Cumulus cells are known to maintain oocyte arrest at prophase I through gap junction-mediated cAMP signalling, but their role after meiotic resumption remains unclear. Here, we show that cumulus cells enhance oocyte genomic quality control by sensitizing oocytes to DNA damage-induced meiotic arrest. Time-lapse imaging of SiR-tubulin-labelled spindles revealed that oocytes from cumulus-oocyte complexes (COCs) matured faster than denuded oocytes (DOs). Upon mild DNA damage induced by low-dose etoposide, COC oocytes arrested at metaphase I, whereas DOs completed maturation despite similar levels of DNA lesions. This arrest required spindle assembly checkpoint (SAC) activity, as Reversine rescued polar body extrusion and BubR1 and Mad2 were elevated in COCs but not DOs. Disruption of gap junctions or inhibition of mTOR signalling abolished the checkpoint response. Notably, cumulus cells did not enhance oocyte response to minor spindle perturbations. These findings reveal a previously unrecognized role of cumulus cells in mediating DNA damage-induced SAC activation, providing post-GVBD genomic surveillance beyond prophase I arrest.

Compound probiotics alleviate aflatoxin B1-induced reproductive toxicity in mice by remodeling gut microbiota and testicular metabolome via NRF2 pathway action.

During spaceflight, the female reproductive system undergoes substantial adaptation to microgravity and faces an increased risk of reproductive impairment. Mammalian oocytes remain arrested for extended periods at the first meiotic prophase, a stage particularly vulnerable to DNA damage, yet whether simulated microgravity (SMG) directly induces oocyte genomic damage and how such damage, together with the associated meiotic arrest, can be mitigated remain unclear. Melatonin (MLT), a pineal hormone with broad physiological roles, has shown benefits in improving oocyte quality in vivo and in vitro. Using SMG as a stress model, we demonstrate that during the prophase-arrested stage, SMG exposure induces DNA double-strand breaks with activation of the ATM-CHK2 DNA damage response, without detectable elevation of global cellular ROS or mitochondrial superoxide. These changes were accompanied by abnormal mitochondrial distribution and increased early apoptosis. In vitro MLT supplementation during SMG exposure alleviated DNA damage through a DNA-PKcs-associated NHEJ repair response, improved mitochondrial distribution, and this protective effect was largely independent of canonical MT1 or MT2 receptor signaling. During meiotic maturation, MLT improved SMG-induced spindle assembly defects, promoted MTOC coalescence, suppressed mitochondrial unfolded protein response overactivation, reduced SMG-induced mitochondrial hyperpolarization, and reduced early apoptosis. Consequently, oocytes exposed to MLT exhibited increased first polar body extrusion, improved spindle integrity, and enhanced oocyte-intrinsic developmental competence, as reflected by increased blastocyst formation after parthenogenetic activation. Together, these findings show that SMG induces DNA damage in prophase-arrested oocytes and identify MLT as a stage- and dose-sensitive modulator of DNA repair and mitochondrial homeostasis, offering a potential strategy to protect female reproductive health during spaceflight.

Do variants in YTH N6-methyladenosine RNA binding protein C2 (YTHDC2) cause male infertility in humans, and what is the underlying pathogenic mechanism? Biallelic pathogenic missense variants in YTHDC2 disrupt the mitotic-to-meiotic transition, causing meiotic arrest and non-obstructive azoospermia (NOA) or severe oligozoospermia in humans. YTHDC2 is a male germ cell-specifically expressed RNA helicase essential for meiotic progression. In mice, loss of Ythdc2 leads to meiotic arrest at the early prophase. However, clinical evidence linking YTHDC2 variants to human male infertility and the underlying mechanisms involved remains to be established. This study utilized a large cohort comprising 56 consanguineous families and 89 sporadic infertile men diagnosed with NOA or severe oligozoospermia. Through extensive genetic screening, we specifically identified five infertile men from three unrelated families who harbored candidate pathogenic variants in the YTHDC2 gene. The overall study design encompassed genetic screening followed by in vivo functional validation using a knock-in mouse model. Whole-exome sequencing (WES) and bioinformatic analyses were performed on the patient cohort to screen for candidate pathogenic variants. Human meiotic defects were characterized via histological analyses and immunofluorescence staining of testicular sections. To validate the pathogenicity of the identified variant, a knock-in mouse model harboring the equivalent variant found in patients was generated by CRISPR/Cas9 technology, and analyzed for spermatogenesis and meiosis using spermatocyte spreading and immunofluorescence staining, quantitative real-time PCR, and western blotting. Two homozygous missense variants in YTHDC2 were identified in four NOA patients from two unrelated consanguineous families (MT1: c.3491A>T, p. E1164V; MT2: c.2639G>A, p. R880H), and compound-heterozygous missense variants were identified in a sporadic patient with severe oligozoospermia (MT3: c.1145A>G, p. D382G; MT4: c.292A>G, p. R98G). The MT1 variant (p.E1164V) is not located in any annotated domains, the other three variants reside within known functional domains of YTHDC2 protein. The knock-in mouse model carrying the MT1 variant recapitulated the patient's phenotype, with both exhibiting meiotic prophase arrest during spermatogenesis. Mechanistically, significantly decreased levels of MEIOC and RBM46, two YTHDC2-interacting proteins required for meiotic transcriptome reprogramming, were observed in the patient's testes. Concurrently, mitotic cell cycle regulators such as CCNA2, CCND1, and WEE1 were aberrantly upregulated in the patient's testicular cells expressing meiosis markers, indicating a failure to silence the mitotic program upon meiotic entry. This study is limited by the small sample size of patients with pathogenic YTHDC2 variants. While the MT1 variant was functionally validated in vivo, the specific pathogenic mechanisms of the other variants identified require further investigation. Our findings provide direct clinical evidence establishing the pathogenicity of YTHDC2 variants in human NOA. The study reveals a conserved role for YTHDC2 in safeguarding the mitotic-to-meiotic transition by suppressing mitotic gene expression while maintaining the meiotic program. These findings expand the genetic spectrum of male infertility and suggest YTHDC2 screening as a promising approach for the genetic diagnosis of male infertility. This work was supported by the National Key Research and Developmental Program of China (2022YFA0806303 to H.Z., and 2024YFC2706801 to H.J.); the National Natural Science Foundation of China (32470898 to H.Z., W2412028 and 32330032 to Q.S., 32470915 to B.S.); the State Key Laboratory of Reproductive Medicine and Offspring Health, Nanjing Medical University (SKLRM-K202405); and the Open Research Project of Fuyang Normal University (FYKFKT24023). The authors declare no competing interests. N/A.

Recurrent assisted reproductive technology failure: the embryo perspective.

The organized chromatin configuration in meiosis prophase I is crucial for spermatogenesis and male fertility, involving a series of tightly coordinated events mediated by numerous proteins. Neddylation, a ubiquitin-like post-translational modification, conjugates NEDD8 to substrate proteins and thus regulates protein degradation via activating Cullin-RING E3 ligases. Despite its importance in other cellular processes, its role in meiosis remains elusive. We inhibited neddylation using intratesticular MLN4924 treatment and generated germ cell-specific Nedd8-depleted male mice to study its meiotic role. Meiotic progression and chromosomal synapsis were assessed via markers of meiotic progression especially the assembly of synaptonemal complexes. Substrate-specific role of neddylation in meiosis was dissected to identify its functional targets both in vivo and in vitro. Nedd8 deficiency in spermatocytes caused meiotic arrest at the zygotene stage, leading to azoospermia and infertility. Spermatocytes showed disrupted synapsis, with persistent HORMAD1 on unsynapsed axes and defective assembly of synaptonemal complex central elements. Mechanistically, neddylation mediated SKP1-Cullin1-FBXO47 complex-dependent ubiquitination and degradation of HORMAD1, a prerequisite for proper synapsis. Our findings demonstrate that neddylation is essential for spermatocyte development and reveal its critical role in regulating synaptonemal complex dynamics, probably depending on SCF complex-mediated HORMAD1 degradation. This study provides insights into post-translational control of protein dynamics in meiosis and potential therapeutic targets for male infertility.

Proper oocyte maturation is critical for female fertility, yet whether cuproptosis, a recently identified copper-dependent cell death pathway, affects meiotic maturation remains unknown. Here, we show that Cu(II)-elesclomol (ELC-Cu(II)) treatment induces dose-dependent metaphase I arrest of mouse oocytes. This arrest results from spindle assembly checkpoint activation caused by defective spindle organization and impaired kinetochore-microtubule attachments. We demonstrate that ELC-Cu(II) triggers changes in canonical cuproptosis markers, including intracellular copper accumulation, FDX1 downregulation, and protein aggregation. Meanwhile, treated oocytes exhibit mitochondrial dysfunction characterized by reduced membrane potential and decreased ATP levels. Integrated transcriptomic and proteomic profiling reveals a predominantly post-transcriptional response, with 223 differentially expressed proteins, while transcriptomic profiles show minimal changes. Pathway analysis identifies dysregulation of lipoic acid metabolism and iron-sulfur cluster biosynthesis as key features. Targeted knockdown of the key lipoyltransferase LIPT1 fails to rescue the meiotic defect, whereas supplementation with the NAD+ precursor nicotinamide mononucleotide (NMN) improves mitochondrial function and partially restores polar body extrusion. These findings establish cuproptosis as a mechanism linking copper toxicity to mitochondrial impairment and meiotic failure in oocytes, and suggest NAD+ metabolism as a potential therapeutic target for protecting oocyte quality.Cuproptosis, via copper accumulation and FDX1 loss, disrupts critical metabolic pathways (such as lipoic acid metabolism and iron-sulfur cluster biosynthesis), causing mitochondrial dysfunction and oocyte meiotic arrest. NMN supplementation effectively mitigates this arrest by restoring cellular energy metabolism and rescuing maturation.

Dynamin-related protein 1 (DRP1) is a central regulator of mitochondrial fission and plays a critical role in maintaining mitochondrial function, distribution, and turnover in reproductive cells. Mitochondrial integrity is essential for oocyte quality, folliculogenesis, fertilization, embryonic development, and ultimately, female reproductive longevity. In this review, we synthesize evidence from mammalian and invertebrate models to illustrate the essential roles of DRP1 in reproductive physiology and aging. Genetic deletion or pharmacologic inhibition of DRP1 results in mitochondrial clustering, energy failure, increased reactive oxygen species production, meiotic arrest, and embryo fragmentation. Furthermore, DRP1 dysfunction has been increasingly implicated in age-associated reproductive decline due to impaired mitophagy and defective organelle crosstalk. Model systems such as mice, pigs, and Caenorhabditis elegans have demonstrated that DRP1 activity is modulated by metabolic and epigenetic pathways, including NAD+/sirtuin signaling and GTP metabolism. Therapeutic interventions aimed at restoring DRP1 function-including nicotinamide mononucleotide, coenzyme Q10 (CoQ10), and dietary modulation-have shown promising effects in delaying reproductive aging and improving oocyte or embryo competence in animal models. Despite the current absence of human interventional efficacy data, DRP1 is a plausible and testable target in reproductive biology, with preclinical findings indicating potential relevance to infertility treatment and reproductive aging. This review highlights DRP1 as a key target in reproductive biology, emphasizing its translational potential for treating infertility and mitigating age-related oocyte deterioration.

Nonobstructive azoospermia (NOA), the most severe form of male infertility, frequently arises from genetic defects that disrupt spermatogenesis. In this study, a novel hemizygous missense variant (NM_001184767.2 [c.G1069A; p.V357I]) is identified in the X-linked BEN domain-containing 2 (BEND2) gene of a patient with NOA characterized by spermatocyte maturation arrest. Whole-exome sequencing and Sanger validation confirmed that this rare variant is absent in fertile controls and that no pathogenic variants were detected in established NOA genes. Computational analysis predicted potential structural alterations via AlphaFold modeling, leading to the hypothesis that the ability of BEND2 to recognize genomic targets may be compromised. The patient's phenotype phenocopies the meiotic arrest observed in Bend2-knockout mice. Expression profiling confirmed predominant BEND2 transcription in human and mouse testes, peaking in early spermatocytes and coinciding with meiotic initiation, with reduced transcript levels detected in the proband's peripheral blood compared with those in an obstructive azoospermia control. This study reports a pathogenic BEND2 variant associated with NOA with spermatocyte arrest, highlighting its critical role in human meiosis and expanding the genetic etiology of male infertility.

The developmental competence of oocytes is crucial for successful fertilization and embryonic development; however, optimizing this capacity remains challenging. Hydrogen sulfide (H2S), a gasotransmitter recognized for its cytoprotective and antioxidant properties, has the potential to enhance oocyte quality. This study investigated the effects of sodium hydrosulfide (NaHS), an exogenous H2S donor, on the developmental competence of ovine oocytes in both conventional (C-IVM) and biphasic invitro maturation (Pre-IVM) systems. Immature ovine oocytes were cultured in C-IVM medium supplemented with increasing NaHS concentrations (0-10 mM) to determine the optimal concentration based on redox state, mitochondrial status, and developmental outcomes. Subsequently, the effects of NaHS during the Pre-IVM phase of the biphasic system were evaluated, focusing on meiotic arrest, cytoplasmic maturation, and developmental competence. The results revealed that 2.5 mM NaHS during C-IVM significantly improved oocyte quality by reducing reactive oxygen species (ROS) levels, increasing glutathione (GSH) content, and enhancing blastocyst rates. Furthermore, a 6-h exposure to 2.5 mM NaHS during Pre-IVM did not alter germinal vesicle (GV) rates and cyclic AMP levels but significantly decreased ROS levels and increased mitochondrial mass index at both 6 and 24 h. In the biphasic system, 2.5 mM NaHS notably elevated blastocyst rates. These findings suggest that NaHS could serve as a promising supplement to improve IVM efficiency and embryonic development, providing valuable insights for assisted reproductive technologies.

Mammalian oogenesis is a precisely orchestrated developmental process, which depends on accurate chromatin remodeling and transcriptional regulation in the absence of DNA replication. The histone variant H2A.Z is required for oogenesis and embryogenesis, yet the chaperone directing its deposition has not been well characterized in mammalian oocytes. Here, we identify Znhit1, a core subunit of the SRCAP chromatin remodeling complex, as the essential factor mediating H2A.Z deposition in oocytes. Oocyte-specific depletion of Znhit1 impairs H2A.Z incorporation and leads to severe ovarian phenotype, characterized by follicle loss, homologous chromosome segregation defects and meiotic arrest, which ultimately leads to female infertility. On a molecular level, integrated Smart-seq2 and H2A.Z CUT&Tag analyses demonstrate that Znhit1 depletion severely reduces genome-wide H2A.Z deposition, particularly at promoter regions of key meiotic genes such as Aurkb, Tpm3 and Zar1, resulting in transcriptional dysregulation and aberrant meiotic gene expression. Our findings pinpoint Znhit1 as the histone chaperone essential for accurate deposition of histone variant H2A.Z, ensuring meiotic progression and oocyte development in mice.

At fertilization, two highly specialized gametic genomes must rapidly reprogram into a single totipotent nucleus. In mammals, this transformation is marked by a dramatic protamine-to-histone exchange and pronounced asymmetry between the parental pronuclei. Zebrafish offer a contrasting vertebrate model in which sperm chromatin is already organized in nucleosomes and carries both active and repressive histone modifications, eliminating the need for protamine replacement. This histone-based configuration provides a unique opportunity to examine how parental chromatin transitions are initiated and coordinated directly in vivo. Although zebrafish is a well-established model organism in developmental biology, the detailed cellular and molecular steps of its fertilization process, particularly the timing and coordination of parental chromatin remodelling, remain largely unknown. Using immunofluorescence coupled with spinning-disk confocal microscopy, we mapped histone variants and post-translational modifications with temporal sampling at minute-level intervals across 3,549 zebrafish embryos from fertilization to the first cleavage. This analysis resolved the main steps of fertilization, from sperm entry and chromatin decondensation to the formation, expansion, and apposition of the parental pronuclei. Throughout these stages, maternal and paternal genomes were remodelled almost synchronously. Activation-associated marks (H3K4me1/2/3, H3K9ac, H4K12ac) appeared shortly after fertilization, spread to both pronuclei by apposition, and disappeared before mitosis. The histone modification H3K9me3, commonly associated with compact or heterochromatin-like chromatin states, was present from the earliest time point examined and became more prominent during pronuclear maturation, whereas H3K27me3 was not detectable at any stage. H2A.Z showed strong paternal enrichment after sperm entry and was later detected in both pronuclei during apposition with weaker maternal signal. Polar bodies did not show evidence of undergoing the extensive chromatin remodelling observed in the zygotic pronuclei. In gametes, sperm contained core histones and methylation marks but lacked acetylation, indicating a compact, semi-active chromatin state, while unfertilized eggs displayed H3K9me3 and H3S10ph but no activating marks, consistent with meiotic arrest. Zebrafish fertilization involves direct, histone-based chromatin remodelling that occurs without protamine replacement and proceeds almost simultaneously in both parental pronuclei. The sperm genome enters fertilization already in a semi-active, histone-bound state, enabling rapid and coordinated remodelling of both parental chromatin. This process results in a transient change in chromatin configuration that is more symmetrical between the parental genomes than that reported in mammalian systems.

Meiosis is regulated by phase-specific genes to orchestrate nuclear and cytoskeletal dynamics essential for sexual reproduction. The ciliate Paramecium tetraurelia exhibits nuclear dimorphism, harboring two germline micronuclei (MICs) and one somatic macronucleus (MAC) within a single cell during vegetative growth. During sexual reproduction, the MICs undergo meiosis and the MAC deforms and fragments, providing a unique model to study the regulation of diverse nuclear events. Transcription factor DP (TFDP), which heterodimerizes with E2Fs, can bind to specific DNA motifs in promoters of cell cycle-regulated genes to activate or repress their expression. Here, we identified 16 TFDP homologs in P. tetraurelia, representing an exceptional gene family expansion accompanied by functional domain diversification. The functions of Tfdp1a and Tfdp1b, which are specifically expressed during sexual reproduction and localize in the old and new MACs, were further investigated. Their depletion resulted in meiotic arrest at metaphase in the MIC, failure of old MAC fragmentation, and abortive cytokinesis. Transcriptomic analysis revealed that TFDP1A/1B knockdown primarily causes gene downregulation, with > 60% of downregulated genes being specifically highly expressed during sexual reproduction. Functional annotation and enrichment analyses demonstrated significant downregulation of proteins involved in meiosis, DNA replication, and DNA repair. Critically, multiple downregulated meiotic regulators are essential for proper homologous chromosome segregation and sister chromatid separation, providing a mechanistic basis for the observed MIC meiotic arrest. This study uncovers Tfdp1a/1b as essential regulators of the distinctive meiotic process in P. tetraurelia, providing crucial insights into nuclear dynamics and the regulation of sexual reproduction in binucleate systems.Supplementary InformationThe online version contains supplementary material available at 10.1007/s42995-026-00378-1.

O-GlcNAc transferase orchestrates oocyte maturation by modulating the activity of mitochondrial respiratory chain complex I.

Taurine restores oocyte quality by enhancing mitochondrial function in mice exposed to dibutyl phthalate during adolescence.

Background: Male infertility is increasingly being conceived as a biomarker of general male health status other than being a significant reproductive health issue. Nonobstructive azoospermia (NOA) patients have the worst health status impairment as well as highest risk of developing cancer. It’s well known that cancer susceptibility gene, MLH1 have roles in homologous recombination during meiosis. Mlh1 mutant mice have azoospermia with meiotic arrest. Aim: We aimed to identify risk alleles in MLH1 gene in males with nonobstructive azoospermia related maturation arrest (MA) to investigate further whether this cancer susceptibility gene is also related to male infertility. Patients and Methods: MLH1 variants were genotyped in 42 patients with MA. The variants of interest then compared to GnomAD v.3.1.2 (non-cancer) male population database and to the repository exome data of a national genetic disease evaluation center (NGDEC). Results: rs1800734 allele frequencies were significantly higher in the patient group compared to GnomAD (p

During spermatogenesis, the unsynapsed XY chromosomes undergo meiotic sex chromosome inactivation (MSCI) and form a heterochromatic XY body. Defects in MSCI lead to meiotic arrest and male infertility. Although DNA damage response (DDR) factors are established as key initiators of MSCI, how transcriptional silencing is subsequently achieved remains elusive. Here, we identify the nucleolar components NPM1, SENP3, and rRNA as essential downstream effectors of DDR signaling in MSCI. During pachytene, these components migrate to and transiently cover the XY body during MSCI establishment, before becoming restricted to a corner of the XY body. Genetic deletion of Npm1 or Senp3, or inhibition of rRNA transcription severely impairs MSCI. Mechanistically, SENP3-mediated deSUMOylation of NPM1 promotes its interaction with rRNA, enabling liquid-liquid phase separation, via which they exclude Pol II from the XY body. Together, these data reveal a critical role of nucleolar components in the transcriptional regulation of MSCI in mammalian spermatogenesis.

Cyclin B3 (CCNB3) plays a critical regulatory role in mammalian meiosis. Studies in mice have demonstrated that CCNB3 interacts with CDK1 to modulate the activity of MPF, thereby driving meiotic progression. However, the functional mechanisms of CCNB3 in porcine oocytes remain unclear. In this study, we reveal for the first time that knockdown of CCNB3 in porcine oocytes induces meiotic arrest at metaphase I, accompanied by impaired degradation of cyclin B1 and securin. Further investigation identifies that the antisense long non-coding RNA CCNB3-AS forms a double-stranded RNA (dsRNA) structure with the CCNB3 mRNA, significantly enhancing its stability by resisting PAT1 homolog 1 (PATL1)-mediated degradation. Mechanistically, CCNB3-AS interacts with the scaffold protein Vimentin (VIM). Structural analysis reveals that VIM binds to the PAT1 domain of PATL1 and is capable of influencing the ability of CNOT7, the core subunit of the CCR4-NOT complex, to bind to PATL1, ultimately maintaining stable CCNB3 mRNA expression. Our study elucidates the molecular mechanism by which the CCNB3-AS/CCNB3 dsRNA duplex cooperates with VIM and PATL1 to collectively regulate meiosis in porcine oocytes. Furthermore, we reveal the non-canonical role of VIM in mRNA degradation, providing new theoretical support for understanding the mechanisms underlying porcine oocyte meiosis.

Oocyte development requires tight regulation of transcription and RNA metabolism, which is coordinated by RNA-binding proteins, whose roles in mammalian oogenesis remain incompletely understood. Here, we identify the DEAD-box RNA helicase DDX5 as a key regulator of RNA homeostasis in oocytes. Oocyte-specific deletion of DDX5 leads to female sterility, which is characterized by defective chromatin remodeling, meiotic arrest, increased aneuploidy, and fertilization failure. Mechanistically, DDX5 maintains RNA homeostasis through three interconnected processes: (1) promoting transcription via interaction with RNA polymerase II in nonsurrounded nucleolus-stage germinal vesicle oocytes; (2) clearing retrotransposon RNAs to safeguard transcriptome integrity; and (3) supporting maternal mRNA storage by coordinating nuclear export, mitochondrial organization, and mitochondria-associated ribonucleoprotein domain assembly. Our study establishes DDX5 as a master regulator that integrates transcriptional and post-transcriptional programs to ensure oocyte competence and fertility.

Bovine oocyte quality is crucial for successful in vitro embryo production; therefore, elucidating the mechanisms underlying oocyte maturation is essential. N6-methyladenosine (m6A) modification plays a pivotal role in gametogenesis and embryonic development. However, the dynamic landscape and functional significance of m6A modification during bovine oocyte maturation remain poorly understood. We performed transcriptome-wide m6A profiling analysis on bovine oocytes at the GV and MII stages using MeRIP-seq and RNA-seq. A total of 6323 differential m6A peaks were identified between the two groups, with 4422 significantly upregulated and 1901 significantly downregulated. The combined analysis of MeRIP-seq and RNA-seq identified 262 genes with significant differences in both methylation modification and transcriptome levels. GO and KEGG enrichment analyses of these genes indicated that they were significantly enriched in biological processes and signaling pathways, including endocytosis, thermogenesis, ribosome biogenesis, cellular metabolism, the Apelin signaling pathway, and the HIF-1 signaling pathway. Finally, we assessed the roles of m6A modification in bovine oocyte maturation. The results demonstrated that inhibition of m6A modification impairs both nuclear and cytoplasmic maturation of bovine oocytes. Our results provide a comprehensive m6A modification profile during bovine oocyte maturation. Moreover, we found that inhibition of m6A modification results in meiotic arrest and impaired oocyte maturation. These findings offer a theoretical foundation for further studies on the function of RNA modifications in oocyte maturation.