Rate Of Germinal Vesicle Breakdown Research Articles

Epigen enhances the developmental potential of in vitro fertilized embryos by improving cytoplasmic maturation

Numerous growth factors contribute to oocyte maturation and embryonic development in vivo; however, only a few are understood. One such factor is epigen, a new member of the epidermal growth factor (EGF) family that is secreted by the granulosa cells of immature oocytes. We hypothesized that epigen may play a role in oocyte maturation, specifically in the nuclear and cytoplasmic aspects. This study aimed to investigate the effects of epigen on porcine oocyte maturation and embryo development in vitro. In this study, three different concentrations of epigen (3, 6, and 30 ng/mL) were added to tissue culture medium-199 (TCM-199) during in vitro maturation of porcine oocytes. A control group that did not receive epigen supplementation was also included. Mature porcine oocytes were fertilized, and the resulting zygotes were cultured until day 7. The levels of intracellular glutathione (GSH) and reactive oxygen species (ROS) were measured in the in vitro matured oocytes. At the same time, the expression patterns of genes related to apoptosis were detected in day 7 blastocysts (BLs) using real-time quantitative PCR Apoptosis was detected by annexin-V assays in mature oocytes. Data were analyzed using ANOVA and Duncan's test on SPSS, and results are presented as mean ± SEM. The group that received 6 ng/mL epigen had a significantly lower rate of germinal vesicle breakdown (GVBD) than the control group without affecting the nuclear maturation among the experimental groups. Among the treatment groups, the 6 ng/mL epigen group showed significantly higher levels of intracellular GSH and lower ROS production. Supplementation with 6 ng/mL epigen significantly improved blastocyst (BL) formation rates compared to those in the control and 3 ng/mL groups. Additionally, the blastocyst expansion rate was significantly higher with epigen supplementation (6 ng/mL). In the fertilization experiment, the group supplemented with 6 ng/mL epigen exhibited significantly higher levels of monospermy and fertilization efficiency and lower levels of polyspermy than the control group. This study indicated that adding epigen at a concentration of 6 ng/mL can significantly enhance the developmental potential of porcine oocytes fertilized in vitro. Specifically, the study found that epigen improves cytoplasmic maturation, which helps prevent polyspermy and emulates monospermic penetration.

Focusing on the accumulation of chromatin/chromosomes around nucleoli and optimizing the timing of ICSI to facilitate the rescue in vitro maturation of denuded GV stage oocytes.

This study aims to achieve the methodological improvement of rescue IVM by predicting germinal vesicle breakdown (GVBD) and optimizing the timing of ICSI. Time lapse analysis was performed retrospectively to evaluated the relationship between the presence of AC around the nucleoli and GVBD. To find the optimal timing of ICSI, the time from the initiation of the first polar body extrusion to ICSI were measured, and the rates of fertilization at each point were calculated. The GVBD rate of GV stage oocytes with AC around the nucleoli was significantly higher than that of GV stage oocytes without AC. The GV stageoocytes required more time for nuclear maturation after polar body extrusion than MI oocytes, with GV stageoocytes taking 400-600min from polar body extrusion to the optimal timing of ICSI, while the MI stageoocytes took 200-400min. The GV stage oocytes resulted in the birth of healthy babies with the appropriate timing of ICSI. It was found that GV stageoocytes with AC around nucleoli can initiate GVBD and reach the MII stage with a high rate, and that GV stage oocytes required more time than MI stageoocytes to reach the optimal timing of ICSI. Considering these factors, ART laboratories may employ immature GV stageoocytes in routine ART procedures rather than discarding them.

HDAC6 decreases H4K16 and α-tubulin acetylation during porcine oocyte maturation

ABSTRACT HDAC6 is an essential factor in mouse oocyte maturation. However, the roles of HDAC6 in porcine oocyte maturation are still unclear. Therefore, we analyzed the roles of HDAC6 in porcine oocyte maturation by treatment with Tubastatin A (TubA) which is an HDAC6 inhibitor. Our results showed that treatment with 10 μg/ml TubA significantly decreased the rate of porcine oocyte maturation, but it did not influence the rate of germinal vesicle breakdown (GVBD). Then, we found that TubA treatment disrupted spindle organization by increasing the α-tubulin acetylation level during porcine oocyte maturation. Moreover, TubA treatment significantly increased H4K16 acetylation, which may compromise kinetochore and microtubule (K-MT) attachment during meiosis in porcine oocytes. We also analyzed the effects of TubA on meiosis-related (H3T3pho and H3S10pho) and transcription-related histone modifications (H3K4me3, H3K9me3 and H3K4ac) during porcine oocyte maturation. The results showed that TubA treatment increased H3S10pho and H3K4ac levels, but no influence was seen in H3T3pho, H3K4me3 and H3K9me3 levels in porcine oocytes. TubA treated oocytes also showed a compromised ability to develop after parthenogenetic activation. Finally, we found that HDAC6 exhibited higher mRNA levels and lower DNA methylation levels in porcine oocytes than it did in porcine embryonic fibroblasts (PEFs). These results indicate that the low level of DNA methylation in HDAC6 promoter ensures high expression. HDAC6 regulates the deacetylation of α-tubulin and H4K16, which promotes correct spindle organization and meiotic apparatus assembly during porcine oocyte maturation. This study illustrates a new pathway by which HDAC6 modulates mammalian oocyte maturation.

P-650 Follicular extracellular vesicles of older women inhibit oocyte maturation

Abstract Study question Whether follicular extracellular vesicles of older women interfere with the quality of oocytes. Summary answer OLD-EVs induced the oxidative stress in the oocytes and inhibited oocyte maturation by increasing the abnormal mitochondria distribution and abnormal spindle rates. What is known already Ovary is a critical regulator of female fertility and the source of oocytes. With the modern tendency of delaying childbearing, ovarian aging has become an age-related disease. Ovarian aging is generally characterized by a gradual decrease in both the quantity and quality of oocytes. The pregnancy outcomes of older women were far from satisfying with higher abortion rates, birth defects rates, and lower live birth rates even after the assistance of IVF. However, the detailed mechanisms of decreased oocyte quality of aging women have not been clarified. Study design, size, duration Extracellular vesicles (EVs) were isolated from follicular fluid of older women (aged 40-50, n = 7, OLD-EVs) and younger women (aged 25-30, n = 6, YOUNG-EVs) via ultracentrifugation. Germinal vesicle (GV) oocytes from female ICR mice were co-cultured with OLD-EVs, YOUNG-EVs, or PBS (blank control), respectively. GV breakdown (GVBD) rate and maturation rate were calculated at two-hour and fourteen-hour of co-culture, respectively. Besides, oocyte mitochondria distribution, meiosis spindle morphology, and oxidative status were assessed in different groups. Participants/materials, setting, methods EVs were determined by western blotting, nanoparticle tracking analysis, and transmission electron microscopy. Fluorescence labeled EVs were used to visualize internalization by oocytes. Oocytes mitochondria and mitosis spindles were stained with fluorescence, and abnormal mitochondria rate or abnormal spindle rate was calculated. Reactive oxygen species (ROS) level was detected in the differently treated oocytes. Moreover, the expression of CAT, GSS, and SOD was determined in the oocytes using quantitative reverse transcription polymerase chain reaction. Main results and the role of chance Both OLD-EVs and YOUNG-EVs are bilayered vesicles, ranging from 100 to 150 nm and enriched in Alix, TSG101, and CD9. EVs could be internalized by oocytes within one hour. After coculture, GVBD rate was similar among the three groups; whereas maturation rate was significantly decreased in the OLD-EV group compared with YOUNG-EV group or PBS group. In addition, the abnormal mitochondria distribution rate or abnormal spindle rate were significantly increased in the OLD-EV group compared with PBS or YOUNG-EV group. The ROS level was increased in the PCOS-EV group compared with YOUNG-EV group, and the expression of CAT, GSS, and SOD was increased in the OLD-EV-treated oocytes. Limitations, reasons for caution Our study did not identify the contents of OLD-EVs and YOUNG-EVs, and the molecular mechanisms of dysregulations induced by OLD-EVs need further researches to investigate. Wider implications of the findings This work confirmed that EV-conducted cellular communication played an important role in oocyte maturation. The dysregulation of oocytes induced by OLD-EVs might be related to the poor oocyte quality of aging women, which provide a novel target to improve pregnancy outcomes of these patients. Trial registration number not applicable

Growth Hormone Promotes Oocyte Maturation In Vitro by Protecting Mitochondrial Function and Reducing Apoptosis.

Some studies have been conducted to explore the influence of growth hormone (GH) on oocytes in in vitro maturation (IVM); however, previous studies reporting showed different results, and the specific mechanisms were not clear. In the present study, GH supplementation improved oocyte maturation rate. The rate of germinal vesicle breakdown (GVBD) in the GH group was 83.9%, which was significantly higher than that (72.1%) in the control group (p = 0.001). The maturation rate of the GH group (79.2%) was significantly higher than that (65.4%) of the control group (p = 0.000). The fertilization (68.6 vs. 59.3%) and blastocyst (30 vs. 25.3%) rates showed an increasing trend in the GH group compared to those in controls. The dynamic parameters of nuclear maturation of oocytes were recorded by time-lapse monitoring system; oocytes in the GH group completed nuclear maturation earlier than did those in the control group. GH reduced cAMP levels to promote oocyte maturation. Single-cell RNA sequencing analysis revealed that the majority of differentially expressed genes (DEGs) involved in mitochondrial oxidative phosphorylation was upregulated in the GH group. Furthermore, the mitochondrial membrane potential of oocytes significantly increased, and the levels of intracellular reactive oxygen species (ROS) and Ca2+ largely decreased in the GH group. Finally, single-oocyte transcriptome analysis indicated that GH decreased the expression of apoptosis-related genes in oocytes. GH treatment reduced the expression of γH2AX and caspase-3. Therefore, GH improves the developmental potential of immature oocytes by reducing cAMP levels more rapidly within 0.5h, protecting mitochondrial function, and reducing DNA damage and apoptosis.

GVBD rate is an independent predictor for pregnancy in ICSI patients with surplus immature oocytes.

It was reported that there were still up to 30% immature retrieved oocyte at germinal vesicle (GV) or metaphase I (MI) stage. Whether the spontaneous maturity competency of immature oocytes associated to the clinical outcome of in vitro fertilization (IVF) cycles remains unclear and unexplored. This study aimed to investigate how the oocyte developmental parameters in in vitro maturation (IVM) affect clinical outcomes of intracytoplasmic sperm injection (ICSI) cycles. This retrospective cohort study included couples undergoing ICSI in a university-affiliated hospital. Surplus immature oocytes during ICSI were collected and cultured in vitro. The numbers of germinal vesicle (GV) oocytes undergoing GV breakdown (GVBD) and polar body 1 extrusion within 24 h culture were recorded. The main outcome measurements were demographic baselines and oocyte developmental parameters in IVM associated with pregnancy outcomes. A total of 191 couples were included with an overall GVBD rate of 63.7% (327/513) and oocyte maturation rate of 46.8% (240/513). 53.4% (102/191) of them had embryos transferred freshly, which originated from metaphase II oocytes that matured spontaneously in vivo, and 60.8% (62/102) got pregnant. Among factors with a P-value < 0.2 in univariate logistic regression analyses of pregnancy correlation, GVBD rate (OR 3.220, 95% CI 1.060-9.782, P=0.039) and progesterone level on human chorionic gonadotropin (HCG) day (OR 0.231, 95% CI 0.056-0.949, P=0.042) remained significant in the multivariate model. The area under the curve (AUC) of the predictive nomogram was 0.729 (95% CI 0.632-0.826) with an acceptable calibration. Moreover, decision curve analyses illustrated the superior overall net benefit of models that included the GVBD rate in clinical decisions within a wide range of threshold probabilities. In conclusion, GVBD rate and progesterone level on HCG day may be associated with pregnancy outcomes in infertile couples during the regular ICSI procedure. An elevated GVBD rate within 24 h may greatly increase the likelihood of pregnancy in infertile couples during ICSI. This preliminary study may optimize clinical pregnancy prediction, which provides support in decision-making in clinical practice.

Leptin modulates oocyte maturation by a central and a direct pathway in zebrafish.

Loss of LEPR function (LOF) in mammals leads to diverse phenotypes including morbid obesity and infertility while zebrafish show relatively minor phenotypes. This however allows the study of LEPR LOF in the absence of the detrimental effects of hyperglycemia or obesity. Here, we show evidence that leptin plays a role in the central as well as peripheral regulation of the hypothalamic-pituitary-gonadal (HPG) axis in zebrafish. Animals with a Lepr LOF show dysregulated pituitary HPG genes as well as evidence that oocytes mature slower and/or exhibit an increased rate of atresia. In culture, Lepr LOF attenuates the effect of 17α-20β-dihydroxy-4 pregnen-3-one in promoting germinal vesicle breakdown (GVBD) and increases the rate of GVBD as well as attenuates the rate of oocyte atresia.

MEIOK21 regulates oocyte quantity and quality via modulating meiotic recombination.

The reproductive life span of females is largely determined by the number and quality of oocytes. Previously, we identified MEIOK21 as a meiotic recombination regulator required for male fertility. Here, we characterize the important roles of MEIOK21 in regulating female meiosis and oocyte number and quality. MEIOK21 localizes at recombination sites as a component of recombination bridges in oogenesis like in spermatogenesis. Meiok21-/- female mice show subfertility. Consistently, the size of the primordial follicle pool in Meiok21-/- females is only ~40% of wild-type females because a great number of oocytes with defects in meiotic recombination and/or synapsis are eliminated. Furthermore, the numbers of primordial and growing follicles show a more marked decrease in an age-dependent manner compared with wild-type females. Further analysis shows Meiok21-/- oocytes also have reduced rates of germinal vesicle breakdown and the first polar body extrusion when cultured in vitro, indicating poor oocyte quality. Additionally, Meiok21-/- oocytes have more chromosomes bearing a single distally localized crossover (chiasmata), suggesting a possible defect in crossover maturation. Taken together, our findings indicate critical roles for MEIOK21 in ensuring the number and quality of oocytes in the follicles.

P–129 Follicular extracellular vesicles of women with polycystic ovarian syndrome inhibit oocyte maturation

Abstract Study question Does follicular extracellular vesicles of women with polycystic ovarian syndrome (PCOS-EVs) interfere with the quality of oocytes? Summary answer PCOS-EVs induced oxidative stress in the oocytes and inhibited oocyte maturation by increasing the abnormal mitochondria distribution and abnormal spindle rates. What is known already Polycystic ovarian syndrome (PCOS) is a common endocrine disorder in women of reproductive age, with a prevalence up to 10%. Women with PCOS are characterized by a clustering of features, including hyperandrogenism, polycystic ovarian morphology, and notably, anovulation. Although international guidelines recommend assisted reproduction techniques to be an effective resort for PCOS patients to conceive. However, even after overcoming ovulatory dysfunction via ovulation induction, the pregnancy outcomes of patients with PCOS were far from satisfying with lower fertilization, cleavage, and implantation rates, implicating that theoocyte quality of these patients are affected. Whereas the mechanisms have not been elucidated yet. Study design, size, duration Follicular fluid of PCOS patients (n = 10) and healthy controls (n = 10) were collected and used for extracellular vesicles (EVs) isolation via ultracentrifugation. Germinal vesicle (GV) oocytes collected from female ICR mice were cocultured with RIF-EVs or FER-EVs, respectively, and PBS served as a blank control. GV breakdown (GVBD) rate and maturation rate were calculated at two-hour and fourteen-hour of co-culture, respectively. Besides, oocyte mitochondria distribution, meiosis spindle morphology, and oxidative status were assessed in different groups. Participants/materials, setting, methods EVs were determined by western blotting, nanoparticle tracking analysis, and transmission electron microscopy. Fluorescence labeled EVs were used to visualize internalization by oocytes. Oocytes mitochondria and mitosis spindles were stained with fluorescence, and abnormal mitochondria rate or abnormal spindle rate was calculated. Reactive oxygen species (ROS) level was detected in the differently treated oocytes. Moreover, the expression of CAT, GSS, and SOD was determined in the oocytes using quantitative reverse transcription polymerase chain reaction. Main results and the role of chance Both PCOS-EVs and CTRL-EVs are bilayered vesicles, ranging from 100 to 150 nm, and enriched in Alix, TSG101, and CD9. EVs could be internalized by oocytes within one hour. After coculture, the GVBD rate was similar among the three groups; whereas the maturation rate was significantly decreased in the PCOS-EV group compared with CTRL-EV group or PBS group. In addition, the abnormal mitochondria distribution rate or abnormal spindle rate were significantly increased in the PCOS-EV group compared with PBS or CTRL-EV group. The ROS level was increased in the PCOS-EV group compared with CTRL-EV group, and the expression of CAT, GSS, and SOD was increased in the PCOS-EV-treated oocytes. Limitations, reasons for caution Our study did not identify the contents of PCOS-EVs and CTRL-EVs, and the molecular mechanisms of dysregulations induced by PCOS-EVs need further researches to investigate. Wider implications of the findings: This work confirmed that EV-conducted cellular communication played an important role in oocyte development in women with PCOS. The dysregulation of oocytes induced by PCOS-EVs might be related to the poor oocyte quality of women with PCOS, which may provide a novel target to improve pregnancy outcomes of these patients. Trial registration number Not applicable

P–556 NAT10-mediated N4-acetylcytidine in RNA regulates mouse oocyte maturation in vitro

Abstract Study question Does NAT10-mediated N4-acetylcytidine (ac4C) in RNA, a newly identified mRNA epigenetic modification, participate in modulating in vitro maturation(IVM) of oocytes? Summary answer NAT10-mediated ac4C modification is an important regulatory factor during oocyte maturation in vitro, by regulating genes associated with translation, mitochondrial functions and protein destabilization. What is known already Unlike somatic cells, transcription and translation are uncoupled during oocyte maturation and gene expression is mainly regulated by post-transcriptional modulation, including mRNA degradation, translation and posttranslational modification, which are complex and have not been fully investigated. RNA ac4C is a newly identified mRNA modification and a key determinant of post-transcriptional regulation, which has been shown to promote mRNA stability and translation, and NAT10 is the only known RNA acetyltransferase. Therefore, NAT10-mediated ac4C represents a possible epigenetic regulator in oocyte maturation. Study design, size, duration Oocytes at different stages from mice were collected to detect the changing levels of ac4C and NAT10 during maturation. NAT10 in GV-stage oocytes was knocked down before IVM, to confirm the regulatory role of NAT10-mediated ac4C in meiotic process, followed by further exploration of cellular mechanisms. Each experiment was repeated at least three times, and data were analyzed by chi-square test, one-way ANOVA or unpaired-sample t-test. Participants/materials, setting, methods The expression of ac4C and NAT10 was detected by immunohistochemistry. NAT10 was knocked down in GV-stage oocytes by RNA interference through electroporation. The efficacy of knockdown was confirmed by qPCR and immunohistochemistry targeting ac4C and NAT10, and the percentages of oocytes maturated in vitro were compared among groups. High-throughput sequencing and RNA immunoprecipitation were performed to reveal the modulated genes. Proteins specifically binding to ac4C sites were identified by RNA pulldown and mass spectrometry. Main results and the role of chance We first retrieved publicly available data from GEO and found that transcripts with potential ac4C sites were enriched in genes downregulated during IVM (P &amp;lt; 0.001). The biased distribution of ac4C implicated a possible regulatory role. Then immunohistochemistry revealed significantly decreasing trends of ac4C and NAT10 expression from immature to mature oocytes. With NAT10 knockdown, ac4C modification was reduced and meiotic progression was significantly retarded. Specifically, the rate of first body extrusion was significantly decreased with NAT10 knockdown (34.6%) compared to control oocytes without transfection (74.6%) and oocytes transfected with control siRNA (72.6%) (p &amp;lt; 0.001), while rates of germinal vesicle breakdown were not affected (P = 0.6531). High-throughput sequencing and RNA immunoprecipitation revealed that the modulated genes were enriched in biological processes known to be associated with oocyte maturation, including translation, mitochondrial translational elongation and termination, and protein destabilization. Also, we identified a series of proteins specifically binding to ac4C probes by RNA pulldown and mass spectrometry, through which ac4C modification may exert its function in post-transcriptional modulation. Limitations, reasons for caution This study was performed in vitro. The role of NAT10-mediated ac4C in vivo remains to be elucidated. Also, limited by current techniques, ac4C modification in oocytes cannot be detected. Our exploration of regulated genes and ac4C binding proteins were performed in somatic cell lines. Wider implications of the findings: Post-transcriptional modulation is crucial in oocyte maturation. Our study using in-vitro systems for mouse oocyte identified NAT10-mediated ac4C as an important regulator in IVM. It provided a new insight into the epigenetic mechanisms of IVM, which may lead to improvement of clinical IVM systems. Trial registration number Not applicable

P-556 Pre-selected for an award: NAT10-mediated N4-acetylcytidine in RNA regulates mouse oocyte maturation in vitro

Abstract Study question Does NAT10-mediated N4-acetylcytidine (ac4C) in RNA, a newly identified mRNA epigenetic modification, participate in modulating in vitro maturation(IVM) of oocytes? Summary answer NAT10-mediated ac4C modification is an important regulatory factor during oocyte maturation in vitro, by regulating genes associated with translation, mitochondrial functions and protein destabilization. What is known already Unlike somatic cells, transcription and translation are uncoupled during oocyte maturation and gene expression is mainly regulated by post-transcriptional modulation, including mRNA degradation, translation and posttranslational modification, which are complex and have not been fully investigated. RNA ac4C is a newly identified mRNA modification and a key determinant of post-transcriptional regulation, which has been shown to promote mRNA stability and translation, and NAT10 is the only known RNA acetyltransferase. Therefore, NAT10-mediated ac4C represents a possible epigenetic regulator in oocyte maturation. Study design, size, duration Oocytes at different stages from mice were collected to detect the changing levels of ac4C and NAT10 during maturation. NAT10 in GV-stage oocytes was knocked down before IVM, to confirm the regulatory role of NAT10-mediated ac4C in meiotic process, followed by further exploration of cellular mechanisms. Each experiment was repeated at least three times, and data were analyzed by chi-square test, one-way ANOVA or unpaired-sample t-test. Participants/materials, setting, methods The expression of ac4C and NAT10 was detected by immunohistochemistry. NAT10 was knocked down in GV-stage oocytes by RNA interference through electroporation. The efficacy of knockdown was confirmed by qPCR and immunohistochemistry targeting ac4C and NAT10, and the percentages of oocytes maturated in vitro were compared among groups. High-throughput sequencing and RNA immunoprecipitation were performed to reveal the modulated genes. Proteins specifically binding to ac4C sites were identified by RNA pulldown and mass spectrometry. Main results and the role of chance We first retrieved publicly available data from GEO and found that transcripts with potential ac4C sites were enriched in genes downregulated during IVM (P &amp;lt; 0.001). The biased distribution of ac4C implicated a possible regulatory role. Then immunohistochemistry revealed significantly decreasing trends of ac4C and NAT10 expression from immature to mature oocytes. With NAT10 knockdown, ac4C modification was reduced and meiotic progression was significantly retarded. Specifically, the rate of first body extrusion was significantly decreased with NAT10 knockdown (34.6%) compared to control oocytes without transfection (74.6%) and oocytes transfected with control siRNA (72.6%) (p &amp;lt; 0.001), while rates of germinal vesicle breakdown were not affected (P = 0.6531). High-throughput sequencing and RNA immunoprecipitation revealed that the modulated genes were enriched in biological processes known to be associated with oocyte maturation, including translation, mitochondrial translational elongation and termination, and protein destabilization. Also, we identified a series of proteins specifically binding to ac4C probes by RNA pulldown and mass spectrometry, through which ac4C modification may exert its function in post-transcriptional modulation. Limitations, reasons for caution This study was performed in vitro. The role of NAT10-mediated ac4C in vivo remains to be elucidated. Also, limited by current techniques, ac4C modification in oocytes cannot be detected. Our exploration of regulated genes and ac4C binding proteins were performed in somatic cell lines. Wider implications of the findings Post-transcriptional modulation is crucial in oocyte maturation. Our study using in-vitro systems for mouse oocyte identified NAT10-mediated ac4C as an important regulator in IVM. It provided a new insight into the epigenetic mechanisms of IVM, which may lead to improvement of clinical IVM systems. Trial registration number not applicable

P–269 Effect of well-of-the-well culture as in vitro maturation system for human oocytes

Abstract Study question Can the Well-of-the-Well system (WOW), applied on denuded oocytes, improve germinal vesicle breakdown (GVBD) and maturation rate? Summary answer In vitro maturation (IVM) of denuded germinal vesicle (GV) oocyte using WOW culture system increases nuclear maturation competence when compared with droplet conventional culture What is known already Further research remains necessary to address the mechanism of oocyte maturation in order to refine culture conditions and improve the implantation rate of in vitro matured oocytes. Several studies on bovine oocytes have shown that oocyte-secreted factors (an uncharacterized mix of growth factors secreted by the oocyte) enhance oocyte developmental competence during in vitro maturation. These oocyte-secreted factors may accumulate at the bottom of the micro-well, as suggested for the WOW culture system. Previous reports suggested that diffusible factors secreted by individual oocytes probably accumulated in a micro-well WOW dish, may provide a suitable microenvironment for their in vitro maturation. Study design, size, duration A total of 879 GV collected between 2017 and 2019 were included in this study. They were randomly allocated into two experimental groups: (1) single-cultured oocytes (SC) that were cultured individually in micro-droplets, and (2) group-cultured oocytes (WOW) that were cultured in a microwell culture system using the WOW dish (culture dish for time lapse incubator). The nuclear maturation was assessed after 24 hours and 48 hours of IVM Participants/materials, setting, methods GV oocytes were obtained from 609 patients undergoing controlled ovarian stimulation cycles. Oocytes from the experimental group (1) were placed individually in conventional 25μl micro-droplets in a 35 mm dish. Oocytes from the experimental group (2) were placed in 80 μl droplet individually in each of 9 microwells of WOW dish. All GV oocytes were matured in a single step embryo culture medium, supplemented with human menopausal gonadotropin and synthetic serum substitute. Main results and the role of chance Mature oocyte (MII) was considered when we observed rupture of the GV and the presence of a first polar body in the perivitelline space during the first 24 or 48 hours of culture under inverted optical microscope. GVBD noted significant differences (p-valor = 0.000) between the study groups after culturing of 24 hours [GVBD: SC group; 70% (318/455) vs. WOW group; 83% (352/424)] and 48 hours [GVBD: SC group; 77% (319/416) vs. WOW group; 94% (398/424)]. The maturation rates (MR) showed significant differences (p-valor = 0.000) between the study groups after culturing of 24 hours [MR: SC group; 51% (233/455) vs. WOW group; 80% (338/424)] and 48 hours [MR: SC group; 71% (295/416) vs. WOW group; 91% (387/424)]. Limitations, reasons for caution There is no data on cleavage and blastocyst rates. There are no previous reports comparing the maturation rates in denuded human oocytes single-cultured in individually droplet or group-cultured in WOW dish. Wider implications of the findings: Our results must be taken into account in order to improve the culture conditions for the optimization of the in vitro maturation technique in human oocytes from stimulated cycles. We now provide evidence that group-cultured oocytes in WOW dish increase GVBD and maturation rates. Trial registration number Not applicable

NAT10-Mediated N4-Acetylcytidine of RNA Contributes to Post-transcriptional Regulation of Mouse Oocyte Maturation in vitro.

N4-acetylcytidine (ac4C), a newly identified epigenetic modification within mRNA, has been characterized as a crucial regulator of mRNA stability and translation efficiency. However, the role of ac4C during oocyte maturation, the process mainly controlled via post-transcriptional mechanisms, has not been explored. N-acetyltransferase 10 (NAT10) is the only known enzyme responsible for ac4C production in mammals and ac4C-binding proteins have not been reported yet. In this study, we have documented decreasing trends of both ac4C and NAT10 expression from immature to mature mouse oocytes. With NAT10 knockdown mediated by small interfering RNA (siRNA) in germinal vesicle (GV)-stage oocytes, ac4C modification was reduced and meiotic maturation in vitro was significantly retarded. Specifically, the rate of first polar body extrusion was significantly decreased with NAT10 knockdown (34.6%) compared to control oocytes without transfection (74.6%) and oocytes transfected with negative control siRNA (72.6%) (p < 0.001), while rates of germinal vesicle breakdown (GVBD) were not significantly different (p = 0.6531). RNA immunoprecipitation and high-throughput sequencing using HEK293T cells revealed that the modulated genes were enriched in biological processes associated with nucleosome assembly, chromatin silencing, chromatin modification and cytoskeletal anchoring. In addition, we identified TBL3 as a potential ac4C-binding protein by a bioinformatics algorithm and RNA pulldown with HEK293T cells, which may mediate downstream cellular activities. Taken together, our results suggest that NAT10-mediated ac4C modification is an important regulatory factor during oocyte maturation in vitro and TBL3 is a potential ac4C-binding protein.

Melatonin Alleviates the Suppressive Effect of Hypoxanthine on Oocyte Nuclear Maturation and Restores Meiosis via the Melatonin Receptor 1 (MT1)-Mediated Pathway.

It is well known that hypoxanthine (HX) inhibits nuclear maturation of oocytes by elevating the intracellular cAMP level, while melatonin (MT) is a molecule that reduces cAMP production, which may physiologically antagonize this inhibition and restore the meiosis process. We conducted in vitro and in vivo studies to examine this hypothesis. The results showed that 10–3 M MT potentiated the inhibitory effect of HX on mouse oocyte meiosis by lowering the rate of germinal vesicle breakdown (GVBD) and the first polar body (PB1). However, 10–5 M and 10–7 M MT significantly alleviated the nuclear suppression induced by HX and restored meiosis in 3- and 6-week-old mouse oocytes, respectively. We identified that the rate-limiting melatonin synthetic enzyme AANAT and melatonin membrane receptor MT1 were both expressed in oocytes and cumulus cells at the GV and MII stages. Luzindole, a non-selective melatonin membrane receptor antagonist, blocked the activity of MT on oocyte meiotic recovery (P < 0.05). This observation indicated that the activity of melatonin was mediated by the MT1 receptor. To understand the molecular mechanism further, MT1 knockout (KO) mice were constructed. In this MT1 KO animal model, the PB1 rate was significantly reduced with the excessive expression of cAPM synthases (Adcy2, Adcy6, Adcy7, and Adcy9) in the ovaries of these animals. The mRNA levels of Nppc and Npr2 were upregulated while the genes related to progesterone synthesis (Cyp11a11), cholesterol biosynthesis (Insig1), and feedback (Lhcgr, Prlr, and Atg7) were downregulated in the granulosa cells of MT1 KO mice (P < 0.05). The altered gene expression may be attributed to the suppression of oocyte maturation. In summary, melatonin protects against nuclear inhibition caused by HX and restores oocyte meiosis via MT1 by reducing the intracellular concentration of cAMP.

Gefitinib reduces oocyte quality by disturbing meiotic progression

Gefitinib is a first-line anti-cancer drug for the treatment of advanced non-small cell lung cancer (NSCLC). It has been reported that gefitinib can generate several drug-related adverse effects, including nausea, peripheral edema, decreased appetite and rash. However, the reproductive toxicity of gefitinib has not been clearly defined until now. Here we assessed the effects of gefitinib on oocyte quality by examining the critical events and molecular changes of oocyte maturation. Gefitinib at 1, 2, 5 or 10 μM concentration was added to culture medium (M2). We found that gefitinib at its median peak concentration of 1 μM did not affect oocyte maturation, but 5 μM gefitinib severely blocked oocyte meiotic progression as indicated by decreased rates of germinal vesicle breakdown (GVBD) and polar body extrusion (PBE). We further showed that gefitinib treatment increased phosphorylation of CDK1 at the site of Try15, inhibited cyclin B1 entry into the nucleus, and disrupted normal spindle assembly, chromosome alignment and mitochondria dynamics, finally leading to the generation of aneuploidy and early apoptosis of oocytes. Our study reported here provides valuable evidence for reproductive toxicity of gefitinib administration employed for the treatment of cancer patients.

Immunization with CENP-C Causes Aberrant Chromosome Segregation during Oocyte Meiosis in Mice.

Anticentromere antibodies (ACA) were associated with lower oocyte maturation rates and cleavage rates, while the mechanism was not clear. Aims of this study were to examine whether active immunization with centromere protein C could elicit the CENP-C autoantibody in mice and the impacts of the CENP-C autoantibody on oocyte meiosis. Mice were divided into two groups, one was the experimental group immunized with human centromere protein C and Freund's adjuvant (CFA), and the other was the control group injected with CFA only. Serum and oocytes of BALB/c mice immunized with human centromere protein C (CENP-C) in complete Freund's adjuvant (CFA) or injected with only CFA were studied for the development of the CENP-C antibody. Rates of germinal vesicle breakdown (GVBD), first polar body (Pb1) extrusion, abnormal spindle morphology, and chromosome misalignment were compared between the experimental group and the control group. The CENP-C antibody was only observed in serum and oocytes of mice immunized with the centromere protein C antigen. The first polar body (Pb1) extrusion rate was lower in the experimental group (P < 0.01). A higher percentage of spindle defects and chromosome congression failure were also detected in the experimental group (spindle defects: 64.67 ± 1.16% vs. 9.27 ± 2.28% control; chromosome misalignment: 50.80 ± 2.40% vs. 8.30 ± 1.16% control; P < 0.01 for both). Oocyte meiosis was severely impaired by the CENP-C antibody, which may be the main mechanism of adverse reproductive outcomes for ACA-positive women who have no clinical symptoms of any autoimmune diseases.

The toxic effects and possible mechanisms of decabromodiphenyl ethane on mouse oocyte

Decabromodiphenyl ethane (DBDPE), a widely used new brominated flame retardant, is added into flammable materials to achieve fire retardation. As it is continuously detected in the environment, it has become an emerging environmental pollutant. However, the effects of DBDPE exposure on oocyte maturation and its underlying mechanisms remain unknown. This study found that DBDPE exposure inhibited the rate of germinal vesicle breakdown (GVBD), first polar body extrusion (PBE) and fertilization of mouse oocytes. After 14 h of exposure to DBDPE, metaphase II (MII) oocytes showed that the hardness of zona pellucida (ZP) markedly increased and that the spindle morphology was abnormal. Moreover, DBDPE exposure induced abnormal mitochondrial distribution, mitochondrial dysfunction, and ATP deficiency. Simultaneously, DBDPE exposure down-regulated the expression of antioxidant-related genes (Sod2, Gpx1) and increased the level of reactive oxygen species (ROS) in oocytes. The results of immunofluorescence and qRT-PCR revealed that autophagy occurred in DBDPE-treated oocytes with high expression of autophagy-related protein (LC3) and genes (Lc3, Beclin1). Meanwhile, DBDPE significantly up-regulated the protein (Bax) and mRNA (Bax, Caspase3) levels of pro-apoptosis genes. However, the protein and mRNA expression of anti-apoptosis genes Bcl-2 was dramatically down-regulated in DBDPE-exposed oocytes. Collectively, DBDPE exposure impaired mitochondrial function, causing oxidative damage, autophagy and apoptosis in oocytes.

The toxic effects and possible mechanisms of glyphosate on mouse oocytes

Glyphosate is a high-efficiency, low-toxicity, broad-spectrum herbicide. The residues of glyphosate-based herbicides are frequent pollutants in the environment. However, the effects of glyphosate on oocyte maturation, as well as its possible mechanisms, remain unclear. The present study revealed that mouse oocytes had reduced rates of germinal vesicle breakdown (GVBD) and first polar body extrusion (PBE) after treatment with 500 μM glyphosate. Reactive oxygen species (ROS) were found in mouse oocytes exposed to glyphosate, as shown by changes in the mRNA expression of related antioxidant enzyme genes (cat, sod2, gpx). After 14 h of exposure to glyphosate, metaphase II (MII) mouse oocytes displayed an abnormal spindle morphology and DNA double-strand breaks (DNA-DSBs). Simultaneously, mitochondria showed an aggregated distribution and decreased membrane potential in mouse oocytes exposed to glyphosate. The protein expression levels of apoptosis factors (Bax, Bcl-2) and the mRNA expression levels of apoptosis-related genes (bax, bcl-2, caspase3) were measured by Western blot and qRT-PCR, respectively. Meanwhile, the expression levels of autophagy-related genes (lc3, atg14, mtor) and proteins (LC3, Atg12) were significantly decreased in the glyphosate treatment group compared with the control group. Collectively, our results indicated that glyphosate exposure could interfere with mouse oocyte maturation by generating oxidative stress and early apoptosis.

Serotonergic Mechanisms of Oocyte Germinal Vesicle Breakdown in the Mud Crab, Scylla paramamosain.

The mechanism of serotonin (5-HT)-induced oocyte germinal vesicle breakdown (GVBD) in the mud crab, Scylla paramamosain, was investigated in this study. Histological staining showed that there were two meiotic arrests in oocyte, appearing at prophase I and metaphase I. This result indicated that meiosis I arrest at prophase I in S. paramamosain was similar to that of vertebrates, but meiosis II arrest at metaphase I was different from that of vertebrates. Resumption of oocytes arrest at meiosis prophase I could be induced by 5-HT rapidly within 5 min in S. paramamosain. We obtained the sequence of the 5-HT receptor type 1A (5-HTR1A) from the NCBI database, and found that 5-HTR1A was expressed in oocytes and follicle cells. In addition, we found that an agonist 8-OH-DPAT which binds 5-HTR1A induced GVBD and an antagonist WAY100635 which inhibited 5-HT induced GVBD in S. paramamosain. This result showed that 5-HTR1A mediated the regulation of oocyte GVBD by 5-HT. To explore the functional mechanism of 5-HT in inducing oocyte GVBD, forskolin, a cAMP agonist was used. Results showed that, forskolin significantly blocked 5-HT-induced GVBD, and there was a negative correlation between GVBD rate and cAMP level. Our data indicate that there are two meiotic arrests in S. paramamosain, and the resumption of prophase I arrest can be induced by 5-HT, which binds to 5-HTR1A, and this process is mediated by cAMP, which acts as negative regulator via cAMP signaling pathway.

Expression and function of exportin 6 in full-grown and growing porcine oocytes.

Exportin 6, which functions specifically in the nuclear export of actin family proteins, has been reported to be absent in immature Xenopus oocytes, which have a huge nucleus containing a large amount of actin. In mammalian oocytes, however, the presence and the function of exportin 6 remain uninvestigated. In this study, we assessed the expression and effects of exportin 6 on meiotic resumption in porcine oocytes after cloning porcine exportin 6 cDNA and carrying out overexpression and expression inhibition by mRNA and antisense RNA injection, respectively. We found for the first time that exportin 6 was expressed in mammalian full-grown germinal-vesicle-stage oocytes and was involved in the nuclear export of actin. In contrast, exportin 6 was absent from the growing oocytes, which are meiotically incompetent and maintain the germinal-vesicle structure in the long term; the regulatory mechanism appeared to be active degradation. We examined the effects of exportin 6 on meiotic resumption of porcine oocytes and noted that its expression did not affect the onset time but increased the rate of germinal vesicle breakdown at 24 h via regulation of the nuclear actin level, which directly influences the physical strength of the germinal-vesicle membrane. Our results suggest that exportin 6 affects the nuclear transport of actin and meiotic resumption in mammalian oocytes.