Abstract
This study examined the effect of continuous dark exposure (DD) on male reproductive maturation in a rat model. Male rats were reared without light from postnatal day (pnd) 21 and analyzed at pnd 35 (juvenile), 42 (peripubertal), and 90 (adult), periods related to key stages of Leydig cell development. DD disrupted circadian locomotor activity and reduced overall physical activity. Early reductions in seminal vesicle and dorsal prostate weights suggested delayed androgen-dependent development. DD exposure significantly lowered circulating androgens and reduced the testosterone-to-corticosterone (T/C) ratio. Circadian profiling revealed flattened rhythms of testosterone, corticosterone, and melatonin, as well as misaligned activity patterns in adulthood, indicating impaired coordination between the circadian and endocrine systems. At the molecular level, DD suppressed the pubertal rise of gonadotropins and Leydig cell-specific gene expression and disrupted transcriptional coordination within pathways regulating steroidogenesis, mitochondrial function, and circadian timing. Key steroidogenic genes (Scarb1, Star, Hsd3b1/2, Cyp17a1) were downregulated under DD, particularly in adulthood. Despite elevated mitochondrial membrane potential, ATP levels were reduced, suggesting functional uncoupling. Genes involved in mitochondrial remodeling (Opa1, Mfn1, Drp1) and mitophagy (Pink1) were upregulated, suggesting an adaptive response to DD. Clock gene expression showed progressive activation during development under normal light conditions but was dysregulated under DD, especially during the peripubertal period. Accordingly, DD delayed spermatid maturation, downregulated differentiation markers (Tnp1, Prm2), and reduced both epididymal sperm count and progesterone-supported acrosome reaction. These findings highlight the important role of environmental light in synchronizing endocrine, mitochondrial, and circadian pathways that regulate Leydig cell development and male fertility.