Single-cell transcriptome analyses reveal critical regulators of spermatogonial stem cell fate transitions.

BACKGROUND: Spermatogonial stem cells (SSCs) are the foundation cells for continual spermatogenesis and germline regeneration in mammals. SSC activities reside in the undifferentiated spermatogonial population, and currently, the molecular identities of SSCs and their committed progenitors remain unclear. RESULTS: We performed single-cell transcriptome analysis on isolated undifferentiated spermatogonia from mice to decipher the molecular signatures of SSC fate transitions. Through comprehensive analysis, we delineated the developmental trajectory and identified candidate transcription factors (TFs) involved in the fate transitions of SSCs and their progenitors in distinct states. Specifically, we characterized the Asingle spermatogonial subtype marked by the expression of Eomes. Eomes+ cells contained enriched transplantable SSCs, and more than 90% of the cells remained in the quiescent state. Conditional deletion of Eomes in the germline did not impact steady-state spermatogenesis but enhanced SSC regeneration. Forced expression of Eomes in spermatogenic cells disrupted spermatogenesis mainly by affecting the cell cycle progression of undifferentiated spermatogonia. After injury, Eomes+ cells re-enter the cell cycle and divide to expand the SSC pool. Eomes+ cells consisted of 7 different subsets of cells at single-cell resolution, and genes enriched in glycolysis/gluconeogenesis and the PI3/Akt signaling pathway participated in the SSC regeneration process. CONCLUSIONS: In this study, we explored the molecular characteristics and critical regulators of subpopulations of undifferentiated spermatogonia. The findings of the present study described a quiescent SSC subpopulation, Eomes+ spermatogonia, and provided a dynamic transcriptional map of SSC fate determination.

Pancreatic lipase-related protein 2 is selectively expressed by peritubular myoid cells in the murine testis and sustains long-term spermatogenesis.

Spermatogenesis is a complicated process of germ cell differentiation that occurs within the seminiferous tubule in the testis. Peritubular myoid cells (PTMCs) produce major components of the basement membrane that separates and ensures the structural integrity of seminiferous tubules. These cells secrete niche factors to promote spermatogonial stem cell (SSC) maintenance and mediate androgen signals to direct spermatid development. However, the regulatory mechanisms underlying the identity and function of PTMCs have not been fully elucidated. In the present study, we showed that the expression of pancreatic lipase-related protein 2 (Pnliprp2) was restricted in PTMCs in the testis and that its genetic ablation caused age-dependent defects in spermatogenesis. The fertility of Pnliprp2 knockout animals (Pnliprp2-/-) was normal at a young age but declined sharply beginning at 9 months. Pnliprp2 deletion impaired the homeostasis of undifferentiated spermatogonia and severely disrupted the development and function of spermatids. Integrated analyses of single-cell RNA-seq and metabolomics data revealed that glyceride metabolism was changed in PTMCs from Pnliprp2-/- mice. Further analysis found that 60 metabolites were altered in the sperm of the Pnliprp2-/- animals; notably, lipid metabolism was significantly dysregulated. Collectively, these results revealed that Pnliprp2 was exclusively expressed in PTMCs in the testis and played a novel role in supporting continual spermatogenesis in mice. The outcomes of these findings highlight the function of lipid metabolism in reproduction and provide new insights into the regulation of PTMCs in mammals.

Single-cell transcriptomic characterization of sheep conceptus elongation and implantation.

Bidirectional communication between the developing conceptus and endometrium is essential for pregnancy recognition and establishment in ruminants. We dissect the transcriptomic dynamics of sheep conceptus and corresponding endometrium at pre- and peri-implantation stages using single-cell RNA sequencing. Spherical blastocysts contain five cell types, with 68.62% trophectoderm cells. Strikingly, elongated conceptuses differentiate into 17 cell types, indicating dramatic cell fate specifications. Cell-type-specific gene expression delineates the features of distinctive trophectoderm lineages and indicates that the transition from polar trophectoderm to trophoblast increases interferon-tau expression and likely drives elongation initiation. We identify 13 endometrium-derived cell types and elucidate their molecular responses to conceptus development. Integrated analyses uncover multiple paired transcripts mediating the dialogues between extraembryonic membrane and endometrium, including IGF2-IGF1R, FGF19-FGFR1, NPY-NPY1R, PROS1-AXL, and ADGRE5-CD55. These data provide insight into the molecular regulation of conceptus elongation and represent a valuable resource for functional investigations of pre- and peri-implantation ruminant development.

ARHGEF15 is expressed in undifferentiated spermatogonia but is not required for spermatogenesis in mice.

Spermatogenesis is a continual process that relies on the activities of undifferentiated spermatogonia, which contain spermatogonial stem cells (SSCs) that serve as the basis of spermatogenesis. The gene expression pattern and molecular control of fate decisions of undifferentiated spermatogonia are not well understood. Rho guanine nucleotide exchange factor 15 (ARHGEF15, also known as EPHEXIN5) is a guanine nucleotide-exchange factor (GEF) that activates the Rho protein. Here, we reported that ARHGEF15 was expressed in undifferentiated spermatogonia and spermatocytes in mouse testes; however, its deletion did not affect spermatogenesis. Arhgef15-/- mice were fertile, and histological examination of the seminiferous tubules of Arhgef15-/- mice revealed complete spermatogenesis with the presence of all types of spermatogenic cells. Proliferation and differentiation of the undifferentiated spermatogonia were not impacted; however, further analysis showed that Arhgef15 deletion resulted in decreased expression of Nanos2, Lin28a and Ddx4. Together, these findings suggest that ARHGEF15 was specifically enriched in undifferentiated spermatogonia and regulated gene expression but dispensable for spermatogenesis in mice.

Ldha-Dependent Metabolic Programs in Sertoli Cells Regulate Spermiogenesis in Mouse Testis.

Sertoli cells play indispensable roles in spermatogenesis by providing the advanced germ cells with structural, nutritional, and regulatory support. Lactate is regarded as an essential Sertoli-cell-derived energy metabolite that nurses various types of spermatogenic cells; however, this assumption has not been tested using genetic approaches. Here, we have reported that the depletion of lactate production in Sertoli cells by conditionally deleting lactate dehydrogenase A (Ldha) greatly affected spermatogenesis. Ldha deletion in Sertoli cells significantly reduced the lactate production and resulted in severe defects in spermatogenesis. Spermatogonia and spermatocytes did not show even mild impairments, but the spermiogenesis of Ldha conditional knockout males was severely disrupted. Further analysis revealed that 2456 metabolites were altered in the sperm of the knockout animals, and specifically, lipid metabolism was dysregulated, including choline, oleic acid, and myristic acid. Surprisingly, choline supplementation completely rescued the spermiogenesis disorder that was caused by the loss of Ldha activities. Collectively, these data have demonstrated that the interruption of Sertoli-cell-derived lactate impacted sperm development through a choline-mediated mechanism. The outcomes of these findings have revealed a novel function of lactate in spermatogenesis and have therapeutic applications in treating human infertility.

Paternal hypoxia exposure impairs fertilization process and preimplantation embryo development.

Environmental hypoxia exposure causes fertility problems in human and animals. Compelling evidence suggests that chronic hypoxia impairs spermatogenesis and reduces sperm motility. However, it is unclear whether paternal hypoxic exposure affects fertilization and early embryo development. In the present study, we exposed male mice to high altitude (3200 m above sea level) for 7 or 60 days to evaluate the effects of hypoxia on sperm quality, zygotic DNA methylation and blastocyst formation. Compared with age-matched controls, hypoxia-treated males exhibited reduced fertility after mating with normoxic females as a result of defects in sperm motility and function. Results of in vitro fertilization (IVF) experiments revealed that 60 days' exposure significantly reduced cleavage and blastocyst rates by 30% and 70%, respectively. Immunohistochemical staining of pronuclear formation indicated that the pronuclear formation process was disturbed and expression of imprinted genes was reduced in early embryos after paternal hypoxia. Overall, the findings of this study suggested that exposing male mice to hypoxia impaired sperm function and affected key events during early embryo development in mammals.