Flavopiridol inhibits oocyte maturation, reduces oocyte quality and blocks cumulus cell function.

Flavopiridol (FP) is a plant-derived flavonoidis and used to treat cancers, fungal infections and inflammation-related diseases. However, it is not clear whether it has side effects on the female reproductive system. In this study, we aimed to investigate the toxic effects and potential underlying mechanisms of FP on oocyte maturation and cumulus cell expansion in mice. Cumulus-oocyte complexes (COCs) were cultured in vitro with FP of gradient concentration (50-1000nM), according to the plasma concentration of FP in the clinical trial. The maturation rate and cumulus expansion index of oocytes were counted and studied by immunofluorescence staining, qRT-PCR, oocyte chromosome preparation and so on. The results showed that the FP-exposed COCs inhibited the oocyte maturation and cumulus cell expansion, leading to cell apoptosis in a dose dependent way. Oocytes exposed to 500nM FP showed abnormalities in the spindle structure and chromosome arrangement, ultimately leading to the oocyte maturation arrest and aneuploidy. This may be due to the excessive oxidative stress caused by mitochondrial membrane potential damage and mislocalization. Therefore, when FP is used for cancer treatment, its side effects on the female reproductive system should be seriously considered.

CtIP regulates G2/M transition and bipolar spindle assembly during mouse oocyte meiosis.

CtBP-interacting protein (CtIP) is known for its multifaceted roles in DNA repair and genomic stability, directing the homologous recombination-mediated DNA double-stranded break (DSBs) repair pathway via DNA end resection, an essential error-free repair process vital for genome stability. Mammalian oocytes are highly prone to DNA damage accumulation due to prolonged G2/prophase arrest. Here, we explore the functions of CtIP in meiotic cell cycle regulation via a mouse oocyte model. Depletion of CtIP by siRNA injection results in delayed germinal vesicle breakdown and failed polar body extrusion. Mechanistically, CtIP deficiency increases DNA damage and decreases the expression and nuclear entry of CCNB1, resulting in marked impairment of meiotic resumption, which can be rescued by exogenous CCNB1 overexpression. Furthermore, depletion of CtIP disrupts MTOCs coalescence at spindle poles as indicated by failed accumulation of γ-tubulin, p-Aurora kinase A, Kif2A, and TPX2, leading to abnormal spindle assembly and prometaphase arrest. These results provide valuable insights into the important roles of CtIP in the G2/M checkpoint and spindle assembly in mouse oocyte meiotic cell cycle regulation.

Protein phosphatase 4 maintains the survival of primordial follicles by regulating autophagy in oocytes.

In mammalian ovary, the primordial follicle pool serves as the source of developing follicles and fertilizable ova. To maintain the normal length of female reproductive life, the primordial follicles must have adequate number and be kept in a quiescent state before menopause. However, the molecular mechanisms underlying primordial follicle survival are poorly understood. Here, we provide genetic evidence showing that lacking protein phosphatase 4 (PPP4) in oocytes, a member of PP2A-like subfamily, results in infertility in female mice. A large quantity of primordial follicles has been depleted around the primordial follicle pool formation phase and the ovarian reserve is exhausted at about 7 months old. Further investigation demonstrates that depletion of PPP4 causes the abnormal activation of mTOR, which suppresses autophagy in primordial follicle oocytes. The abnormal primordial follicle oocytes are eventually erased by pregranulosa cells in the manner of lysosome invading. These results show that autophagy prevents primordial follicles over loss and PPP4-mTOR pathway governs autophagy during the primordial follicle formation and dormant period.

MEIKIN expression and its C-terminal phosphorylation by PLK1 is closely related the metaphase-anaphase transition by affecting cyclin B1 and Securin stabilization in meiotic oocyte.

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.

P300 regulates histone crotonylation and preimplantation embryo development.

Histone lysine crotonylation, an evolutionarily conserved modification differing from acetylation, exerts pivotal control over diverse biological processes. Among these are gene transcriptional regulation, spermatogenesis, and cell cycle processes. However, the dynamic changes and functions of histone crotonylation in preimplantation embryonic development in mammals remain unclear. Here, we show that the transcription coactivator P300 functions as a writer of histone crotonylation during embryonic development. Depletion of P300 results in significant developmental defects and dysregulation of the transcriptome of embryos. Importantly, we demonstrate that P300 catalyzes the crotonylation of histone, directly stimulating transcription and regulating gene expression, thereby ensuring successful progression of embryo development up to the blastocyst stage. Moreover, the modification of histone H3 lysine 18 crotonylation (H3K18cr) is primarily localized to active promoter regions. This modification serves as a distinctive epigenetic indicator of crucial transcriptional regulators, facilitating the activation of gene transcription. Together, our results propose a model wherein P300-mediated histone crotonylation plays a crucial role in regulating the fate of embryonic development.

Vinorelbine administration impedes the timely progression of meiotic maturation and induces aneuploidy in mouse oocytes.

Vinorelbine is a commonly used drug to treat various malignancies, such as breast cancer, non-small cell lung cancer, and metastatic pleural mesothelioma. Its side effects include severe neutropenia, local phlebitis, gastrointestinal reactions, and neurotoxicity. In view of the scarcity of research on vinorelbine's reproductive toxicity, this study evaluated the impact of vinorelbine ditartrate, a commonly used form of vinorelbine, on oocyte maturation in vitro. Our investigation revealed that vinorelbine ditartrate had no effect on oocyte meiotic resumption. However, it did reduce the rate of first polar body extrusion, suggesting that it could significantly impede the meiotic maturation of oocytes. Vinorelbine ditartrate exposure was found to disturb the regular spindle assembly and chromosome alignment, leading to the continuous activation of the spindle assembly checkpoint (SAC) and a delayed activation of the anaphase-promoting complex/cyclosome (APC/C), ultimately causing aneuploidy in oocytes. Consequently, the administration of vinorelbine is likely to result in oocyte aneuploidy, which can be helpful in providing a drug reference and fertility guidance in a clinical context.

The transgenerational effects of maternal low-protein diet during lactation on offspring.

Environmental factors such as diet and lifestyle can influence the health of both mothers and offspring. However, its transgenerational transmission and underlying mechanisms remain largely unknown. Here, using a maternal lactation-period low-protein diet (LPD) mouse model, we show that maternal LPD during lactation causes decreased survival and stunted growth, significantly reduces ovulation and litter size, and alters the gut microbiome in the female LPD-F1 offspring. The transcriptome of LPD-F1 metaphase II (MII) oocytes shows that differentially expressed genes are enriched in female pregnancy and multiple metabolic processes. Moreover, maternal LPD causes early stunted growth and impairs metabolic health, which is transmitted over two generations. The methylome alteration of LPD-F1 oocytes can be partly transmitted to the F2 oocytes. Together, our results reveal that LPD during lactation transgenerationally affects offspring health, probably via oocyte epigenetic changes.

Loss of UBE2S causes meiosis I arrest with normal spindle assembly checkpoint dynamics in mouse oocytes.

The timely degradation of proteins that regulate the cell cycle is essential for oocyte maturation. Oocytes are equipped to degrade proteins via the ubiquitin-proteasome system. In meiosis, anaphase promoting complex/cyclosome (APC/C), an E3 ubiquitin-ligase, is responsible for the degradation of proteins. Ubiquitin-conjugating enzyme E2 S (UBE2S), an E2 ubiquitin-conjugating enzyme, delivers ubiquitin to APC/C. APC/C has been extensively studied, but the functions of UBE2S in oocyte maturation and mouse fertility are not clear. In this study, we used Ube2s knockout mice to explore the role of UBE2S in mouse oocytes. Ube2s-deleted oocytes were characterized by meiosis I arrest with normal spindle assembly and spindle assembly checkpoint dynamics. However, the absence of UBE2S affected the activity of APC/C. Cyclin B1 and securin are two substrates of APC/C, and their levels were consistently high, resulting in the failure of homologous chromosome separation. Unexpectedly, the oocytes arrested in meiosis I could be fertilized and the embryos could become implanted normally, but died before embryonic day 10.5. In conclusion, our findings reveal an indispensable regulatory role of UBE2S in mouse oocyte meiosis and female fertility.

RNF20 Regulates Oocyte Meiotic Spindle Assembly by Recruiting TPM3 to Centromeres and Spindle Poles.

Previously a ring finger protein 20 (RNF20) is found to be essential for meiotic recombination and mediates H2B ubiquitination during spermatogenesis. However, its role in meiotic division is still unknown. Here, it is shown that RNF20 is localized at both centromeres and spindle poles, and it is required for oocyte acentrosomal spindle organization and female fertility. RNF20-depleted oocytes exhibit severely abnormal spindle and chromosome misalignment caused by defective bipolar organization. Notably, it is found that the function of RNF20 in spindle assembly is not dependent on its E3 ligase activity. Instead, RNF20 regulates spindle assembly by recruiting tropomyosin3 (TPM3) to both centromeres and spindle poles with its coiled-coil motif. The RNF20-TPM3 interaction is essential for acentrosomal meiotic spindle assembly. Together, the studies uncover a novel function for RNF20 in mediating TPM3 recruitment to both centromeres and spindle poles during oocyte spindle assembly.

Maternal exposure to 4-vinylcyclohexene diepoxide during pregnancy leads to disorder of gut microbiota and bile acid metabolism in offspring.

Our previous study reveals that maternal exposure to 4-vinylcyclohexene diepoxide (VCD) during pregnancy causes insufficient ovarian follicle reserve and decreased fertility in offspring. The present study aims to further explore the reasons for the significant decline of fecundity in mice caused by VCD, and to clarify the changes of gut microbiota and microbial metabolites in F1 mice. The ovarian metabolomics, gut microbiota and microbial metabolites were analyzed. The results of ovarian metabolomics analysis showed that maternal VCD exposure during pregnancy significantly reduced the concentration of carnitine in the ovaries of F1 mice, while supplementation with carnitine (isovalerylcarnitine and valerylcarnitine) significantly increased the number of ovulation. The results of 16 S rDNA-seq and microbial metabolites analysis showed that maternal VCD exposure during pregnancy caused disordered gut microbiota, increased abundance of Parabacteroides and Flexispira bacteria that are involved in secondary bile acid synthesis. The concentrations of NorDCA, LCA-3S, DCA and other secondary bile acids increased significantly. Our results indicate that maternal exposure to VCD during pregnancy leads to disorder in gut microbiota and bile acid metabolism in F1 mice, accompanying with decreased ovarian function, providing further evidence that maternal exposure to VCD during pregnancy has intergenerational deleterious effects on offspring.

Cordycepin delays postovulatory aging of oocytes through inhibition of maternal mRNAs degradation via DCP1A polyadenylation suppression.

Postovulatory aging leads to the decline in oocyte quality and subsequent impairment of embryonic development, thereby reducing the success rate of assisted reproductive technology (ART). Potential preventative strategies preventing oocytes from aging and the associated underlying mechanisms warrant investigation. In this study, we identified that cordycepin, a natural nucleoside analogue, promoted the quality of oocytes aging in vitro, as indicated by reduced oocyte fragmentation, improved spindle/chromosomes morphology and mitochondrial function, as well as increased embryonic developmental competence. Proteomic and RNA sequencing analyses revealed that cordycepin inhibited the degradation of several crucial maternal proteins and mRNAs caused by aging. Strikingly, cordycepin was found to suppress the elevation of DCP1A protein by inhibiting polyadenylation during postovulatory aging, consequently impeding the decapping of maternal mRNAs. In humans, the increased degradation of DCP1A and total mRNA during postovulatory aging was also inhibited by cordycepin. Collectively, our findings demonstrate that cordycepin prevents postovulatory aging of mammalian oocytes by inhibition of maternal mRNAs degradation via suppressing polyadenylation of DCP1A mRNA, thereby promoting oocyte developmental competence.

SRSF1-mediated alternative splicing is required for spermatogenesis.

Alternative splicing (AS) plays significant roles in a multitude of fundamental biological activities. AS is prevalent in the testis, but the regulations of AS in spermatogenesis is only little explored. Here, we report that Serine/arginine-rich splicing factor 1 (SRSF1) plays critical roles in alternative splicing and male reproduction. Male germ cell-specific deletion of Srsf1 led to complete infertility by affecting spermatogenesis. Mechanistically, by combining RNA-seq data with LACE-seq data, we showed that SRSF1 affected the AS of Stra8 in a direct manner and Dazl, Dmc1, Mre11a, Syce2 and Rif1 in an indirect manner. Our findings demonstrate that SRSF1 has crucial functions in spermatogenesis and male fertility by regulating alternative splicing.

SRSF2 is required for mRNA splicing during spermatogenesis.

BACKGROUND: RNA splicing plays significant roles in fundamental biological activities. However, our knowledge about the roles of alternative splicing and underlying mechanisms during spermatogenesis is limited. RESULTS: Here, we report that Serine/arginine-rich splicing factor 2 (SRSF2), also known as SC35, plays critical roles in alternative splicing and male reproduction. Male germ cell-specific deletion of Srsf2 by Stra8-Cre caused complete infertility and defective spermatogenesis. Further analyses revealed that deletion of Srsf2 disrupted differentiation and meiosis initiation of spermatogonia. Mechanistically, by combining RNA-seq data with LACE-seq data, we showed that SRSF2 regulatory networks play critical roles in several major events including reproductive development, spermatogenesis, meiotic cell cycle, synapse organization, DNA recombination, chromosome segregation, and male sex differentiation. Furthermore, SRSF2 affected expression and alternative splicing of Stra8, Stag3 and Atr encoding critical factors for spermatogenesis in a direct manner. CONCLUSIONS: Taken together, our results demonstrate that SRSF2 has important functions in spermatogenesis and male fertility by regulating alternative splicing.

Gut microbiota-bile acid-vitamin D axis plays an important role in determining oocyte quality and embryonic development.

OBJECTIVE: To reveal whether gut microbiota and their metabolites are correlated with oocyte quality decline caused by circadian rhythm disruption, and to search possible approaches for improving oocyte quality. DESIGN: A mouse model exposed to continuous light was established. The oocyte quality, embryonic development, microbial metabolites and gut microbiota were analyzed. Intragastric administration of microbial metabolites was conducted to confirm the relationship between gut microbiota and oocyte quality and embryonic development. RESULTS: Firstly, we found that oocyte quality and embryonic development decreased in mice exposed to continuous light. Through metabolomics profiling and 16S rDNA-seq, we found that the intestinal absorption capacity of vitamin D was decreased due to significant decrease of bile acids such as lithocholic acid (LCA), which was significantly associated with increased abundance of Turicibacter. Subsequently, the concentrations of anti-Mullerian hormone (AMH) hormone in blood and melatonin in follicular fluid were reduced, which is the main reason for the decline of oocyte quality and early embryonic development, and this was rescued by injection of vitamin D3 (VD3). Secondly, melatonin rescued oocyte quality and embryonic development by increasing the concentration of lithocholic acid and reducing the concentration of oxidative stress metabolites in the intestine. Thirdly, we found six metabolites that could rescue oocyte quality and early embryonic development, among which LCA of 30 mg/kg and NorDCA of 15 mg/kg had the best rescue effect. CONCLUSION: These findings confirm the link between ovarian function and gut microbiota regulation by microbial metabolites and have potential value for improving ovary function.

A heterozygous ZP2 mutation causes zona pellucida defects and female infertility in mouse and human.

The zona pellucida (ZP) is an extracellular glycoprotein matrix surrounding mammalian oocytes. Recently, numerous mutations in genes encoding ZP proteins have been shown to be possibly related to oocyte abnormality and female infertility; few reports have confirmed the functions of these mutations in living animal models. Here, we identified a novel heterozygous missense mutation (NM_001376231.1:c.1616C>T, p.Thr539Met) in ZP2 from a primary infertile female. We showed that the mutation reduced ZP2 expression and impeded ZP2 secretion in cell lines. Furthermore, we constructed the mouse model with the mutation (Zp2T541M) using CRISPR-Cas9. Zp2WT/T541M female mice had normal fertility though generated oocytes with the thin ZP, whereas Zp2T541M female mice were completely infertile due to degeneration of oocytes without ZP. Additionally, ZP deletion impaired folliculogenesis and caused female infertility in Zp2T541M mice. Our study not only expands the spectrum of ZP2 mutation sites but also, more importantly, increases the understanding of pathogenic mechanisms of ZP2 mutations.

Maternal SMC2 is essential for embryonic development via participating chromosome condensation in mice.

During the oocyte growth, maturation and zygote development, chromatin structure keeps changing to regulate different nuclear activities. Here, we reported the role of SMC2, a core component of condensin complex, in oocyte and embryo development. Oocyte-specific conditional knockout of SMC2 caused female infertility. In the absence of SMC2, oocyte meiotic maturation and ovulation occurred normally, but chromosome condensation showed defects and DNA damages were accumulated in oocytes. The pronuclei were abnormally organized and micronuclei were frequently observed in fertilized eggs, their activity was impaired, and embryo development was arrested at the one-cell stage, suggesting that maternal SMC2 is essential for embryonic development.

LSM14B controls oocyte mRNA storage and stability to ensure female fertility.

Controlled mRNA storage and stability is essential for oocyte meiosis and early embryonic development. However, how to regulate mRNA storage and stability in mammalian oogenesis remains elusive. Here we showed that LSM14B, a component of membraneless compartments including P-body-like granules and mitochondria-associated ribonucleoprotein domain (MARDO) in germ cell, is indispensable for female fertility. To reveal loss of LSM14B disrupted primordial follicle assembly and caused mRNA reduction in non-growing oocytes, which was concomitant with the impaired assembly of P-body-like granules. 10× Genomics single-cell RNA-sequencing and immunostaining were performed. Meanwhile, we conducted RNA-seq analysis of GV-stage oocytes and found that Lsm14b deficiency not only impaired the maternal mRNA accumulation but also disrupted the translation in fully grown oocytes, which was closely associated with dissolution of MARDO components. Moreover, Lsm14b-deficient oocytes reassembled a pronucleus containing decondensed chromatin after extrusion of the first polar body, through compromising the activation of maturation promoting factor, while the defects were restored via WEE1/2 inhibitor. Together, our findings reveal that Lsm14b plays a pivotal role in mammalian oogenesis by specifically controlling of oocyte mRNA storage and stability.

NLRP14 Safeguards Calcium Homeostasis via Regulating the K27 Ubiquitination of Nclx in Oocyte-to-Embryo Transition.

Sperm-induced Ca2+ rise is critical for driving oocyte activation and subsequent embryonic development, but little is known about how lasting Ca2+ oscillations are regulated. Here it is shown that NLRP14, a maternal effect factor, is essential for keeping Ca2+ oscillations and early embryonic development. Few embryos lacking maternal NLRP14 can develop beyond the 2-cell stage. The impaired developmental potential of Nlrp14-deficient oocytes is mainly caused by disrupted cytoplasmic function and calcium homeostasis due to altered mitochondrial distribution, morphology, and activity since the calcium oscillations and development of Nlrp14-deficient oocytes can be rescued by substitution of whole cytoplasm by spindle transfer. Proteomics analysis reveal that cytoplasmic UHRF1 (ubiquitin-like, containing PHD and RING finger domains 1) is significantly decreased in Nlrp14-deficient oocytes, and Uhrf1-deficient oocytes also show disrupted calcium homeostasis and developmental arrest. Strikingly, it is found that the mitochondrial Na+ /Ca2+ exchanger (NCLX) encoded by Slc8b1 is significantly decreased in the Nlrp14mNull oocyte. Mechanistically, NLRP14 interacts with the NCLX intrinsically disordered regions (IDRs) domain and maintain its stability by regulating the K27-linked ubiquitination. Thus, the study reveals NLRP14 as a crucial player in calcium homeostasis that is important for early embryonic development.

CircKDM5B sponges miR-128 to regulate porcine blastocyst development by modulating trophectoderm barrier function.

Circular RNAs (circRNAs), which exert critical functions in the regulation of transcriptional and post-transcriptional gene expression, are found in mammalian cells but their functions in mammalian preimplantation embryo development remain poorly understood. Here, we showed that circKDM5B mediated miRNA-128 (miR-128) to regulate porcine early embryo development. We screened circRNAs potentially expressed in porcine embryos through an integrated analysis of sequencing data from mouse and human embryos, as well as porcine oocytes. An authentic circRNA originating from histone demethylase KDM5B (referred to as circKDM5B) was abundantly expressed in porcine embryos. Functional studies revealed that circKDM5B knockdown not only significantly reduced blastocyst formation but also decreased the number of total cells and trophectoderm (TE) cells. Moreover, the knockdown of circKDM5B resulted in the disturbance of tight junction assembly and impaired paracellular sealing within the TE epithelium. Mechanistically, miR-128 inhibitor injection could rescue the early development of circKDM5B knockdown embryos. Taken together, the findings revealed that circKDM5B functions as a miR-128 sponge, thereby facilitating early embryonic development in pigs through the modulation of gene expression linked to tight junction assembly.