Developmental stage-specific transcriptomics of the hypothalamus-pituitary-testis axis reveals gene networks governing testicular maturation in Xianghuang chickens.

Sexual maturity significantly impacts poultry production efficiency, yet data on testicular development and regulatory mechanisms in indigenous breeds remain limited. In this study, we examined Xianghuang chickens, a indigenous early-maturing breed, to investigate the hypothalamus-pituitary-testis (HPT) axis. Hypothalamus, pituitary, and testis tissues were collected at 80 and 120 days post-hatch (dph) for analysis. Results showed that testicular weight and index were significantly higher in 120dph compared to 80dph chickens. Histologically, the testes at 120dph contained multi-layered spermatocytes and mature sperm while the testes at 80dph only had 1-2 layers of spermatocytes. Weighted Gene Co-expression Network Analysis of transcriptome data clustered gene sets into 11 modules including those with testis-specific high expression, 80dph testis-specific high expression, and 120dph testis high expression. Differential expression analysis identified 26, 681, and 11,999 differentially expressed genes (DEGs) in the hypothalamus, pituitary, and testes respectively between the two age groups with 16 DEGs shared among the three tissues. DEGs in all three tissues were enriched in pathways such as cytoskeleton in muscle cells, ECM-receptor interaction, and focal adhesion highlighting the importance of HPT axis tissue remodeling during this period. Hormone-related pathways (GnRH signaling, melanogenesis, and progesterone-mediated oocyte maturation) were enriched with DEGs in the pituitary and testes between 80dph and 120dph . Concurrently the pituitary and testes exhibited distinct lipid metabolic adaptations through enrichment of PPAR, adipocytokine, and insulin signaling pathways. These adaptations provided substrates for sperm membrane formation and testosterone synthesis while supporting spermatogenesis and cellular functions. Collectively these findings clarify the morphological, histological, and molecular regulatory mechanisms underlying testicular development and sexual maturation in Xianghuang chickens and offer a theoretical basis for optimizing poultry breeding strategies.