The quantity of Sertoli cells in the adult testis decides the daily gamete formation, and accumulating evidence indicates that epigenetic factors regulate the proliferation of Sertoli cells. Research on the function and regulatory mechanism of microRNAs (miRNAs) in Sertoli cells has not been comprehensive yet, especially on domestic animals. In this article, we report that miR-126 controls the proliferation and apoptosis of immature porcine Sertoli cells based on previous studies. Our results confirmed that miR-126 elevation promotes cell cycle progression, cell proliferation and represses cell apoptosis; on the contrary, the inhibitory effects of miR-126 result in the opposite. The phosphoinositide-3-kinase regulatory subunit 2 (PIK3R2) gene, a member of the PI3K family, was verified as a direct target of miR-126 using the dual-luciferase reporter analysis. miR-126 negatively regulated the mRNA and protein expression level of PIK3R2 in immature porcine Sertoli cells. siRNA-induced PIK3R2 inhibition caused similar effects as miR-126 overexpression and eliminated the influences of miR-126 knockdown in immature porcine Sertoli cells. In addition, both miR-126 overexpression and PIK3R2 inhibition elevated the phosphorylation of PI3K and AKT, whereas the miR-126 knockdown demonstrated the contrary result. In short, miR-126 controls the proliferation and apoptosis of immature porcine Sertoli cells by targeting the PIK3R2 gene through the PI3K/AKT signaling pathway. The research supplies a theoretical and practical foundation for exploring the functional parts of miR-126 in swine sperm by defining the destiny of immature Sertoli cells.

RNA N⁶-methyladenosine (m⁶A) modification is one of the principal post-transcriptional modifications and plays a dynamic role in testicular development and spermatogenesis. However, the role of m⁶A in porcine testis is understudied. Here, we performed a comprehensive analysis of the m⁶A transcriptome-wide profile in Shaziling pig testes at birth, puberty, and maturity. We analyzed the total transcriptome m⁶A profile and found that the m⁶A patterns were highly distinct in terms of the modification of the transcriptomes during porcine testis development. We found that key m⁶A methylated genes (AURKC, OVOL, SOX8, ACVR2A, and SPATA46) were highly enriched during spermatogenesis and identified in spermatogenesis-related KEGG pathways, including Wnt, cAMP, mTOR, AMPK, PI3K-Akt, and spliceosome. Our findings indicated that m⁶A methylations are involved in the complex yet well-organized post-transcriptional regulation of porcine testicular development and spermatogenesis. We found that the m⁶A eraser ALKBH5 negatively regulated the proliferation of immature porcine Sertoli cells. Furthermore, we proposed a novel mechanism of m⁶A modification during testicular development: ALKBH5 regulated the RNA methylation level and gene expression of SOX9 mRNA. In addition to serving as a potential target for improving boar reproduction, our findings contributed to the further understanding of the regulation of m⁶A modifications in male reproduction.

The regulatory role of non-CpG methylation in mammals has been important in whole-genome bisulfite sequencing. It has also been suggested that non-CpG methylation regulates gene expression to affect the development and health of mammals. However, the dynamic regulatory mechanisms of genome-wide, non-CpG methylation during testicular development still require intensive study. In this study, we analyzed the dataset from the whole-genome bisulfite sequencing (WGBS) and the RNA-seq of precocious porcine testicular tissues across two developmental stages (1 and 75 days old) in order to explore the regulatory roles of non-CpG methylation. Our results showed that genes regulated by non-CpG methylation affect the development of testes in multiple pathways. Furthermore, several hub genes that are regulated by non-CpG methylation during testicular development—such as VEGFA, PECAM1, and FZD7—were also identified. We also found that the relative expression of FZD7 was downregulated by the zebularine-induced demethylation of the first exon of FZD7. This regulatory relationship was consistent with the results of the WGBS and RNA-seq analysis. The immature porcine Sertoli cells were transfected with RNAi to mimic the expression patterns of FZD7 during testicular development. The results of the simulation test showed that cell proliferation was significantly impeded and that cell cycle arrest at the G2 phase was caused by the siRNA-induced FZD7 inhibition. We also found that the percentage of early apoptotic Sertoli cells was decreased by transfecting them with the RNAi for FZD7. This indicates that FZD7 is an important factor in linking the proliferation and apoptosis of Sertoli cells. We further demonstrated that Sertoli cells that were treated with the medium collected from apoptotic cells could stimulate proliferation. These findings will contribute to the exploration of the regulatory mechanisms of non-CpG methylation in testicular development and of the relationship between the proliferation and apoptosis of normal somatic cells.

Normal testicular development ensures the process of spermatogenesis, which is a complex biological process. The sustained high productivity of spermatogenesis throughout life is predominantly attributable to the constant proliferation and differentiation of spermatogonial stem cells (SSCs). The self-renewal and differentiation processes of SSCs are strictly regulated by the SSC niche. Therefore, understanding the developmental pattern of SSCs is crucial for spermatogenesis. The Shaziling pig is a medium-sized indigenous pig breed originating from central China. It is renowned for its superior meat quality and early male sexual maturity. The spermatogenic ability of the boars is of great economic importance to the pig industry. To investigate testicular development, particularly the pattern of SSC development in Shaziling pigs, we used single-cell transcriptomics to identify gene expression patterns in 82,027 individual cells from nine Shaziling pig testes at three key postnatal developmental stages. We generated an unbiased cell developmental atlas of Shaziling pig testicular tissues. We elucidated the complex processes involved in the development of SSCs within their niche in the Shaziling pig. Specifically, we identified potential marker genes and cellular signaling pathways that regulate SSC self-renewal and maintenance. Additionally, we proposed potential novel marker genes for SSCs that could be used for SSC isolation and sorting in Shaziling pigs. Furthermore, by immunofluorescence staining of testicular tissues of different developmental ages using marker proteins (UCHL1 and KIT), the developmental pattern of the spermatogonia of Shaziling pigs was intensively studied. Our research enhances the comprehension of the development of SSCs and provides a valuable reference for breeding Shaziling pigs.

Aging is characterized by cellular degeneration and impaired physiological functions, leading to a decline in male sexual desire and reproductive capacity. Oxidative stress (OS) lead to testicular aging by impairing the male reproductive system, but the potential mechanisms remain unclear. In the present study, the functional status of testicular tissues from young and aged boars was compared, and the transcriptional responses of Leydig cells (LCs) to hydrogen peroxide (H₂O₂)-induced senescence were explored, revealing the role of OS in promoting aging of the male reproductive system. 601 differentially expressed genes (DEGs) associated with OS, cell cycle regulation, and intracellular processes were identified. These DEGs were significantly enriched in critical aging pathways, including the p53 signaling pathway, autophagy, and cellular senescence. Protein-protein interaction (PPI) network analysis unveiled 15 key genes related to cell cycle and DNA replication, with polo-like kinase 3 (PLK3) exhibiting increased expression under OS. In vitro, PLK3 knockdown significantly enhanced the viability and antioxidant capacity of LCs under OS. This study deepens our understanding of how LCs respond to OS and provides new therapeutic targets for enhancing cellular resistance to oxidative damage and promoting tissue health.