Mouse Oocyte Maturation Research Articles

Gossypol exposure induces mitochondrial dysfunction and oxidative stress during mouse oocyte in vitro maturation

Gossypol is a yellow natural polyphenolic compound extracted from the seeds, leaves, stems, and flower buds of the cotton plant. Several studies have shown that exposure to gossypol impacts reproductive health in both humans and animals. However, whether gossypol exposure would influence oocyte quality has not yet been determined. Here, we studied the effects of gossypol on the meiotic maturation of mouse oocytes in vitro. The results revealed that gossypol exposure did not affect germinal vesicle breakdown (GVBD) but significantly reduced polar body extrusion (PBE) rates. Moreover, we observed meiotic spindle organization and chromosome alignment were entirely disturbed after gossypol exposure. Further, gossypol exposure also caused mitochondrial dysfunction and abruptly decreased the levels of cellular ATP, and diminished the mitochondrial membrane potential (MMP). Accordingly, gossypol-induced oxidative stress was confirmed through an increased level of reactive oxygen species (ROS). Early apoptosis incidence also increased as identified by positive Annexin-V signaling. Collectively, the above findings provide evidence that gossypol exposure impaired oocyte meiotic maturation, disturbed spindle structure and chromosome dynamics, disrupted mitochondrial function, induced oxidative stress, and triggered early apoptosis. These findings emphasize gossypol's adverse effects on oocyte maturation and thus on female fertility.

PSPC1 regulates CHK1 phosphorylation through phase separation and participates in mouse oocyte maturation.

Liquid-liquid phase separation (LLPS) underlies the formation of membraneless compartments in mammal cells. However, there are few reports that focus on the correlation of mouse oocyte maturation with LLPS. Previous studies have reported that paraspeckle component 1 (PSPC1) is related to the occurrence and development of tumors, but whether PSPC1 functions in mouse oocyte maturation is still unclear. Sequence analysis of PSPC1 protein showed that it contains a prion-like domain (PrLD) that is required for phase separation of proteins. In this study, we found that PSPC1 could undergo phase separation. Moreover, the loss of PrLD domain of PSPC1 could greatly weaken its phase separation ability. The immunofluorescence assays showed that PSPC1 is present in mouse oocytes in the germinal vesicle (GV) stage. Knockdown of PSPC1 significantly impeded the maturation of mouse oocytes in vitro. CHK1 has been reported to play important roles in the GV stage of mouse oocytes. Co-IP experiment revealed that PSPC1 could interact with phosphatase serine/threonine-protein phosphatase 5 (PPP5C), which regulates CHK1 phosphorylation. Western blot analysis revealed that PSPC1 could regulate the phosphorylation of CHK1 through PPP5C; however, PSPC1 without PrLD domain was inactive, suggesting that the lack of phase separation ability led to the abnormal function of PSPC1 in regulating CHK1 phosphorylation. Thus, we conclude that PSPC1 may undergo phase separation to regulate the phosphorylation level of CHK1 via PPP5C and participate in mouse oocyte maturation. Our study provides new insights into the mechanism of mouse oocyte maturation.

MicroRNA-21 is involved in oocyte maturation, blastocyst formation, and pre-implantation embryo development

Follicular fluid is one source of microRNAs (miRNAs). These miRNAs originate from oocytes and their neighboring cells. The changes in the miRNAs profile in the follicular fluid could alter folliculogenesis and oocyte maturation, and lead to infertility. Polycystic ovary syndrome (PCOS) patients have increased miR-21 levels in their sera, granulosa cells, and follicular fluid, and this mi-RNA plays a role in the pathophysiology and follicular dysfunction of PCOS patients. In the current study, we intend to examine whether expression levels of miR-21 influence oocyte maturation and embryo development. We examined miR-21 over-expression and down-regulation of miR-21 by miR-off 21 during in vitro maturation (IVM) to assess its influence on oocyte maturation and embryo development in mice. Over-expression of miR-21 in cumulus cells decreased expansion, meiotic progression, Glutathione-S-transferase GSH levels, and decreased expressions of Bmpr2 and Ptx3 genes. Subsequently, we noted that in vitro fertilization, and the cleavage rate and blastocyst formation significantly increased in cumulus oocyte complexes (COCs) that over-expressed miR-21. Inhibition of miR-21 by miR-off 21 led to increased cumulus expansion and GSH levels, along with decreased cleavage rate and blastocyst formation by alterations in Cdk2ap1 and Oct4 gene expressions. However, oocyte progression from the germinal vesicle (GV) to the metaphase II (MII) stage was not significant. miR-21 altered the gene expression levels in cumulus cells and influenced cytoplasmic oocyte maturation, cumulus expansion, and subsequent embryonic development in mice.

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 < 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 < 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 < 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 < 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

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.

RAB14 GTPase is essential for actin-based asymmetric division during mouse oocyte maturation.

ObjectivesRAB14 is a member of small GTPase RAB family which localizes at the endoplasmic reticulum (ER), Golgi apparatus and endosomal compartments. RAB14 acts as molecular switches that shift between a GDP‐bound inactive state and a GTP‐bound active state and regulates circulation of vesicles between the Golgi and endosomal compartments. In present study, we investigated the roles of RAB14 during oocyte meiotic maturation.Materials and methodsMicroinjection with siRNA and exogenous mRNA for knock down and rescue, and immunofluorescence staining, Western blot and real‐time RT‐PCR were utilized for the study.ResultsOur results showed that RAB14 localized in the cytoplasm and accumulated at the cortex during mouse oocyte maturation, and it was also enriched at the spindle periphery. Depletion of RAB14 did not affect polar body extrusion but caused large polar bodies, indicating the failure of asymmetric division. We found that absence of RAB14 did not affect spindle organization but caused the spindle migration defects, and this might be due to the regulation on cytoplasmic actin assembly via the ROCK‐cofilin signalling pathway. We also found that RAB14 depletion led to aberrant Golgi apparatus distribution. Exogenous Myc‐Rab14 mRNA supplement could significantly rescue these defects caused by Rab14 siRNA injection.ConclusionsTaken together, our results suggest that RAB14 affects ROCK‐cofilin pathway for actin‐based spindle migration and Golgi apparatus distribution during mouse oocyte meiotic maturation.

Perfluorononanoic acid impedes mouse oocyte maturation by inducing mitochondrial dysfunction and oxidative stress

Perfluorononanoic acid (PFNA), a member of PFAS, is frequently detected in human blood and tissues, even in follicular fluid of women. The exposure of PFNA, but not PFOA and PFOS, is positively correlated with miscarriage and increased time to pregnancy. Toxicological studies indicated that PFNA exposure is associated with immunotoxicity, hepatotoxicity, developmental toxicity, and reproductive toxicity in animals. However, there is little information regarding the toxic effects of PFNA on oocyte maturation. In this study, we investigated the toxic effects of PFNA exposure on mouse oocyte maturation in vitro. Our results showed that 600 μM PFNA significantly inhibited germinal vesicle breakdown (GVBD) and polar body extrusion (PBE) in mouse oocytes. Our further study revealed that PFNA induced abnormal metaphase I (MI) spindle assembly, evidenced by malformed spindles and mislocalization of p-ERK1/2 in PFNA-treated oocytes. We also found that PFNA induced abnormal mitochondrial distribution and increased mitochondrial membrane potential. Consequently, PFNA increased reactive oxygen species (ROS) levels, leading to oxidative stress, DNA damage, and eventually early-stage apoptosis in oocytes. In addition, after 14 h culture, PFNA disrupted the formation of metaphase II (MII) spindle in most PFNA-treated oocytes with polar bodies. Collectively, our results indicate that PFNA interferes with oocyte maturation in vitro via disrupting spindle assembly, damaging mitochondrial functions, and inducing oxidative stress, DNA damage, and early-stage apoptosis.

Ral GTPase is essential for actin dynamics and Golgi apparatus distribution in mouse oocyte maturation

BackgroundRal family is a member of Ras-like GTPase superfamily, which includes RalA and RalB. RalA/B play important roles in many cell biological functions, including cytoskeleton dynamics, cell division, membrane transport, gene expression and signal transduction. However, whether RalA/B involve into the mammalian oocyte meiosis is still unclear. This study aimed to explore the roles of RalA/B during mouse oocyte maturation.ResultsOur results showed that RalA/B expressed at all stages of oocyte maturation, and they were enriched at the spindle periphery area after meiosis resumption. The injection of RalA/B siRNAs into the oocytes significantly disturbed the polar body extrusion, indicating the essential roles of RalA/B for oocyte maturation. We observed that in the RalA/B knockdown oocytes the actin filament fluorescence intensity was significantly increased at the both cortex and cytoplasm, and the chromosomes were failed to locate near the cortex, indicating that RalA/B regulate actin dynamics for spindle migration in mouse oocytes. Moreover, we also found that the Golgi apparatus distribution at the spindle periphery was disturbed after RalA/B depletion.ConclusionsIn summary, our results indicated that RalA/B affect actin dynamics for chromosome positioning and Golgi apparatus distribution in mouse oocytes.

Single-cell RNA sequencing identifies molecular targets associated with poor in vitro maturation performance of oocytes collected from ovarian stimulation.

What is the transcriptome signature associated with poor performance of rescue IVM (rIVM) oocytes and how can we rejuvenate them? The GATA-1/CREB1/WNT signalling axis was repressed in rIVM oocytes, particularly those of poor quality; restoration of this axis may produce more usable rIVM oocytes. rIVM aims to produce mature oocytes (MII) for IVF through IVM of immature oocytes collected from stimulated ovaries. It is not popular due to limited success rate in infertility treatment. Genetic aberrations, cellular stress and the absence of cumulus cell support in oocytes could account for the failure of rIVM. We applied single-cell RNA sequencing (scRNA-seq) to capture the transcriptomes of human in vivo oocytes (IVO) (n = 10) from 7 donors and rIVM oocytes (n = 10) from 10 donors. The effects of maternal age and ovarian responses on rIVM oocyte transcriptomes were also studied. In parallel, we studied the effect of gallic acid on the maturation rate of mouse oocytes cultured in IVM medium with (n = 84) and without (n = 85) gallic acid. Human oocytes were collected from donors aged 28-41 years with a body mass index of <30. RNA extraction, cDNA generation, library construction and sequencing were performed in one preparation. scRNA-seq data were then processed and analysed. Selected genes in the rIVM versus IVO comparison were validated by quantitative real-time PCR. For the gallic acid study, we collected immature oocytes from 5-month-old mice and studied the effect of 10-μM gallic acid on their maturation rate. The transcriptome profiles of rIVM/IVO oocytes showed distinctive differences. A total of 1559 differentially expressed genes (DEGs, genes with at least 2-fold change and adjusted P < 0.05) were found to be enriched in metabolic processes, biosynthesis and oxidative phosphorylation. Among these DEGs, we identified a repression of WNT/β-catenin signalling in rIVM when compared with IVO oocytes. We found that oestradiol levels exhibited a significant age-independent correlation with the IVO mature oocyte ratio (MII ratio) for each donor. rIVM oocytes from women with a high MII ratio were found to have over-represented cellular processes such as anti-apoptosis. To further identify targets that contribute to the poor clinical outcomes of rIVM, we compared oocytes collected from young donors with a high MII ratio with oocytes from donors of advanced maternal age and lower MII ratio, and revealed that CREB1 is an important regulator. Thus, our study identified that GATA-1/CREB1/WNT signalling was repressed in both rIVM oocytes versus IVO oocytes and in rIVM oocytes of lower versus higher quality. Consequently we investigated gallic acid, as a potential antioxidant substrate in human rIVM medium, and found that it increased the mouse oocyte maturation rate by 31.1%. Raw data from this study can be accessed through GSE158539. In the rIVM oocytes of the high- and low-quality comparison, the number of samples was limited after data filtering with stringent selection criteria. For the oocyte stage identification, we were unable to predict the presence of oocyte spindle, so polar body extrusion was the only indicator. This study showed that GATA-1/CREB1/WNT signalling was repressed in rIVM oocytes compared with IVO oocytes and was further downregulated in low-quality rIVM oocytes, providing us the foundation of subsequent follow-up research on human oocytes and raising safety concerns about the clinical use of rescued oocytes. This work was supported by the Collaborative Research Fund, Research Grants Council, C4054-16G, and Research Committee Funding (Research Sustainability of Major RGC Funding Schemes), The Chinese University of Hong Kong. The authors have no conflicts of interest to declare.

Oocyte meiosis-coupled poly(A) polymerase α phosphorylation and activation trigger maternal mRNA translation in mice.

Mammalian oocyte maturation is driven by strictly regulated polyadenylation and translational activation of maternal mRNA stored in the cytoplasm. However, the poly(A) polymerase (PAP) that directly mediates cytoplasmic polyadenylation in mammalian oocytes has not been determined. In this study, we identified PAPα as the elusive enzyme that catalyzes cytoplasmic mRNA polyadenylation implicated in mouse oocyte maturation. PAPα was mainly localized in the germinal vesicle (GV) of fully grown oocytes but was distributed to the ooplasm after GV breakdown. Inhibition of PAPα activity impaired cytoplasmic polyadenylation and translation of maternal transcripts, thus blocking meiotic cell cycle progression. Once an oocyte resumes meiosis, activated CDK1 and ERK1/2 cooperatively mediate the phosphorylation of three serine residues of PAPα, 537, 545 and 558, thereby leading to increased activity. This mechanism is responsible for translational activation of transcripts lacking cytoplasmic polyadenylation elements in their 3′-untranslated region (3′-UTR). In turn, activated PAPα stimulated polyadenylation and translation of the mRNA encoding its own (Papola) through a positive feedback circuit. ERK1/2 promoted Papola mRNA translation in a 3′-UTR polyadenylation signal-dependent manner. Through these mechanisms, PAPα activity and levels were significantly amplified, improving the levels of global mRNA polyadenylation and translation, thus, benefiting meiotic cell cycle progression.

Ribonucleic Acid Export 1 Is a Kinetochore-Associated Protein That Participates in Chromosome Alignment in Mouse Oocytes.

Ribonucleic acid export 1 (Rae1) is an important nucleoporin that participates in mRNA export during the interphase of higher eukaryotes and regulates the mitotic cell cycle. In this study, small RNA interference technology was used to knockdown Rae1, and immunofluorescence, immunoblotting, and chromosome spreading were used to study the role of Rae1 in mouse oocyte meiotic maturation. We found that Rae1 is a crucial regulator of meiotic maturation of mouse oocytes. After the resumption of meiosis (GVBD), Rae1 was concentrated on the kinetochore structure. The knockdown of Rae1 by a specific siRNA inhibited GVBD progression at 2 h, finally leading to a decreased 14 h polar body extrusion (PBE) rate. However, a comparable 14 h PBE rate was found in the control, and the Rae1 knockdown groups that had already undergone GVBD. Furthermore, we found elevated PBE after 9.5 h in the Rae1 knockdown oocytes. Further analysis revealed that Rae1 depletion significantly decreased the protein level of securin. In addition, we detected weakened kinetochore–microtubule (K-MT) attachments, misaligned chromosomes, and an increased incidence of aneuploidy in the Rae1 knockdown oocytes. Collectively, we propose that Rae1 modulates securin protein levels, which contribute to chromosome alignment, K-MT attachments, and aneuploidy in meiosis.

Functional roles of the chromatin remodeler SMARCA5 in mouse and bovine preimplantation embryos†.

Upon fertilization, extensive chromatin reprogramming occurs during preimplantation development. Growing evidence reveals species-dependent regulations of this process in mammals. ATP-dependent chromatin remodeling factor SMARCA5 (also known as SNF2H) is required for peri-implantation development in mice. However, the specific functional role of SMARCA5 in preimplantation development and if it is conserved among species remain unclear. Herein, comparative analysis of public RNA-seq datasets reveals that SMARCA5 is universally expressed during oocyte maturation and preimplantation development in mice, cattle, humans, and pigs with species-specific patterns. Immunostaining analysis further describes the temporal and spatial changes of SMARCA5 in both mouse and bovine models. siRNA-mediated SMARCA5 depletion reduces the developmental capability and compromises the specification and differentiation of inner cell mass in mouse preimplantation embryos. Indeed, OCT4 is not restricted into the inner cell mass and the formation of epiblast and primitive endoderm disturbed with reduced NANOG and SOX17 in SMARCA5-deficient blastocysts. RNA-seq analysis shows SMARCA5 depletion causes limited effects on the transcriptomics at the morula stage, however, dysregulates 402 genes, including genes involved in transcription regulation and cell proliferation at the blastocyst stage in mice. By comparison, SMARCA5 depletion does not affect the development through the blastocyst stage but significantly compromises the blastocyst quality in cattle. Primitive endoderm formation is greatly disrupted with reduced GATA6 in bovine blastocysts. Overall, our studies demonstrate the importance of SMARCA5 in fostering the preimplantation development in mice and cattle while there are species-specific effects.

Bisphenol A Exposure Disrupts Organelle Distribution and Functions During Mouse Oocyte Maturation.

Bisphenol A (BPA) is one of the ubiquitous environmental endocrine disruptors (EEDs). Previous studies have shown that the reproduction toxicity of BPA could cause severe effects on the mammal oocytes and disturb the quality of mature oocytes. However, the toxic effects of BPA on the organelles of mouse oocytes have not been reported. In this study, to investigate whether BPA can be toxic to the organelles, we used different concentrations of BPA (50, 100, and 200 μM) to culture mouse oocytes in vitro. The results showed that 100 μM BPA exposure could significantly decrease the developmental capacity of oocytes. Then, we used the immunofluorescence staining, confocal microscopy, and western blotting to investigate the toxic effects of BPA on the organelles. The results revealed that mitochondrial dysfunction is manifested by abnormal distribution and decreased mitochondrial membrane potential. Moreover, the endoplasmic reticulum (ER) is abnormally distributed which is accompanied by ER stress showing increased expression of GRP78. For the Golgi apparatus, BPA-exposed dose not disorder the Golgi apparatus distribution but caused abnormal structure of Golgi apparatus, which is manifested by the decrease of GM130 protein expression. Moreover, we also found that BPA-exposed led to the damage of lysosome, which were shown by the increase of LAMP2 protein expression. Collectively, our findings demonstrated that the exposure of BPA could damage the normal function of the organelles, which may explain the reduced maturation quality of oocytes.

Highly successful production of viable mice derived from vitrified germinal vesicle oocytes.

The vitrification of immature germinal vesicle (GV) oocytes is an important way to preserve genetic resources and female fertility. However, it is well known that cryopreserved GV oocytes have very poor developmental ability and that further improvement in this technique is needed. We previously reported the successful vitrification of matured mouse oocytes with enclosed cumulus cells using the calcium-free vitrification solution supplemented with ethylene glycol (EG) by the minimal volume cooling (MVC) method. In this study, we investigated whether our method is applicable to the vitrification of mouse oocytes at the GV stage (GV oocytes). Following maturation and fertilization in vitro, vitrified GV oocytes showed high survival (94.3 ± 2.0%) and maturation (94.3 ± 2.1%) rates. Although the fertilization and blastocyst rates of vitrified oocytes (fertilization: 46.6 ± 4.9% and blastocyst: 46.6 ± 3.0%) were significantly lower than those of fresh oocytes (fertilization: 73.0 ± 7.1% and blastocyst: 71.6 ± 8.0%) (P < 0.01), there were no differences in the ability to develop to term between fresh oocytes (50.0 ± 8.4%) and vitrified oocytes (37.5 ± 4.6%) (P > 0.05). In conclusion, we here show, for the first time, the efficient production of live mice derived from vitrified GV oocytes.

Increased Environment-Related Metabolism and Genetic Expression in the In Vitro Matured Mouse Oocytes by Transcriptome Analysis.

Infertility in humans at their reproductive age is a world-wide problem. Oocyte in vitro maturation (IVM) is generally used in such cases to acquire the embryo in assisted reproductive technology (ART). However, the differences between an in vivo (IVO) and IVM culture environment in the RNA expression profile in oocytes, remains unclear. In this study, we compared the global RNA transcription pattern of oocytes from in vitro and in vivo maturation. Our results showed that 1,864 genes differentially expressed between the IVO and IVM oocytes. Among these, 1,638 genes were up-regulated, and 226 genes were down-regulated, and these changes were mainly divided into environmental adaption, metabolism, and genetic expression. Our detailed analysis showed that the expression of genes that belonged to metabolism-related processes such as energy metabolism, nucleotide metabolism, and carbohydrate metabolism was changed; and these genes also belonged to organismal systems including environmental adaptation and the circulatory system; moreover, we also found that the relative gene expression of genetic expression processes, such as protein synthesis, modification, and DNA replication and repair were also altered. In conclusion, our data suggests that in vitro maturation of mouse oocyte resulted in metabolism and genetic expression changes due to environmental changes compared with in vivo matured oocytes.

Melatonin rescues the reproductive toxicity of low-dose glyphosate-based herbicide during mouse oocyte maturation via the GPER signaling pathway.

Glyphosate-based herbicides (GBHs) are a group of widely used broad-spectrum agricultural pesticides. Due to the recalcitrance of GBH, it has been found in food and environment as a contaminant, posing a threat to public health. The health risks associated with GBH have been indicated by reporting acute toxicity data (an acute exposure of GBH at a 0.5% dose), which primarily discuss toxicity in relation to accidental high-rate exposure. Currently, there is little information regarding the toxicity of GBH at environmentally relevant levels. In this study, we used mature mouse oocytes to study the toxic effects of low-dose GBH exposure in vitro (0.00001%-0.00025%) and in vivo (0.0005%, orally administered through daily drinking water) during meiotic maturation. GBH exposure led to meiotic maturation failure with spindle defects and chromosome misalignment. In addition, GBH treatment severely reduced sperm-binding ability and disrupted early embryo cleavage. Moreover, GBH exposure significantly increased the reactive oxygen species (ROS) levels and apoptotic rates. Evidence indicates that such effects in GBH-exposed oocytes are likely due to overexpression of the G-protein estrogen receptor (GPER/GPR30). Remarkably, we found that melatonin administration elicited significant protection against GBH-induced oocyte deterioration via preserving the expression of GPR30, along with activation of its downstream signaling event (pERK/ERK). Taken together, these results revealed that low-dose glyphosate has a certain adverse effect on oocyte maturation and early embryo cleavage, and highlight the protective roles of melatonin.

Paraquat Reduces the Female Fertility by Impairing the Oocyte Maturation in Mice.

Paraquat (PQ) is a widely used non-selective and oxidizing herbicide in farmland, orchards, flower nursery, and grassland. Overuse of PQ will accumulate in the body and affect the reproduction in mammals. In this study, we found that PQ could reduce the female fertility by oral administration for 21 days in mice. PQ exposure could impair the nuclear maturation by perturbing the spindle assembly and kinetochore–microtubule attachment to cause the misaligned chromosomes during meiosis. In the meantime, PQ exposure disturbed the mitochondrial distribution and enhanced the level of reactive oxygen species and early apoptosis, which thereby deteriorated the early embryo development. Also, PQ administration could cause some changes in epigenetic modifications such as the level of H3K9me2 and H3K27me3. Therefore, PQ administration reduces the female fertility by impairing the nuclear and cytoplasmic maturation of oocytes in mice.

RBM14 Modulates Tubulin Acetylation and Regulates Spindle Morphology During Meiotic Maturation in Mouse Oocytes.

RBM14 is an RNA-binding protein that regulates spindle integrity in mitosis; however, its functions during meiosis are still unclear. In this study, we discovered that RBM14 expression was down-regulated in oocytes from old mice. The RBM14 distribution at different stages of meiosis was explored, while it presents overlapped localization patterns with α-tubulin in MI- and MII-stage oocytes. Treatment of MI-stage oocytes with spindle-perturbing agents revealed that RBM14 was co-localized with microtubules. RBM14 knockdown with RBM14-specific morpholino showed that RBM14-depleted oocytes underwent symmetric division compared to the controls. RBM14 knockdown also resulted in spindle defects and chromosome abnormalities during oocyte maturation, presumably due to α-tubulin hyperacetylation. Co-immunoprecipitation analysis demonstrated that RBM14 is interacted with endogenous α-tubulin in mammalian cells. These findings indicate that RBM14 is an essential modulator of oocyte meiotic maturation by regulating α-tubulin acetylation to affect spindle morphology and chromosome alignment. Consequently, RBM14 represents a potential biomarker of oocyte quality and a novel therapeutic target in women with oocyte maturation failure.

Fbf1 regulates mouse oocyte meiosis by influencing Plk1

Fas binding factor 1 (Fbf1) is one of the distal appendage proteins in the centriole, located at its distal and proximal ends. It influences the duplication and separation of centrosomes, thereby affecting the progression of the cell cycle during mitosis. However, the function of Fbf1 in meiosis has remained unclear. To explore the role of Fbf1 in the in vitro maturation of mouse oocyte, immunofluorescence staining was used to examine the Fbf1 location in the oocyte and their phenotype after protein deletion. Western blot was used to examine the protein abundance. This study showed that mouse oocytes express Fbf1 which locates at the spindle poles and around the microtubules. Through taxol and nocodazole treatment, and microinjection of siRNA, it was demonstrated that Fbf1 had an important role in the spindle assembly and chromosome separation during mouse oocyte meiosis In particular, microinjection of Fbf1-siRNA resulted in severe abnormalities in the spindle and chromosome arrangement, decreased aggregation of microtubules, disrupted the first oocyte meiosis, and the extrusion of the first polar body. Furthermore, in the Fbf1-siRNA group, there was reduced expression of Plk1 and its agglutination at the spindle poles, along with retarded chromosome segregation due to the activation of the spindle assembly checkpoint (SAC) component BubR1. These results indicate that Fbf1 may function in microtubule depolymerization and agglutination, control the microtubule dynamics, spindle assembly and chromosome arrangement and, thus, influence the mouse oocyte meiotic maturation.