Knowledge about the relationship between steroidogenesis and the regulation of the mitochondrial bioenergetics and dynamics, in steroidogenic cells, is not completely elucidated. Here we employed in vivo and ex vivo experimental models to analyze mitochondrial physiology in Leydig cells depending on the different LH-cAMP environments. Activation of LH-receptor in rat Leydig cells ex and in vivo triggered cAMP, increased oxygen consumption, mitoenergetic and steroidogenic activities. Increased mitoenergetic activity i.e., ATP production is achieved through augmented glycolytic ATP production and a small part of oxidative phosphorylation (OXPHOS). Transcription of major genes responsible for mitochondrial dynamics was upregulated for Ppargc1a (regulator of mitogenesis and function) and downregulated for Drp1 (main fission marker), Prkn, Pink1 and Tfeb (mitophagy markers). Leydig cells from gonadotropin-treated rats show increased mitogenesis confirmed by increased mitochondrial mass, increased mtDNA, more frequent mitochondria observed by a transmission electron microscope and increased expression of subunits of respiratory proteins Cytc/CYTC and COX4. Opposite, Leydig cells from hypogonadotropic-hypogonadal rats characterized by low LH-cAMP, testosterone, and ATP production, reduced markers of mitogenesis and mitofusion (Mfn1/2, Opa1) associated with reduced mtDNA content. Altogether results underline LH-cAMP signaling as an important regulator of mitochondrial physiology arranging mitochondrial dynamics, bioenergetic and steroidogenic function in Leydig cells.

Circadian desynchrony, caused by a misalignment between the internal biological clock and environmental light cues, is increasingly prevalent in adolescents due to irregular light exposure and social pressures. However, its impact on reproductive maturation remains poorly understood. In this study, the effects of chronic circadian disruption, induced by the 223 light regimen (two days of constant light, two days of constant darkness, and three days of a 14:10 h light-dark cycle), were examined in juvenile and peripubertal male rats (postnatal days 21-49). Gene expression profiles associated with Leydig cell maturation, including steroidogenic, mitochondrial, and clock-related genes, as well as markers of germ cell differentiation, were analyzed alongside functional mitochondrial parameters in Leydig cells. Under control conditions, Leydig cell maturation was marked by increased expression of core clock genes, steroidogenic enzymes (Star, Cyp11a1, Hsd3b1/2), and mitochondrial biogenesis and dynamics markers (Tfam, Nrf1, Cytc, Opa1, Mfn2). These transcriptional changes coincided with rising mitochondrial content, membrane potential, ATP levels, serum androgens, and progression of spermatogenesis. Conversely, the 223-regimen disrupted behavioral rhythms, reduced circulating melatonin, blunted expression of maturation-associated genes, and shifted the acrophase of key steroidogenic and circadian transcripts in 49-day-old rats, indicating altered Leydig cell rhythmicity. These molecular disruptions were accompanied by decreased testosterone levels, altered expression of spermatid differentiation genes (Tnp1 and Prm2), and a reduction in the number of elongated spermatids at stage VII of spermatogenesis. In conclusion, circadian misalignment disrupts endocrine and transcriptional coordination during Leydig cell development, underscoring the vulnerability of pubertal reproductive maturation to environmental light disturbances.

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.