Low sperm motility is a significant contributor to male infertility. beta-defensins have been implicated in host defence and the acquisition of sperm motility; however, the regulatory mechanisms governing their gene expression patterns and functions remain poorly understood. In this study, we performed single-cell RNA and spatial transcriptome sequencing to investigate the cellular composition of testicular and epididymal tissues and examined their gene expression characteristics. In the epididymis, we found that epididymal epithelial cells display a region specificity of gene expression in different epididymal segments, including the beta-defensin family genes. In particular, Defb15, Defb18, Defb20, Defb25 and Defb48 are specific to the caput; Defb22, Defb23 and Defb26 to the corpus; Defb2 and Defb9 to the cauda of the epididymis. To confirm this, we performed mRNA fluorescence in situ hybridisation (FISH) targeting certain exon region of beta-defensin genes, and found some of their expression matched the sequencing results and displayed a close connection with epididimosome marker gene Cd63. In addition, we paid attention to the Sertoli cells and Leydig cells in the testis, along with fibroblasts and smooth muscle cells in the epididymis, by demonstrating their gene expression profile and spatial information. Our study provides a single-cell and spatial landscape for analysing the gene expression characteristics of testicular and epididymal environments and has important implications for the study of spermatogenesis and sperm maturation.

Reproductive disorders are a concern in the pig industry. Successful gestation processes are closely related to a suitable endometrial microenvironment, and the physiological mechanisms leading to failed pregnancy during the peri‐implantation period remain unclear. We constructed single‐cell transcriptome profiles of peri‐implantation embryo loss and successful gestation endometrial tissues and identified 22 cell subpopulations, with epithelial and stromal cells being the predominant endometrial cell types. The two tissues showed marked differences in cell type composition, especially among epithelial cell subpopulations. We also observed functional differences between epithelial and stromal cells in tissues from embryonic loss and successful gestation, as well as the expression levels and differentiation trajectories of genes associated with embryo attachment and endometrial receptivity in epithelial and stromal cells. The results of cell communication interactions analysis showed that ciliated cells were more active in endometrial tissue with embryo loss, and there were differences in the types of endometrial cells with major roles in embryo loss and embryo implantation successful tissues for bone morphogenic protein, insulin‐like growth factor, and transforming growth factor‐β signaling networks associated with embryo implantation. In addition, we compared the functional differences in immune cells between the two tissue types and the expression levels of genes related to the inflammatory microenvironment. Overall, the present study revealed the molecular features of endometrial cell transcription in embryo‐lost endometrial tissues, providing deeper insights into the endometrial microenvironment of reproductive disorders, which may inform the etiological, diagnostic, and therapeutic studies of reproductive disorders.

(1) Background: Spermatozoa acquired motility and matured in epididymis after production in the testis. However, there is still limited understanding of the specific characteristics of sperm development across different species. In this study, we employed a comprehensive approach to analyze cell compositions in both testicular and epididymal tissues, providing valuable insights into the changes occurring during meiosis and spermiogenesis in mouse and pig models. Additionally, we identified distinct gene expression signatures associated with various spermatogenic cell types. (2) Methods: To investigate the differences in spermatogenesis between mice and pigs, we constructed a single-cell RNA dataset. (3) Results: Our findings revealed notable differences in testicular cell clusters between these two species. Furthermore, distinct gene expression patterns were observed among epithelial cells from different regions of the epididymis. Interestingly, regional gene expression patterns were also identified within principal cell clusters of the mouse epididymis. Moreover, through analysing differentially expressed genes related to the epididymis in both mouse and pig models, we successfully identified potential marker genes associated with sperm development and maturation for each species studied. (4) Conclusions: This research presented a comprehensive single-cell landscape analysis of both testicular and epididymal tissues, shedding light on the intricate processes involved in spermatogenesis and sperm maturation, specifically within mouse and pig models.

As a crucial component of the male reproductive system, the epididymis plays multiple roles, including sperm storage and secretion of nutritive fluids for sperm development and maturation. The acquisition of fertilization capacity by sperm occurs during their transport through the epididymis. Compared with the testis, little has been realized about the importance of the epididymis. However, with the development of molecular biology and single-cell sequencing technology, the importance of the epididymis for male fertility should be reconsidered. Recent studies have revealed that different regions of the epididymis exhibit distinct functions and cell type compositions, which are likely determined by variations in gene expression patterns. In this research, we primarily focused on elucidating the cellular composition and region-specific gene expression patterns within different segments of the epididymis and provided detailed insights into epididymal function in male fertility.

Twinning trait in donkeys is an important manifestation of high fecundity, but few reports are available elucidating its genetic mechanism. To explore the genetic mechanism underlying the twin colt trait in Dezhou donkeys, DNA from 21 female Dezhou donkeys that had birthed single or twin colts were collected for whole-genome resequencing. F_ST, θπ and Tajima’s D were used to detect the selective sweeps between single and twin colt fecundity in the Dezhou donkey groups. Another set of 20 female Dezhou donkeys with single or multiple follicles during estrus were selected to compare concentrations of reproductive hormone including follicle-stimulating hormone (FSH), luteinizing hormone (LH), estradiol (E₂) and progesterone (P₄). Four candidate genes including ENO2, PTPN11, SOD2 and CD44 were identified in the present study. The CD44 gene had the highest F_ST value, and ENO2, PTPN11 and SOD2 were screened by two joint analyses (F_ST and θπ, θπ and Tajima’s D). There was no significant difference in the LH, FSH and P₄ levels between the two groups (p > 0.05); however, the serum E₂ content in the multi-follicle group was significantly higher than that in the single-follicle group (p < 0.05). The identified candidate genes may provide new insights into the genetic mechanism of donkey prolificacy and may be useful targets for further research on high reproductive efficiency.