The follicular microenvironment, including intra-follicular granulosa cells (GCs), is responsible for oocyte maturation and subsequent ovulation. However, the functions of GCs and cellular components of the follicular microenvironment in preovulatory follicles have not been extensively explored. Here, we surveyed the single-cell transcriptome of the follicular microenvironment around MII oocytes in six human preovulatory follicles in in vitro fertilization. There were six different cell types in the preovulatory follicles, including GCs and various immune cells. In GCs, we identified nine different functional clusters with different functional transcriptomic profiles, including specific clusters involved in inflammatory responses and adhesive function. Follicular macrophages are involved in immune responses, extracellular matrix remoulding and assist GCs in promoting the oocyte meiotic resumption. Interestingly, we observed that the specific terminal state subcluster of GCs with high levels of adhesive-related molecules should result in macrophage recruitment and residence, further contributing to an obvious heterogeneity of the immune cell proportion in preovulatory follicles from different patients. Our results provide a comprehensive understanding of the transcriptomic landscape of the preovulatory follicular microenvironment at the single-cell level. It provides valuable insights into understanding the regulation of the oocyte maturation and ovulation process, offering potential clues for the diagnosis and treatment of oocyte-maturation-related and ovulation-related diseases.

Summary: Mutation inCFAP43leads to severe asthenozoospermia and multiple morphological abnormalities of the sperm flagellum (MMAF) in both human and mouse. Previous studies have shown that disruption of intra-manchette transport (IMT) caused failure of flagellum assembly and sperm head shaping. In a previous study, therefore, we postulated that disruption of IMT may contribute to the failure of sperm flagellum formation and result in MMAF, however the mechanisms underlying these defects are still poorly understood.Cfap43-deficient mice were studied here to reveal the cellular mechanisms of abnormal sperm head morphology and MMAF. Depletion ofCfap43led to abnormal spermiogenesis and caused MMAF, sperm head abnormality and oligozoospermia. Furthermore, both abnormal manchette and disorganized ectoplasmic specialization (ES) could be observed at the elongated spermatids inCfap43-deficient mice. Therefore, our findings demonstrated that, in mice, CFAP43-mediated IMT is essential for sperm head shaping and sperm flagellum formation.

Myotubularin related protein 7 (MTMR7), a key member of the MTMR family, depicts phosphatase activity and is involved in myogenesis and tumor growth. We have previously identified MTMR7 in the proteomic profile of mouse spermatogonial stem cell (SSC) maturation and differentiation, implying that MTMR7 is associated with neonatal testicular development. In this study, to further explore the distribution and function of MTMR7 in mouse testis, we studied the effect of Mtmr7 knockdown on neonatal testicular development by testicular and SSC culture methods. Our results revealed that MTMR7 is exclusively located in early germ cells. Deficiency of MTMR7 by morpholino in neonatal testis caused excessive SSC proliferation, which was attributable to the aberrant PI3K/AKT signaling activation. Altogether, our study demonstrates that MTMR7 maintains SSC homeostasis by inhibiting PI3K/AKT signaling activation.

The self-renewal of spermatogonial stem cells (SSCs) requires a special microenvironment and is strictly controlled. Previously, we identified BMI1 as a key regulator of spermatogenesis in a knock-out mouse model. However, the mechanisms by which BMI1 regulates SSC maintenance remain largely unknown. Herein, we show that BMI1 is essential for SSC maintenance. BMI1 directs the transcriptional repression of target genes by increasing H2AK119ub and reducing H3K4me3 in SSCs. Furthermore, BMI1 inhibition resulted in the transcriptional activation of Wnt10b and thereby promoted the nuclear translocation of β-catenin in SSCs. Importantly, the suppression of Wnt/β-catenin signaling restored both the cytoplasmic expression of β-catenin and SSC maintenance in BMI1-deficient SSCs. Finally, we demonstrated that Wnt/β-catenin signaling was also involved in BMI1-mediated SSC maintenance in vivo. Altogether, our study not only reveals a novel mechanism for BMI1 in the process of SSC maintenance, but also provides a potential new strategy for treating male infertility.

Background: Retinoic Acid Induced Protein 14 (Rai14) is an evolutionarily conserved gene that is highly expressed in the testis. Previous experiments have reported that small interfering RNA (siRNA)-mediated gene knockdown (KD) of Rai14 in rat testis disrupted spermatid polarity and transport. Of note, a gene knockout (KO) model is considered the “gold standard” for in vivo assessment of crucial gene functions. Herein, we used CRISPR/Cas9-based gene editing to investigate the in vivo role of Rai14 in mouse testis. Methods: Sperm concentration and motility were assayed using a computer-assisted sperm analysis (CASA) system. Histological and immunofluorescence (IF) staining and transmission electron microscopy (TEM) were used to visualize the effects of Rai14 KO in the testes and epididymides. Terminal deoxynucleotidyl transferase-dUTP nick-end labeling (TUNEL) was used to determine apoptotic cells. Gene transcript levels were calculated by real-time quantitative PCR. Results: Rai14 KO in mice depicted normal fertility and complete spermatogenesis, which is in sharp contrast with the results reported previously in a Rai14 KD rat model. Sperm parameters and cellular apoptosis did not appear to differ between wild-type (WT) and KO group. Mechanistically, in contrast to the well-known role of Rai14 in modulating the dynamics of F-actin at the ectoplasmic specialization (ES) junction in the testis, morphological changes of ES junction exhibited no differences between Rai14 KO and WT testes. Moreover, the F-actin surrounded at the ES junction was also comparable between the two groups. Conclusion: In summary, our study demonstrates that Rai14 is dispensable for mouse spermatogenesis and fertility. Although the results of this study were negative, the phenotypic information obtained herein provide an enhanced understanding of the role of Rai14 in the testis, and researchers may refer to these results to avoid conducting redundant experiments.