Sertoli cells play vital roles in normal spermatogenesis, and microRNAs (miRNAs) participate in regulating Sertoli cell development. However, the functions and mechanisms of action of most identified miRNAs in porcine Sertoli cells remain largely unknown. Herein, we primarily explored the regulatory roles of miR‐130a in immature porcine Sertoli cells using EdU‐based high‐content screening assay. The results demonstrated that 27 miRNAs have potential roles in the promotion of immature porcine Sertoli cell proliferation, and miR‐130a was identified as a promising candidate. miR‐130a promoted cell cycle progression and cell proliferation, whereas it impeded cell apoptosis in immature porcine Sertoli cells. It also contributed to Sertoli cell proliferation and testis development in vivo. A TMT‐based proteomics approach revealed that miR‐130a regulated the expression of 91 proteins and multiple pathways, including the TGF‐β and PI3K/AKT signaling. miR‐130a did not directly target the 3′‐UTR of SMAD5 ; however, it increased SMAD5 phosphorylation. Moreover, miR‐130a enhanced TGF‐β signaling by activating SMAD5 protein, and TGF‐β signaling further activated the PI3K/AKT signaling pathway to promote cell proliferation and inhibit cell apoptosis in porcine immature Sertoli cells. Collectively, miR‐130a promoted immature porcine Sertoli cell growth by activating SMAD5 through the TGF‐β‐PI3K/AKT signaling pathway. This study, therefore, provides novel insights into the effects of miR‐130a on porcine spermatogenesis through the regulation of immature Sertoli cell proliferation and apoptosis.

In the genomes of diploid organisms, runs of homozygosity (ROH), consecutive segments of homozygosity, are extended. ROH can be applied to evaluate the inbreeding situation of individuals without pedigree data and to detect selective signatures via ROH islands. We sequenced and analyzed data derived from the whole-genome sequencing of 97 horses, investigated the distribution of genome-wide ROH patterns, and calculated ROH-based inbreeding coefficients for 16 representative horse varieties from around the world. Our findings indicated that both ancient and recent inbreeding occurrences had varying degrees of impact on various horse breeds. However, recent inbreeding events were uncommon, particularly among indigenous horse breeds. Consequently, the ROH-based genomic inbreeding coefficient could aid in monitoring the level of inbreeding. Using the Thoroughbred population as a case study, we discovered 24 ROH islands containing 72 candidate genes associated with artificial selection traits. We found that the candidate genes in Thoroughbreds were involved in neurotransmission (CHRNA6, PRKN, and GRM1), muscle development (ADAMTS15 and QKI), positive regulation of heart rate and heart contraction (HEY2 and TRDN), regulation of insulin secretion (CACNA1S, KCNMB2, and KCNMB3), and spermatogenesis (JAM3, PACRG, and SPATA6L). Our findings provide insight into horse breed characteristics and future breeding strategies.

DNA methylation is one of the key epigenetic regulatory mechanisms in development and spermatogenesis. However, the dynamic regulatory mechanisms of genome-wide DNA methylation during testicular development remain largely unknown. Herein, we generated a single-base resolution DNA methylome and transcriptome atlas of precocious porcine testicular tissues across three developmental stages (1, 75, and 150 days old). The results showed that the dynamic methylation patterns were directly related to the expression of the DNMT3A gene. Conjoint analysis revealed a negative regulatory pattern between promoter methylation and the positive regulation of 3'-untranslated region (3'UTR) methylation. Mechanistically, the decrease in promoter methylation affected the upregulation of meiosis-related genes, such as HORMAD1, SPO11, and SYCE1. Demethylation in the 3'UTR induced the downregulation of the INHBA gene and knockdown of INHBA inhibited cell proliferation by reducing the synthesis of activin A. These findings contribute to exploring the regulatory mechanisms of DNA methylation in testicular development.

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.

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.