Abstract

Male infertility is increasingly linked to impaired spermatogenesis, with emerging evidence implicating the umami taste receptor T1R1/T1R3 in male reproductive regulation. This study explored its role in regulating autophagy during spermatogenesis in Xiang pigs. Immunofluorescence confirmed T1R1/T1R3 co-localization in spermatogenic cells. Knockdown of TAS1R3 by RNAi triggered robust autophagy, characterized by increased autophagosomes, mitochondrial swelling, a significant accumulation of 2   C (diploid, spermatogonia) cells (increasing by ~21%), a corresponding reduction 4   C (tetraploid, spermatocytes) cells (decreasing by ~11%), a marked upregulation of Beclin 1 protein (by ~8.0-fold) and the LC3-II/LC3-I ratio (by ~2.9-fold), downregulation of mTOR , and suppression of key spermatogenic genes ( POU5F1 , SYCP3 , STRA8 ). Conversely, the receptors activation by its natural ligand L-glutamate (L-Glu) suppressed autophagy, reducing Beclin 1 levels and the LC3-II/LC3-I ratio to approximately 0.7-fold of the control. This activation improved cellular function, upregulated mTOR, increased 1   C (haploid, spermatids) cell populations, and downregulated STRA8 . Under H₂O₂-induced autophagy, L-Glu restored T1R3 expression, thereby reversing excessive autophagy and calcium overload, and rescuing the expression of autophagy-related ( BECN1 , mTOR ) and spermatogenesis-related genes ( SYCP3 , STRA8 ). In vivo experiments, dietary supplementation with 0.5% L-Glu significantly increased seminiferous tubule diameter and epithelium thickness in Xiang pig testes, upregulating T1R1 and the autophagy substrate p62/SQSTM1. In conclusion, T1R1/T1R3 regulates spermatogenesis by modulating autophagy. Activation by L-Glu mitigates autophagic damage and promotes spermatogenesis, offering a novel strategy for enhancing male fertility by targeting T1R1/T1R3.