ABSTRACT Humanin (HN), a mitochondrial-derived peptide with cytoprotective functions, has not been thoroughly investigated in the bull gonad. This study characterized the developmental localization of HN in the bull testis and its relationship with hormonal status, oxidative stress, and key miRNAs. Immunofluorescence analysis of testicular tissues from pre-pubertal, pubertal, mature, and aged bulls (N = 10/group) revealed a distinct age-dependent expression pattern. HN immunoreactivity was faint in pre-pubertal and aged groups, moderate in pubertal animals, and most intense in the somatic and germ cells of mature adults (P ≤ 0.0001). Quantitatively, HN levels correlated positively with serum testosterone (r = 0.5852, p < 0.01) and the antioxidant enzyme SOD (r = 0.8208, p < 0.001), and negatively with the oxidative stress marker MDA (r = - 0.7140, p < 0.001). Furthermore, a strong positive correlation was found between HN and miR-202-5p expression (r = 0.8196, p < 0.001), while both miRNAs (miR-202-5p and miR-21) were highly expressed pre-pubertally and declined with maturation. These results provide the first evidence of developmentally-specific HN expression in the bull testis, strongly associating it with peak reproductive maturity, hormonal balance, and redox homeostasis. The correlation with miR-202-5p suggests a potential novel regulatory network, positioning HN as a key peptide in testicular function and aging. The findings from this investigation could provide fundamental insights aligned with previous research on the expression of HN across various species and could support further studies involve the distribution of HN during gonad development for improving the reproductive performance of livestock.
