Leydig cells (LCs) are testosterone-generating endocrine cells that are located outside the seminiferous tubules in the testis, and testosterone is fundamental for retaining spermatogenesis and male fertility. In buffalo, adult Leydig cells (ALCs) are developed by immature Leydig cells (ILCs) in the postnatal testes. However, the genes/pathways associated to the regulation of testosterone secretion function during the development of postnatal LCs remains comprehensively unidentified. The present study comparatively analyzed the transcriptome profiles of ILC and ALC in buffalo with significant differences in testosterone secretion. Differentially expressed genes (DEGs) analysis identified 972 and 1,091 annotated genes that were significantly up- and down-regulated in buffalo ALC. Functional enrichment analysis showed that cAMP signaling being the most significantly enriched pathway, and testosterone synthesis and lipid transport-related genes/pathways were upregulated in ALC. Furthermore, gene set enrichment analysis (GSEA) shows that cAMP signaling and steroid hormone biosynthesis were activated in ALC, demonstrating that cAMP signaling may serve as a positive regulatory pathway in the maintenance of testosterone function during postnatal development of LCs. Protein-protein interaction (PPI) networks analysis highlighted that ADCY8, ADCY2, POMC, CHRM2, SST, PTGER3, SSTR2, SSTR1, NPY1R, and HTR1D as hub genes in the cAMP signaling pathway. In conclusion, this study identified key genes and pathways associated in the regulation of testosterone secretion function during the ILC-ALC transition in buffalo based on bioinformatics analysis, and these key genes might be deeply involved in cAMP generation to influencing testosterone levels in LCs. The results suggest that ALCs might increase testosterone levels by enhancing cAMP production than ILCs. Our data will enhance the understanding of developmental mechanism studies related to testosterone function and provide preliminary evidence for molecular mechanisms of LCs regulating spermatogenesis.

Leydig 细胞 (LC) 是产生睾酮的内分泌细胞，位于睾丸中的生精小管外，而睾酮是保持精子发生和男性生育能力的基础。在水牛中，成年 Leydig 细胞 (ALC) 由出生后睾丸中的未成熟 Leydig 细胞 (ILC) 发育而成。然而，在出生后 LC 的发展过程中与睾酮分泌功能调节相关的基因/途径仍未完全确定。本研究比较分析了水牛ILC和ALC的转录组谱，睾酮分泌存在显着差异。差异表达基因 (DEG) 分析确定了 972 和 1,091 个注释基因，这些基因在水牛 ALC 中显着上调和下调。功能富集分析表明，cAMP 信号是最显着富集的途径，并且睾酮合成和脂质转运相关基因/途径在 ALC 中上调。此外，基因集富集分析 (GSEA) 显示，在 ALC 中 cAMP 信号传导和类固醇激素生物合成被激活，表明 cAMP 信号传导可能在 LC 出生后发育过程中作为维持睾酮功能的正调控途径。蛋白质-蛋白质相互作用 (PPI) 网络分析强调，证明 cAMP 信号可能在 LC 的出生后发育过程中作为维持睾酮功能的正调节途径。蛋白质-蛋白质相互作用 (PPI) 网络分析强调，证明 cAMP 信号可能在 LC 的出生后发育过程中作为维持睾酮功能的正调节途径。蛋白质-蛋白质相互作用 (PPI) 网络分析强调，ADCY8，ADCY2，POMC，CHRM2，SST，PTGER3，SSTR2，SSTR1，NPY1R和HTR1D作为 cAMP 信号通路中的枢纽基因。总之，本研究基于生物信息学分析确定了与水牛ILC-ALC转变过程中睾酮分泌功能调节相关的关键基因和途径，这些关键基因可能与cAMP的产生密切相关，从而影响LCs中的睾酮水平。结果表明，与 ILC 相比，ALC 可能通过增强 cAMP 的产生来提高睾酮水平。我们的数据将增强对与睾酮功能相关的发育机制研究的理解，并为 LC 调节精子发生的分子机制提供初步证据。