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.

Spermatogenesis is a cellular differentiation process that includes three major events: mitosis of spermatogonia, meiosis of spermatocytes and spermiogenesis. Steady-state spermatogenesis relies on functions of spermatogonial stem cells (SSCs). Establishing and maintaining a foundational SSC pool is essential for continued spermatogenesis in mammals. Currently, our knowledge about SSC and spermatogenesis is severely limited in domestic animals. In the present study, we examined transcriptomes of testes from domestic yaks at four different stages (3, 5, 8 and 24 months of age) and attempted to identify genes that are associated with key developmental events of spermatogenesis. Histological analyses showed that the most advanced germ cells within seminiferous tubules of testes from 3, 5, 8 and 24 months old yaks were gonocytes, spermatogonia, spermatocytes and elongated spermatids, respectively. RNA-sequencing (RNA-seq) analyses revealed that 11904, 4381 and 2459 genes were differentially expressed during the gonocyte to spermatogonia transition, the mitosis to meiosis transition and the meiosis to post-meiosis transition. Further analyses identified a list of candidate genes than may regulate these important cellular processes. CXCR4, a previously identified SSC niche factor in mouse, was one of the up-regulated genes in the 5 months old yak testis. Results of immunohistochemical staining confirmed that CXCR4 was exclusively expressed in gonocytes and a subpopulation of spermatogonia in the yak testis. Together, these findings demonstrated histological changes of postnatal testis development in the domestic yak. During development of spermatogonial lineage, meiotic and haploid germ cells are supported by dynamic transcriptional regulation of gene expression. Our transcriptomic analyses provided a list of candidate genes that potentially play crucial roles in directing the establishment of SSC and spermatogenesis in yak.

Heat stress can affect the reproductive function of livestock and cause harm to animal production, which can seriously damage the economic interests of livestock producers. Therefore, it is important to explore the effect of heat stress on reproductive function to improve livestock production. In this study, the experimental animals Turpan black sheep and Suffolk sheep were selected as controls, each with 10 sheep, and the reproductive physiological performance was measured in Turpan, China from April to August when there was no heat stress to strong heat stress. The results showed that the sperm density, vitality, and kinematic parameters of Suffolk sheep were significantly lower than that in Turpan black sheep (p < 0.01) after heat stress, while the sperm acrosome malfunctions and DNA damage were significantly higher in Suffolk sheep (p < 0.01). In addition, the endogenous levels of reproductive hormones and oxidative stress indicators in the blood of Turpan black sheep were stable before and after heat stress treatment, while Suffolk sheep showed different degrees of fluctuations. There was no significant difference in testicular histomorphology between the two after heat stress treatment. However, Suffolk sheep showed a significantly decreased number of spermatocytes after heat stress treatment (p < 0.05). It was found that during meiosis, the proportion of cells in the meiotic zygotene stage of Suffolk sheep was significantly higher than that of Turpan black sheep. To investigate the mechanism of normal spermatogenesis in Turpan black sheep under heat stress, we performed RNA-Seq analysis on the testis. The results showed that there were 3,559 differential genes in Turpan black sheep before and after heat stress, with 2,118 up-regulated genes and 1,441 down-regulated genes. The enrichment analysis of GO and KEGG showed that the differential genes are mainly involved in cellular component organization or biogenesis, cell cycle process, mitotic cell cycle process, meiotic cell cycle process, double-strand break repair and Rap1 signaling pathway, Ras signaling pathway, Cell cycle, signaling pathways regulating pluripotency of stem cells Oocyte meiosis. Genes related to spermatogenesis, SYCP2, TDRD9, BRDT, CEP120, BRCA1, etc. were significantly up-regulated in Turpan black sheep after heat stress. In summary, our results showed that the up-regulation of genes involved in spermatogenesis protects the normal production of sperm in Turpan black sheep under HS, thereby achieving normal reproductive function.Our research systematically elucidated the mechanism of heat stress resistance during spermatogenesis in Turpan black sheep and provided potential possibilities for the subsequent breeding of new heat-resistant breeds.

The homeostasis of mitochondrial calcium ([Ca²⁺]_mt) in oocytes plays a critical role in maintaining normal reproductive cellular progress such as meiosis. However, little is known about the association between [Ca²⁺]_mt homeostasis and early embryonic development. Two in vitro mouse MII oocyte models were established by using a specific agonist or inhibitor targeting mitochondrial calcium uniporters (MCU) to upregulate or downregulate [Ca²⁺]_mt concentrations. The imbalance of [Ca²⁺]_mt in MII oocytes causes mitochondrial dysfunction and morphological abnormity, leading to an abnormal spindle/chromosome structure. Oocytes in drug‐treated groups are less likely to develop into blastocyst during in vitro culture. Abnormal [Ca²⁺]_mt concentrations in oocytes hindered epigenetic modification and regulated mitogen‐activated protein kinase (MAPK) signaling that is associated with gene expression. We also found that MAPK/ERK signaling is regulating DNA methylation in MII oocytes to modulate epigenetic modification. These data provide a new insight into the protective role of [Ca²⁺]_mt homeostasis in early embryonic development and also demonstrate a new mechanism of MAPK signaling regulated by [Ca²⁺]_mt that influences epigenetic modification.

Spermatogenesis is a highly orchestrated germ cell differentiation process involving the dynamic regulation of cell fate transitions. Dissecting the molecular landscapes of spermatogenic cell types is crucial for identifying fertility-related problems and improving the reproductive performance of farm animals. Here, we conducted transcriptomic and chromosome spreading across meiotic stages of testicular cells from taurine cattle (Bos taurus), yak (Bos grunniens), and their hybrid progenies to describe the transcriptional landscape of normal spermatogenesis and identify potential regulators that are involved in hybrid sterility. The results revealed seven types of spermatogonia, ten spermatocytes and ten types of spermatids in the cattle or yak testes. In sharp contrast, the testes of the cattle–yak hybrids contained only seven spermatogonial subtypes and six types of spermatocytes. Notably, the arrest of spermatocytes at the diplotene-to-diakinesis transition was accompanied by defects in double-strand break repair. In the testes of backcrossed offspring, spermatogenic arrest was partially rescued, and round spermatozoa were produced. By performing joint analysis, we identified 115 genes that exhibited differential protein abundance in spermatocytes of cattle-yak. Among them, 24 genes carrying genomic structural variations were differentially expressed in spermatocytes of cattle-yak but recovered in those of backcrossed offspring. This work provides important insights into spermatogenesis in large animals and serves as a valuable resource for identifying the factors determining reproductive isolation.