The spindle apparatus is essential for both mitotic and meiotic cell division. In contrast to its mitotic counterpart, the meiotic spindle of mammalian oocytes lacks centrosomes and thus relies on acentrosomal microtubule organizing centers to assemble the spindle. In addition, the positioning of spindle in oocytes is mediated by the F-actin network, not the astral microtubules (MTs). Overall, the role of astral MTs in meiotic spindle positioning has been overlooked due to the lack of centrosomes. In this study, we present an optimized method for staining and super-resolution imaging of astral MTs during meiosis. Though lacking centrosomes, both interpolar and astral MTs were present in the spindle through meiosis. The growth of astral MTs was inhibited by CDK1 and actin-related protein 2/3. Premature extension of astral MTs at metaphase I impaired the positioning of meiotic spindle, resulting in symmetric division of oocytes after meiosis I. Collectively, these results provide novel insights into the regulation of astral MT growth to facilitate the positioning of meiotic spindle in oocytes.

STUDY QUESTION:Does nucleoporin 205 (NUP205) deficiency caused by a novel de novo truncation mutation underlie the pathogenesis of premature ovarian insufficiency (POI)? SUMMARY ANSWER:NUP205 plays a critical role in ovarian development, and mutations in NUP205 represent a key factor in the pathogenesis of POI. WHAT IS KNOWN ALREADY:POI is a highly heterogeneous disorder with a significant genetic basis. The nuclear pore complex (NPC) is a fundamental channel mediating nucleocytoplasmic transport, with NUP205 serving as a key scaffold component of the NPC. STUDY DESIGN, SIZE, DURATION:This study employed a multilevel genetic and functional investigation, starting with whole-exome sequencing (WES) of a POI pedigree. Clinical significance was further assessed through a large-scale cohort involving 1030 POI cases. Functional validation was performed using the in vitro human cell line COV434 and an in vivo zebrafish model. PARTICIPANTS/MATERIALS, SETTING, METHODS:A Chinese family with idiopathic POI was recruited. To evaluate the clinical prevalence of NUP205 variants, WES data from a previously published cohort of 1030 POI cases were rescreened. Candidate variants were prioritized based on American College of Medical Genetics and Genomics guidelines and the functional impact of the identified mutation was further evaluated through protein structural modeling. The expression of NUP205 in follicles was determined by reanalyzing public ovarian single-cell RNA sequencing datasets and confirmed via immunofluorescence on human ovarian tissues. Functional assays were performed through siRNA-mediated knockdown in COV434 cells, complementing phenotypic and ultrastructural analyses of a CRISPR/Cas9-generated nup205 (p.R1057*) truncation zebrafish model. MAIN RESULTS AND THE ROLE OF CHANCE:A novel heterozygous nonsense mutation in NUP205 c.3160C>T (p.R1054*) was identified in the index pedigree, which was absent in public genomic databases. Expanded screening of the cohort identified five additional families carrying NUP205 variants (three heterozygous and two compound heterozygous) affecting highly conserved residues. In vitro, NUP205 knockdown in COV434 cells impaired the protein stability of NUP93 and NUP62, ultimately leading to NPC structural defects. In vivo, a zebrafish model carrying the equivalent nup205 (p.R1057*) mutation exhibited impaired oogenesis, compromised fertility, and lower fertilization rates. Transmission electron microscopy revealed abnormal NPC morphology in the theca cells of the mutant follicles. These findings demonstrate that NUP205 is essential for ovarian development and suggest that its deficiency is a key factor in POI pathogenesis, indicating that the observed association is unlikely to be due to chance. LIMITATIONS, REASONS FOR CAUTION:Although we identified additional NUP205 variants in a large POI cohort, the detailed molecular mechanisms of these specific variants remain to be further investigated. WIDER IMPLICATIONS OF THE FINDINGS:These findings identify NUP205 as a novel genetic contributor to POI, expanding the spectrum of nucleoporin-related reproductive disorders. STUDY FUNDING/COMPETING INTEREST(S):This work was supported by the National Natural Science Foundation of China (U24A20659 and 82500964). The authors declare that they have no conflicts of interest. TRIAL REGISTRATION NUMBER:N/A.

Nuclear pore complexes (NPCs), massive assemblies of approximately 30 distinct nucleoporins (NUPs), serve as the major gateways for nucleocytoplasmic transport. Recent research highlights that NUP aberrations-ranging from gene fusions and mutations to pathological protein accumulation-are increasingly implicated in the pathogenesis of a broad spectrum of human diseases. The underlying pathogenic mechanisms are highly multifactorial, encompassing the structural collapse of the NPC, transport network paralysis, epigenetic hijacking via liquid-liquid phase separation, and off-pore transcriptional dysregulation. This review synthesizes the multifaceted molecular etiology of NUP-associated pathologies. A deeper understanding of these diverse functions will shed light on the broader biological roles of NUPs and guide future research into targeted and personalized therapeutic strategies for NUP-associated disorders.

In mammals, the epididymis is composed of pseudostratified epithelium that forms the post-testicular milieu for spermatozoa. To date, the role of epithelial cells in shaping the luminal microenvironment for spermatozoa remains poorly understood. Here, we generated a conditional knockout mouse model of Nudt21 in the epididymis. NUDT21 is a key component of mammalian cleavage factor I (CFIm) that participates in the 3' end processing of pre-mRNAs. We found that Nudt21 deletion occurred mainly in principal cells of the corpus and the cauda. The loss of NUDT21 in principal cells resulted in male infertility, owing to sperm abnormalities present in the cauda. Pseudotime analysis revealed that the differentiation of Nudt21-null principal cells was blocked, preventing them from exerting their physiological function. Amid transcripts with shortened 3' untranslated regions (3'UTRs) after Nudt21 deletion, Dicer1 transcripts not only had shortened 3'UTR, but also skipped exon 1, resulting in downregulation of full-length DICER1 protein. Together, these results demonstrate the essential role of CFIm-mediated 3'UTR dynamics in preserving sperm integrity and provide insights into the intricate communication between the epididymal epithelium and spermatozoa.

Spermiogenesis is a unique process, in which round spermatids undergo morphological changes to form spermatozoa. Serine/Threonine Kinase 33 (STK33), a member of the serine/threonine protein kinase family, plays a pivotal role in spermiogenesis, manifested by the infertile phenotype of Stk33 knockout mice and patients carrying STK33 mutations. To date, the mechanism by which STK33 promotes spermiogenesis is not fully understood. Here we aimed to identify germ cell-specific proteins that interact with STK33. Using immunoprecipitation and mass spectrometry, 13 proteins were identified that potentially interact with STK33 in testicular germ cells. By comparing the expression patterns of the candidate genes in testicular germ cells, we selected Y-Box Binding Protein 2 (YBX2) and Testis Specific Serine Kinase Substrate (TSKS) for validation. When co-expressed in cultured cells, TSKS was immunoprecipitated by STK33, and vice versa. Furthermore, STK33 was recruited to the TSKS foci, likely through interaction with TSKS. Although proximity ligation assay demonstrated that STK33 and YBX2 form the complex in germ cells, their interaction was not recapitulated in cultured cells. Phosphorylation assays showed that STK33 was unable to phosphorylate both YBX2 and TSKS in vitro. Overall, these results suggest that STK33 regulates spermiogenesis through TSKS and YBX2, which warrants further investigation in vivo.

Quantification of transcription activities in mammalian preimplantation embryos is challenging due to a huge amount of maternally stored transcripts and paucity of research materials. Here, we investigate genomewide transcription activities of mouse and human preimplantation embryos by quantifying elongating RNA polymerase II. Two transcriptional waves are identified in early mouse embryos, with summits at the 2-cell and 8-cell stages. Gene collections with different expression patterns are obtained, with genes mainly transcribed at the mouse early/late 2-cell stage designated as zygotic genome activation-early/late 2-cell (ZGAE2C/L2C). ZGA-E2C genes are short and have low promoter CpG density. Protein translation/degradation not only regulates transcription activity through stepwise orchestration of histone modifications, transcriptional initiation, and elongation in early mouse embryos but also controls on/off switching of ZGA-E2C/ L2C genes in maternal aged mouse embryos. Genes mainly transcribed at the mouse 2-cell stage can also be transcribed as early as the human 2-cell stage.

Diminished ovarian reserve (DOR) is a physiological or pathological condition that progresses in an age-dependent manner, which is characterized by impaired ovarian follicle quality, decreased anti-Müllerian hormone levels, elevated follicle-stimulating hormone levels, and reduced antral follicle counts. Oxidative stress (OS) is one of the culprits of DOR. By imposing OS damage on various kinds of ovarian cells including granulosa cells, OS can result in ovarian hypofunction and eventually lead to female infertility. However, the underlying mechanisms have not been fully elucidated yet. In this study, HIGD1A, a mitochondrial inner membrane component, is found to be downregulated in granulosa cells upon OS exposure. By systematically studying the role of HIGD1A in regulating granulosa cell and ovarian functions as well as its corresponding mechanisms, a novel regulatory mechanism underlying OS-related female infertility is revealed, and provided a potential molecular target for anti-OS therapies.

The senescence of bone marrow mesenchymal stem cells (BMSCs) contributes to the development of degenerative skeletal conditions. To date, the molecular mechanism resulting in BMSC senescence has not been fully understood. In this study, we identified a small non-coding RNA, miR-203-3p, the expression of which was elevated in BMSCs from aged mice. On the other hand, overexpression of miR-203-3p in BMSCs from young mice reduced cell growth and enhanced their senescence. Mechanistically, PDZ-linked kinase (PBK) is predicted to be the target of miR-203-3p. The binding of miR-203-3p to Pbk mRNA could decrease its expression, which in turn inhibited the ubiquitination-mediated degradation of p53. Furthermore, the intravitreal injection of miR-203-3p-inhibitor into the bone marrow cavity of aged mice attenuated BMSC senescence and osteoporosis in aged mice. Collectively, these findings suggest that targeting miR-203-3p to delay BMSC senescence could be a potential therapeutic strategy to alleviate age-related osteoporosis.

Ovarian aging is mainly characterized by a progressive decline in oocyte quantity and quality, which ultimately leads to female infertility. Various therapies have been established to cope with ovarian aging, among which exosome-based therapy is considered a promising strategy that can benefit ovarian functions via multiple pathways. Here, we isolated and characterized exosomes derived from ovarian follicular fluid and profiled the differential expression patterns of noncoding exosomal RNAs in young and aged women. Treatment with young mouse-derived exosomes efficiently rescued ovarian function in aged mice. The follicular fluid exosomes from young mice and miR-320-3p can also promote the proliferation of ovarian granulosa cells and improve mitochondrial function from old mice in vitro. The mechanism may be involve that exosomes transfer miR-320-3p to granulosa cells, and inhibit the expression of FOXQ1. Exosomes also can increase the number of primordial and growing follicles, and improve the developmental ability of oocytes in the old mice in vivo. And hnRNPA2B1 controls miR-320-3p entry into exosomes. This work provides insights into the antiaging potential of follicular fluid-derived exosomes and the underlying molecular mechanisms, which may facilitate prevention of ovarian aging and an improvement in female fertility.

Ovarian aging reduced the quality of oocytes, resulting in age-related female infertility. It is reported that mesenchymal stem cells (MSCs) therapy can improve age-related ovarian function decline and the success rate of in vitro maturation (IVM) in assisted reproductive therapy. In order to investigate the effectiveness and mechanisms of MSCs to enhance oocyte quality of cumulus oocyte complexes (COCs) in advanced age, this study focus on the respective functional improvement of oocytes and granulosa cells (GCs) from aging mice and further to explore and verify the possible mechanisms. Here, we studied a popular but significant protein of follicular development, Forkhead box O-3a (FOXO3a), which is a transcription factor that mediates a variety of cellular processes, but the functions of which in regulating oocyte quality in MSCs therapy still remain inconclusive. In this study, the RNA-seq data of metaphase II (MII) oocytes and GCs isolated from COCs confirmed that, GCs of immature follicles show the most potential to be the targeted cells of bone marrow mesenchymal stem cells (BMSCs) by FOXO3a signaling pathway. Furthermore, we demonstrated the effectiveness of BMSCs co-culture with aging COCs to enhance oocyte quality and found its mechanism to function via ameliorating the biological function of GCs by alleviating FOXO3a levels. These results provide significant fundamental research on MSCs therapy on ovarian aging, as well as offering guidance for raising the success rate of assisted reproductive technology such IVM in clinical and non-clinical settings.

OBJECTIVE:This study aimed to evaluate the combined efficacy of hyperthermia and chemotherapy using a bladder cancer organoid model and to explore hyperthermia-related molecular pathways. METHOD:Tumor organoids were generated by embedding RT4 bladder cancer cells into Matrigel. The resulting organoids were treated with pirarubicin or gemcitabine at 37 °C or 42 °C. Proliferation was determined by Ki67 immunofluorescence staining, and apoptosis was assessed using a TdT-mediated dUTP nick end labeling (TUNEL) assay. RNA sequencing was used to identify the differentially expressed genes. RESULTS:Bladder cancer organoids were successfully established and exhibited robust proliferative abilities. Treatment with gemcitabine or pirarubicin under hyperthermic conditions caused pronounced structural damage to the organoids and increased cell death compared to that in the normothermically treated group. Furthermore, Ki67 labeling and TUNEL assays showed that the hyperthermia chemotherapy group showed a significantly reduced proliferation rate and high level of apoptosis. Finally, RNA sequencing revealed the IFN-γ signaling pathway to be associated with hyperthermia. CONCLUSION:Overall, hyperthermia combined with chemotherapy exerted better therapeutic effects than those of normothermic chemotherapy in grade 1-2 non-muscle-invasive bladder cancer, potentially through activation of the IFN-γ-JAK-STAT pathway.

Although the composition and assembly of stress granules (SGs) are well understood, the molecular mechanisms underlying SG disassembly remain unclear. Here, we identify that heterogeneous nuclear ribonucleoprotein A2/B1 (hnRNPA2B1) is associated with SGs and that its absence specifically enhances the disassembly of arsenite-induced SGs depending on the ubiquitination-proteasome system but not the autophagy pathway. hnRNPA2B1 interacts with many core SG proteins, including G3BP1, G3BP2, USP10, and Caprin-1; USP10 can deubiquitinate G3BP1; and hnRNPA2B1 depletion attenuates the G3BP1-USP10/Caprin-1 interaction but elevates the G3BP1 ubiquitination level under arsenite treatment. Moreover, the disease-causing mutation FUSR521C also disassembles faster from SGs in HNRNPA2B1 mutant cells. Furthermore, knockout of hnRNPA2B1 in mice leads to Sertoli cell-only syndrome (SCOS), causing complete male infertility. Consistent with this, arsenite-induced SGs disassemble faster in Hnrnpa2b1 knockout (KO) mouse Sertoli cells as well. These findings reveal the essential roles of hnRNPA2B1 in regulating SG disassembly and male mouse fertility.

Expression & regulation of TGCT susceptibility genes.

Identification of RHAMM regulators via RNAi screening.