovulatory stage with higher efficiency than canonical FSH and enhanced post-fertilization development rates of the ovulated oocytes. The use of KN015 also allows us to compare the dynamic transcriptome changes in oocytes and granulosa cells at different stages, in vivo and in vitro. In particular, KN015 facilitates mRNA accumulation in growing mouse oocytes and prevents spontaneous luteinization of granulosa cells in vitro. Novel analyses of transcriptome changes in this study reveal that the in vivo oocytes were more efficient than in vitro oocytes in terms of maternal mRNA clearing during meiotic maturation. KN015 promotes the degradation of maternal mRNA during in vitro oocyte maturation, improves cytoplasmic maturation and, therefore, enhances embryonic developmental potential. These findings establish new transcriptome data for oocyte and granulosa cells at the key stages of follicle development, and should help to widen the use of KN015 as a valid and commercially available hormonal support enabling optimized in vitro development of follicles and oocytes.
Assuntos
RNA Mensageiro Estocado , Transcriptoma , Feminino , Camundongos , Animais , RNA Mensageiro Estocado/metabolismo , Oogênese/genética , Oócitos/metabolismo , Células da Granulosa , Hormônio Foliculoestimulante/genética , Hormônio Foliculoestimulante/farmacologia , Hormônio Foliculoestimulante/metabolismo , Meiose , MamíferosRESUMO
With the rapid change of people's lifestyle, more childbearing couples live with irregular schedules (i.e., staying up late) and suffer from decreased fertility and abortion, which can be caused by luteal phase defect (LPD). We used continuous light-exposed mice as a model to observe whether continuous light exposure may affect luteinization and luteal function. We showed that the level of progesterone in serum reduced (p < .001), the number of corpus luteum (CL) decreased (p < .01), and the expressions of luteinization-related genes (Lhcgr, Star, Ptgfr, and Runx2), clock genes (Clock and Per1), and Mt1 were downregulated (p < .05) in the ovaries of mice exposed to continuous light, suggesting that continuous light exposure induces defects in luteinization and luteal functions. Strikingly, injection of melatonin (3 mg/kg) could improve luteal functions in continuous light-exposed mice. Moreover, we found that, after 2 h of hCG injection, the level of pERK1/2 in the ovary decreased in the continuous light group, but increased in the melatonin administration group, suggesting that melatonin can improve LPD caused by continuous light exposure through activating the ERK1/2 pathway. In summary, our data demonstrate that continuous light exposure affects ovary luteinization and luteal function, which can be rescued by melatonin.
Assuntos
Melatonina , Ovário , Feminino , Gravidez , Camundongos , Animais , Ovário/metabolismo , Camundongos Endogâmicos ICR , Melatonina/farmacologia , Melatonina/metabolismo , Corpo Lúteo/metabolismo , Progesterona/metabolismo , LuteinizaçãoRESUMO
Meiotic resumption-coupled degradation of maternal transcripts occurs during oocyte maturation in the absence of mRNA transcription. The CCR4-NOT complex has been identified as the main eukaryotic mRNA deadenylase. In vivo functional and mechanistic information regarding its multiple subunits remains insufficient. Cnot6l, one of four genes encoding CCR4-NOT catalytic subunits, is preferentially expressed in mouse oocytes. Genetic deletion of Cnot6l impaired deadenylation and degradation of a subset of maternal mRNAs during oocyte maturation. Overtranslation of these undegraded mRNAs caused microtubule-chromosome organization defects, which led to activation of spindle assembly checkpoint and meiotic cell cycle arrest at prometaphase. Consequently, Cnot6l-/- female mice were severely subfertile. The function of CNOT6L in maturing oocytes is mediated by RNA-binding protein ZFP36L2, not maternal-to-zygotic transition licensing factor BTG4, which interacts with catalytic subunits CNOT7 and CNOT8 of CCR4-NOT Thus, recruitment of different adaptors by different catalytic subunits ensures stage-specific degradation of maternal mRNAs by CCR4-NOT This study provides the first direct genetic evidence that CCR4-NOT-dependent and particularly CNOT6L-dependent decay of selective maternal mRNAs is a prerequisite for meiotic maturation of oocytes.
Assuntos
Meiose , Oócitos/metabolismo , Estabilidade de RNA , RNA Mensageiro/metabolismo , Ribonucleases/metabolismo , Animais , Exorribonucleases , Feminino , Deleção de Genes , Camundongos , Camundongos Knockout , Oócitos/citologia , Proteínas/genética , Proteínas/metabolismo , RNA Mensageiro/genética , Proteínas Repressoras , Ribonucleases/genética , Tristetraprolina/genética , Tristetraprolina/metabolismoRESUMO
An important event of the maternal-to-zygotic transition (MZT) in animal embryos is the elimination of a subset of the maternal transcripts that accumulated during oogenesis. In both invertebrates and vertebrates, a maternally encoded mRNA decay pathway (M-decay) acts before zygotic genome activation (ZGA) while a second pathway, which requires zygotic transcription, subsequently clears additional mRNAs (Z-decay). To date the mechanisms that activate the Z-decay pathway in mammalian early embryos have not been investigated. Here, we identify murine maternal transcripts that are degraded after ZGA and show that inhibition of de novo transcription stabilizes these mRNAs in mouse embryos. We show that YAP1-TEAD4 transcription factor-mediated transcription is essential for Z-decay in mouse embryos and that TEAD4-triggered zygotic expression of terminal uridylyltransferases TUT4 and TUT7 and mRNA 3'-oligouridylation direct Z-decay. Components of the M-decay pathway, including BTG4 and the CCR4-NOT deadenylase, continue to function in Z-decay but require reinforcement from the zygotic factors for timely removal of maternal mRNAs. A long 3'-UTR and active translation confer resistance of Z-decay transcripts to M-decay during oocyte meiotic maturation. The Z-decay pathway is required for mouse embryo development beyond the four-cell stage and contributes to the developmental competence of preimplantation embryos.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/genética , Proteínas de Ciclo Celular/genética , Proteínas de Ligação a DNA/genética , Genoma/genética , Proteínas Musculares/genética , RNA Mensageiro/genética , Fatores de Transcrição/genética , Zigoto/crescimento & desenvolvimento , Animais , Embrião de Mamíferos , Desenvolvimento Embrionário/genética , Feminino , Regulação da Expressão Gênica no Desenvolvimento/genética , Proteínas de Homeodomínio/genética , Camundongos , Nucleotidiltransferases/genética , Oócitos/crescimento & desenvolvimento , Estabilidade de RNA/genética , Receptores CCR4/genética , Fatores de Transcrição de Domínio TEA , Proteínas de Sinalização YAPRESUMO
Ca2+ participates in many important cellular processes, but the underlying mechanisms are still poorly understood, especially during oocyte maturation. First, we confirmed that calcium in the culture medium was essential for oocyte maturation. Next, various inhibitors of Ca2+ channels were applied to investigate their roles in mitochondrial Ca2+ changes and oocyte maturation. Our results showed that Trmp7, Orai, T-type Ca2+ channels and Na+ /Ca2+ exchanger complex (NCLX) were important for oocyte maturation. Trmp7 inhibition delayed germinal vesicle breakdown. Orai and NCLX inhibition significantly weakened the distribution of mitochondrial Ca2+ around the nucleus compared to the Ctrl group. Interestingly, even T-type Ca2+ channels-specific inhibitor Mibefradil blocked germinal vesicle breakdown; mitochondrial Ca2+ surrounding the nucleus still was maintained at a high level without spindle formation. Two calcium transporter inhibitors, Thapsigargin and Ruthenium Red, which have been confirmed to inhibit oocyte activation, did not significantly affect oocyte maturation. Increasing the knowledge of calcium transport may provide a basis to build on for improving oocyte in vitro maturation in human assisted reproduction clinics.
Assuntos
Cálcio/metabolismo , Técnicas de Maturação in Vitro de Oócitos , Proteínas de Membrana Transportadoras/metabolismo , Mitocôndrias/metabolismo , Oócitos/citologia , Oócitos/metabolismo , Animais , Membrana Celular/metabolismo , Sobrevivência Celular , Citoplasma/metabolismo , Feminino , Camundongos Endogâmicos ICRRESUMO
Obesity causes many reproductive dysfunctions such as reduced conception, infertility, and early pregnancy loss, and this is largely due to the negative effects of obesity on oocyte and embryo quality. In the present study, we employed single-cell RNA transcriptome sequencing to investigate the potential causes for the maternal obesity effects on mouse embryos. Our results showed that the 4-cell and morula/blastocyst rates were all significantly decreased during embryo development in obese mice. Genome-wide analysis indicated that obesity altered the expression of more than 1100 genes in 2-cell embryos, including the genes which were related to the p53 signaling pathway and apoptosis. Further analysis showed that the expression of 47 genes related to DNA damage was changed, and a positive γH2A signal and the altered expression of Rad51 and Tex15 were observed in the obese embryos. Obesity also affected histone methylation, shown by the decrease of the H3K4-me2 level. Besides this, we observed the occurrence of autophagy and apoptosis in the embryos of obese mice. There were 42 genes that were related to autophagy/apoptosis that showed aberrant expression, and the positive LC3 signal and the decrease of Clec16a, Rraga, and Atg10 level were also observed. In summary, our study suggested that obesity affected early embryonic development by inducing DNA damage, aberrant histone methylation, and autophagy levels in mice.
Assuntos
Autofagia/fisiologia , Metilação de DNA/genética , Reparo do DNA/genética , Desenvolvimento Embrionário/fisiologia , Obesidade Materna/patologia , Animais , Apoptose/fisiologia , Blastocisto/fisiologia , Proteínas de Ciclo Celular/biossíntese , Desenvolvimento Embrionário/genética , Feminino , Regulação da Expressão Gênica no Desenvolvimento/genética , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Obesos , Oócitos/citologia , Gravidez , Rad51 Recombinase/biossíntese , Análise de Célula Única , TranscriptomaRESUMO
Germ cell-derived genomic structure variants not only drive the evolution of species but also induce developmental defects in offspring. The genomic structure variants have different types, but most of them are originated from DNA double-strand breaks (DSBs). It is still not well known whether DNA DSBs exist in adult mammalian oocytes and how the growing and fully grown oocytes repair their DNA DSBs induced by endogenous or exogenous factors. In this study, we detected the endogenous DNA DSBs in the growing and fully grown mouse oocytes and found that the DNA DSBs mainly localized at the centromere-adjacent regions, which are also copy number variation hotspots. When the exogenous DNA DSBs were introduced by Etoposide, we found that Rad51-mediated homologous recombination (HR) was used to repair the broken DNA. However, the HR repair caused the chromatin intertwined and impaired the homologous chromosome segregation in oocytes. Although we had not detected the indication about HR repair of endogenous centromere-adjacent DNA DSBs, we found that Rad52 and RNA:DNA hybrids colocalized with these DNA DSBs, indicating that a Rad52-dependent DNA repair might exist in oocytes. In summary, our results not only demonstrated an association between endogenous DNA DSBs with genomic structure variants but also revealed one specific DNA DSB repair manner in oocytes.
Assuntos
Segregação de Cromossomos/fisiologia , Quebras de DNA de Cadeia Dupla , Reparo do DNA/fisiologia , Meiose/fisiologia , Oócitos/metabolismo , Animais , Segregação de Cromossomos/genética , Reparo do DNA/genética , Feminino , Infertilidade Feminina/genética , Masculino , Meiose/genética , CamundongosRESUMO
Within the development of ovarian follicle, in addition to cell proliferation and differentiation, sophisticated cell-cell cross talks are established among follicular somatic cells such as granulosa cells (GCs) and theca cells. To systematically reveal the cell differentiation and signal transductions in follicular somatic cells, we collected the mouse follicular somatic cells from secondary to ovulatory stage, and analyzed the single cell transcriptomes. Having data filtered and screened, we found 6883 high variable genes in 4888 single cells. Then follicular somatic cells were clustered into 26 cell clusters, including 18 GC clusters, 4 theca endocrine cell (TEC) clusters, and 4 other somatic cell clusters, which include immune cells and Acta2 positive theca externa cells. From our data, we found there was metabolic reprogramming happened during GC differentiation. We also found both Cyp19a1 and Cyp11a1 could be expressed in TECs. We analyzed the expression patterns of genes associated with cell-cell interactions such as steroid hormone receptor genes, insulin signaling genes, and cytokine/transformation growth factor beta associated genes in all cell clusters. Lastly, we clustered the highly variable genes into 300 gene clusters, which could be used to search new genes involved in follicle development. These transcriptomes of follicular somatic cells provide us potential clues to reveal how mammals regulating follicle development and could help us find targets to improve oocyte quality for women with low fertility.
Assuntos
Comunicação Celular/genética , Expressão Gênica/fisiologia , Folículo Ovariano/metabolismo , Transdução de Sinais , Transcriptoma , Animais , Feminino , Camundongos , Análise de Sequência de RNA , Análise de Célula ÚnicaRESUMO
Meiosis initiation is a crucial step for the production of haploid gametes, which occurs from anterior to posterior in fetal ovaries. The asynchrony of the transition from mitosis to meiosis results in heterogeneity in the female germ cell populations, which limits the studies of meiosis initiation and progression at a higher resolution level. To dissect the process of meiosis initiation, we investigated the transcriptional profiles of 19 363 single germ cells collected from E12.5, E14.5, and E16.5 mouse fetal ovaries. Clustering analysis identified seven groups and defined dozens of corresponding transcription factors, providing a global view of cellular differentiation from primordial germ cells toward meiocytes. Furthermore, we explored the dynamics of gene expression within the developmental trajectory with special focus on the critical state of meiosis. We found that meiosis initiation occurs as early as E12.5 and the cluster of oogonia_4 is the critical state between mitosis and meiosis. Our data provide key insights into the transcriptome features of peri-meiotic female germ cells, which offers new information not only on meiosis initiation and progression but also on screening pathogenic mutations in meiosis-associated diseases.
Assuntos
Meiose , Oogênese , Oogônios/citologia , Ovário/citologia , Transcriptoma , Animais , Diferenciação Celular , Feminino , Regulação da Expressão Gênica no Desenvolvimento , Camundongos , Camundongos Endogâmicos C57BL , Mitose , Análise de Sequência de RNA , Análise de Célula ÚnicaRESUMO
Oocyte activation deficiency leads to female infertility. [Ca2+ ]i oscillations are required for mitochondrial energy supplement transition from the resting to the excited state, but the underlying mechanisms are still very little known. Three mitochondrial Ca2+ channels, Mitochondria Calcium Uniporter (MCU), Na+ /Ca2+ Exchanger (NCLX) and Voltage-dependent Ca2+ Channel (VDAC), were deactivated by inhibitors RU360, CGP37157 and Erastin, respectively. Both Erastin and CGP37157 inhibited mitochondrial activity significantly while attenuating [Ca2+ ]i and [Ca2+ ]m oscillations, which caused developmental block of pronuclear formation. Thus, NCLX and VDAC are two mitochondria-associated Ca2+ transporter proteins regulating oocyte activation, which may be used as potential targets to treat female infertility. SIGNIFICANCE OF THE STUDY: NCLX and VDAC are two mitochondria-associated Ca2+ transporter proteins regulating oocyte activation.
Assuntos
Canais de Cálcio/metabolismo , Cálcio/metabolismo , Oócitos/metabolismo , Animais , Canais de Cálcio/química , Feminino , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Camundongos , Camundongos Endogâmicos ICR , Mitocôndrias/metabolismo , Oócitos/citologia , Oócitos/efeitos dos fármacos , Compostos de Rutênio/farmacologia , Rutênio Vermelho/farmacologia , Trocador de Sódio e Cálcio/antagonistas & inibidores , Trocador de Sódio e Cálcio/metabolismo , Tiazepinas/farmacologia , Canais de Ânion Dependentes de Voltagem/antagonistas & inibidores , Canais de Ânion Dependentes de Voltagem/metabolismoRESUMO
CxxC-finger protein 1 (CFP1)-mediated trimethylated histone H3 at lysine-4 (H3K4me3) during oocyte development enables the oocyte genome to establish the competence to generate a new organism. Nevertheless, it remains unclear to which extent this epigenetic modification forms an instructive component of ovarian follicle development. We investigated the ovarian functions using an oocyte-specific Cxxc1 knockout mouse model, in which the H3K4me3 accumulation is downregulated in oocytes of developing follicles. CFP1-dependent H3K4 trimethylation in oocytes was necessary to maintain the expression of key paracrine factors and to facilitate the communication between an oocyte and the surrounding granulosa cells. The distinct gene expression patterns in cumulus cells within preovulatory follicles were disrupted by the Cxxc1 deletion in oocytes. Both follicle growth and ovulation were compromised after CFP1 deletion, because Cxxc1 deletion in oocytes indirectly impaired essential signaling pathways in granulosa cells that mediate the functions of follicle-stimulating hormone and luteinizing hormone. Therefore, CFP1-regulated epigenetic modification of the oocyte genome influences the responses of ovarian follicles to gonadotropin in a cell-nonautonomous manner.
Assuntos
Histonas/metabolismo , Oócitos/metabolismo , Folículo Ovariano/metabolismo , Transativadores/metabolismo , Animais , Células do Cúmulo/metabolismo , Feminino , Hormônio Foliculoestimulante/metabolismo , Células da Granulosa/citologia , Células da Granulosa/metabolismo , Hormônio Luteinizante/metabolismo , Metilação , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Folículo Ovariano/crescimento & desenvolvimento , Ovulação , Comunicação Parácrina , Fosfatidilinositol 3-Quinases/química , Fosfatidilinositol 3-Quinases/metabolismo , Transdução de Sinais , Transativadores/deficiência , Transativadores/genéticaRESUMO
Meiotic maturation of mammalian oocytes depends on the temporally and spatially regulated cytoplasmic polyadenylation and translational activation of maternal mRNAs. Cytoplasmic polyadenylation is controlled by cis-elements in the 3'-UTRs of mRNAs including the polyadenylation signal (PAS), which is bound by the cleavage and polyadenylation specificity factor (CPSF) and the cytoplasmic polyadenylation element (CPE), which recruits CPE binding proteins. Using the 3'-UTRs of mouse Cpeb1, Btg4 and Cnot6l mRNAs, we deciphered the combinatorial code that controls developmental stage-specific translation during meiotic maturation: (i) translation of a maternal transcript at the germinal vesicle (GV) stage requires one or more PASs that locate far away from CPEs; (ii) PASs distal and proximal to the 3'-end of the transcripts are equally effective in mediating translation at the GV stage, as long as they are not close to the CPEs; (iii) Both translational repression at the GV stage and activation after germinal vesicle breakdown require at least one CPE adjacent to the PAS; (iv) The numbers and positions of CPEs in relation to PASs within the 3'-UTR of a given transcript determines its repression efficiency in GV oocytes. This study reveals a previously unrecognized non-canonical mechanism by which the proximal PASs mediate 3'-terminal polyadenylation and translation of maternal transcripts.
Assuntos
Proteínas de Ciclo Celular/genética , Oócitos/crescimento & desenvolvimento , Biossíntese de Proteínas , Ribonucleases/genética , Fatores de Transcrição/genética , Fatores de Poliadenilação e Clivagem de mRNA/genética , Regiões 3' não Traduzidas/genética , Animais , Citoplasma/genética , Feminino , Células Germinativas/crescimento & desenvolvimento , Meiose/genética , Camundongos , Oócitos/metabolismo , Oogênese/genética , Poliadenilação/genética , RNA Mensageiro/genéticaRESUMO
Insufficiency of oocyte activation impairs the subsequent embryo development in assisted reproductive technology (ART). Intracellular Ca2+ concentration ([Ca2+]i) oscillations switch the oocytes to resume the second meiosis and initiate embryonic development. However, the [Ca2+]i oscillation patterns in oocytes are poorly characterized. In this study, we investigated the effects of various factors, such as the oocytes age, pH, cumulus cells, in vitro or in vivo maturation, and ER stress on [Ca2+]i oscillation patterns and pronuclear formation after parthenogenetic activation of mouse oocytes. Our results showed that the oocytes released to the oviduct at 17 h post-human chorionic gonadotrophin (hCG) displayed a significantly stronger [Ca2+]i oscillation, including higher frequency, shorter cycle, and higher peak, compared with oocytes collected at earlier or later time points. [Ca2+]i oscillations in acidic conditions (pH 6.4 and 6.6) were significantly weaker than those in neutral and mildly alkaline conditions (pH from 6.8 to 7.6). In vitro-matured oocytes showed reduced frequency and peak of [Ca2+]i oscillations compared with those matured in vivo. In vitro-matured oocytes from the cumulus-oocyte complexes (COCs) showed a significantly higher frequency, shorter cycle, and higher peak compared with the denuded oocytes (DOs). Finally, endoplasmic reticulum stress (ER stress) severely affected the parameters of [Ca2+]i oscillations, including elongated cycles and lower frequency. The pronuclear (PN) rate of oocytes after parthenogenetic activation was correlated with [Ca2+]i oscillation pattern, decreasing with oocyte aging, cumulus removal, acidic pH, and increasing ER stress. These results provide fundamental but critical information for the mechanism of how these factors affect oocyte activation.
Assuntos
Desenvolvimento Embrionário/genética , Estresse do Retículo Endoplasmático/genética , Técnicas de Maturação in Vitro de Oócitos , Oócitos/crescimento & desenvolvimento , Animais , Gonadotropina Coriônica/genética , Células do Cúmulo/metabolismo , Feminino , Meiose/genética , Camundongos , Partenogênese/genética , GravidezRESUMO
During oocyte meiosis, mitochondria usually surround spindle to meet the energy demand of spindle migration and chromosome segregation. Therefore, the mitochondrion surrounding spindle is widely accepted as an important indicator to demonstrate the mitochondrial function in oocyte studies. However, the role of mitochondria surrounding spindle in oocyte quality is not exactly addressed. Mitofusin-2 (MFN2) is a mitochondrial outer membrane GTPase that mediates mitochondrial clustering and fusion. Here, we increased the mitochondria surrounding spindle by overexpression of MFN2 in mouse oocytes. Results indicate that the increase of mitochondria surrounding spindle has little effect on germinal vesicle breakdown (GVBD), spindle migration, mitochondrial membrane potential (MMP), reactive oxygen species (ROS) production and Endoplasmic reticulum (ER) distribution, while blocks chromosome segregation, destroys the spindle, and finally causes most of the oocytes to arrest at metaphase I stage. Collectively, our results demonstrate the mitochondria surrounding spindle is precisely regulated during oocyte maturation, while too much of it may cause abnormal oocyte meiosis. Therefore, although mitochondrion surrounding spindle is a typical biological event during oocyte maturation, utilizing it to demonstrate the mitochondrial function and oocyte quality should be much careful.
Assuntos
Metáfase , Mitocôndrias/metabolismo , Oócitos/citologia , Fuso Acromático/metabolismo , Animais , Células Cultivadas , Feminino , GTP Fosfo-Hidrolases/genética , GTP Fosfo-Hidrolases/metabolismo , Células HeLa , Humanos , Potencial da Membrana Mitocondrial , Camundongos , Camundongos Endogâmicos ICR , Mitocôndrias/genética , Oócitos/metabolismo , Oogênese , Espécies Reativas de Oxigênio/metabolismo , Fuso Acromático/genética , Regulação para CimaRESUMO
During follicle growth, DNA methylation is gradually established, which is important for oocyte developmental competence. Due to the facts that oocytes from prepubertal individuals show reduced developmental outcomes when compared to those from sexually mature individuals, and the fact that oocytes derived from in vitro follicle culture have much lower developmental competence, it is worth exploring whether prepubertal superovulation and in vitro follicle culture will cause changes in DNA methylation imprinting status in oocytes. In this study, we found that the CpG island in maternally imprinted GNAS clusters was hypermethylated in the MII-stage oocytes from sexually mature mice, but was hypomethylated in oocytes from prepuberty individuals. The GNAS clusters in the MII-stage oocytes obtained by in vitro follicle culture showed heterogeneous methylation levels, indicating different qualities of oocytes, however, three other maternally imprinted genes, Peg1, Lot1 and Impact, were all hypermethylated in the MII-stage oocytes derived from both prepubertal superovulation and in vitro follicle culture. Taken together, the findings suggest that the methylation status in GNAS clusters may potentially represent a novel epigenetic marker for oocyte quality detection.
Assuntos
Ilhas de CpG , Metilação de DNA , Impressão Genômica , Oócitos/metabolismo , Fatores Etários , Animais , Biomarcadores , Células Cultivadas , Feminino , Camundongos , Folículo Ovariano/citologiaRESUMO
Before fertilization, ovulated mammalian oocytes are arrested at the metaphase of second meiosis (MII), which is maintained by the so-called cytostatic factor (CSF). It is well known that the continuous synthesis and accumulation of cyclin B is critical for maintaining the CSF-mediated MII arrest. Recent studies by us and others have shown that Ccnb3 is required for the metaphase-to-anaphase transition during the first meiosis of mouse oocytes, but whether Ccnb3 plays a role in MII arrest and exit remains unknown. Here, we showed that the protein level of Ccnb3 gradually decreased during oocyte meiotic maturation, and exogenous expression of Ccnb3 led to release of MII arrest, degradation of securin, separation of sister chromatids, extrusion of the second polar body (PB2), and finally entry into interphase. These phenotypes could be rescued by inhibition of Wee1B or CDK2. Our results indicate that Ccnb3 plays a critical regulatory role in MII arrest and exit in mouse oocytes.
Assuntos
Ciclina B/metabolismo , Meiose/genética , Oócitos/citologia , Oócitos/metabolismo , Animais , Células Cultivadas , Ciclina B/genética , Feminino , Metáfase/genética , Camundongos , Camundongos Endogâmicos ICR , RNA Mensageiro/genética , RNA Mensageiro/metabolismoRESUMO
The genome methylation is globally erased in early fetal germ cells, and it is gradually re-established during gametogenesis. The expression of some imprinted genes is regulated by the methylation status of CpG islands, while the exact time of DNA methylation establishment near maternal imprinted genes during oocyte growth is not well known. Here, growing oocytes were divided into three groups based on follicle diameters including the S-group (60-100 µm), M-group (100-140 µm), and L-group (140-180 µm). The fully grown germinal vesicle (GV)-stage and metaphase II (M2)-stage mature oocytes were also collected. These oocytes were used for single-cell bisulfite sequencing to detect the methylation status of CpG islands near imprinted genes on chromosome 7. The results showed that the CpG islands near Ndn, Magel2, Mkrn3, Peg12, and Igf2 were completely unmethylated, but those of Peg3, Snrpn, and Kcnq1ot1 were hypermethylated in MII-stage oocytes. The methylation of CpG islands near different maternal imprinted genes occurred asynchronously, being completed in later-stage growing oocytes, fully grown GV oocytes, and mature MII-stage oocytes, respectively. These results show that CpG islands near some maternally imprinted genes are not necessarily methylated, and that the establishment of methylation of other maternally imprinted genes is completed at different stages of oocyte growth, providing a novel understanding of the establishment of maternally imprinted genes in oocytes.