Beyond its fundamental biological importance, this work also has translational significance. In modern societies, widespread exposure to artificial light, irregular sleep-wake cycles, and shift work are increasingly prevalent, particularly among adolescents26. Such circadian misalignment during critical developmental windows may disrupt hormonal rhythms, delay sexual maturation, and compromise fertility27. By examining the endocrine and cellular effects of light deprivation during puberty, this study provides new insights into how circadian regulation functions in the absence of external light cues and underscores the importance of chronobiologically informed strategies to prevent light-related reproductive disorders.

The development of adult Leydig cells during puberty is essential for the initiation and maintenance of spermatogenesis, as well as the emergence of male secondary sexual characteristics. To investigate how DD affects Leydig cell maturation, male rats were reared in complete darkness from postnatal day (pnd) 21 and examined at pnd 35, 42, and 90. Age-matched control animals were kept under a standard summer photoperiod of 14 h light and 10 h dark (LD). These time points were selected to represent key phases of Leydig cell development: the juvenile stage (pnd 21-35), marked by proliferative progenitor and immature Leydig cells; the peripubertal stage (pnd 42), reflecting the transitional period toward functional maturation; and the adult stage (pnd 90), characterized by fully differentiated Leydig cells capable of producing testosterone to support full reproductive function.

To assess behavioral circadian patterns, 24-hour locomotor activity was continuously recorded from pnd 60 to 90. In parallel, the expression of genes involved in Leydig cell differentiation and steroidogenic function was measured to evaluate the molecular impact of light deprivation. Additionally, sperm parameters were analyzed in adulthood to assess the potential consequences of disrupted androgen production on male fertility.

Rats are nocturnal animals, typically active during the dark phase and inactive during the light phase of a standard light-dark (LD) cycle (Fig. 1A upper panel). However, exposure to DD altered this pattern, with the onset of activity shifting earlier each day (Fig. 1A, lower panel). This behavioral shift was accompanied by a shortened free-running circadian period (τ = 23.83 ± 0.05 h), reflecting a change in the endogenous clock. Furthermore, rats exposed to DD exhibited significantly increased activity during the subjective day (Fig. 1A, lower panel), suggesting a disruption in circadian organization. Despite this altered temporal distribution of activity, total daily locomotor output was substantially reduced in DD rats by approximately 40% compared to controls (Fig. 1B). Specifically, average wheel rotations per day were 4411 ± 988 in LD rats, versus 1870 ± 637 in those kept under DD conditions.

Body mass measurements at pnd 35, 42, and 90 did not differ significantly between groups (Fig. 1C). However, subtle effects of constant darkness were observed in the development of androgen-dependent organs. While the weights of the testes (Fig. 1D), seminal vesicles (Fig. 1E), and both the dorsal (Fig. 1F) and ventral prostate (Fig. 1G) increased with age in both groups, DD rats showed significantly lower seminal vesicle and dorsal prostate weights at pnd 35 (Fig. 1E, F). This transient delay in accessory sex organ development likely reflects a temporary adjustment during early puberty. By pnd 90, however, the weights of all reproductive organs in DD rats were comparable to those of LD controls (Fig. 1D-G).

To assess the impact of DD on endocrine pathways involved in reproductive maturation, circulating levels of androgens, corticosterone, and melatonin (hormones with well-established circadian rhythms that act as systemic cues for peripheral clocks, including those in Leydig cells were measured. Blood levels of androgens and corticosterone were monitored throughout postnatal development. As expected, androgen concentrations progressively increased from the juvenile period to adulthood, reflecting normal reproductive maturation (Fig. 2A). However, rats exposed to DD exhibited significantly reduced androgen levels, particularly in adulthood (Fig. 2A). Corticosterone concentrations also fluctuated across development, peaking around puberty (Fig. 2B), consistent with its role in supporting metabolic and endocrine adaptation during this critical period. Notably, DD exposure led to an early elevation in corticosterone at pnd 35 (Fig. 2B), suggesting adrenal activation during a sensitive window in reproductive development.

To further evaluate Leydig cell steroidogenic capacity in the context of stress and reduced activity, the testosterone-to-corticosterone (T/C) ratio was calculated. This ratio reflects the balance between androgen and glucocorticoid signaling and was found to increase progressively during postnatal development in control animals (Fig. 2C). In contrast, a significantly reduced T/C ratio was observed in the DD group, which coincided with markedly lower locomotor activity, indicating that circadian disruption may impair androgen output relative to stress hormone levels (Fig. 2C).

To determine whether these effects were linked to altered circadian regulation, hormone rhythms in adult rats were analyzed at five circadian time points (ZT/CT3, 7, 11, 17, and 23) under both LD and DD conditions. Blood levels of testosterone, corticosterone, and melatonin were measured and compared with locomotor activity. Because these hormones exhibit circadian rhythms closely aligned with behavioral states, their temporal profiles could provide insight into how constant darkness affects the coordination of endocrine and behavioral rhythms. In LD conditions, as expected for a nocturnal species, rats displayed a sharp peak in activity at ZT17, coinciding with the onset of the dark phase (Fig. 2D, black line). Testosterone levels began to rise around ZT7 and peaked around ZT11, aligning with the start of the active phase and reflecting the circadian regulation of Leydig cell steroidogenesis (Fig. 2D, red line). The diurnal pattern of corticosterone peaking at ZT11 demonstrated a robust rhythmicity under LD conditions consistent with a preparatory role in energy mobilization before the onset of activity (Fig. 2D, green line). As expected, melatonin levels gradually increased and remained elevated throughout the dark phase, in line with its light-sensitive synthesis in rodents (Fig. 2D, blue line). Together, these coordinated hormonal and behavioral rhythms highlight the strong circadian integration of endocrine function and activity under normal photoperiodic conditions (Fig. 2D).

In contrast, rats exposed to DD exhibited disrupted patterns across all measured parameters. Locomotor activity appeared phase-shifted and more diffusely distributed, lacking the distinct onset observed under LD conditions (Fig. 2E, black line). Testosterone levels showed a flattened rhythm with a markedly reduced peak, indicating a disruption in the circadian regulation of steroidogenesis (Fig. 2E, red line). Similarly, corticosterone lost its typical diurnal pattern, presenting as a blunted profile without a clearly defined peak (Fig. 2E, green line). Although melatonin retained rhythmicity, its pattern was less pronounced than in LD animals, suggesting that while endogenous circadian regulation of melatonin synthesis persists, it is weakened in the absence of photic entrainment (Fig. 2E, blue line). Together, these findings suggest that constant darkness disrupts the temporal synchronization between endocrine output and behavior, underscoring the crucial role of environmental light in maintaining coordinated circadian and reproductive hormone rhythms (Fig. 2E). Additionally, the expression levels of hormone receptors, melatonin receptor 1a (Mtnr1a) and glucocorticoid receptor (Nr3c1), in both the pituitary and Leydig cells of adult rats were not significantly affected by DD exposure (Fig. 2F-I), indicating that the observed androgen changes likely result from circadian modifications in hormone synthesis rather than altered receptor abundance.

To further understand how DD influences the endocrine mechanisms underlying reproductive development, the expression of key developmental markers in the pituitary and Leydig cells during critical windows of puberty was examined. The analysis focused on juvenile, peripubertal, and adult stages. Specifically, the expression of genes encoding pituitary hormones, LH and FSH (Cga, Lhb, Fshb), the LH receptor (Lhcgr), and the GnRH receptor (Gnrhr), as well as key enzymes involved in Leydig cell maturation and androgen metabolism (Srd5a, Akr1c14), was assessed.

As expected, a gradual increase in the expression of pituitary Cga, Lhb, Fshb, and Gnrhr was observed during maturation (Fig. 3A, B, C, D). The maturation of Leydig cells was accompanied by an increase in the expression of Insl3 (Fig. 3F), a marker of Leydig cell maturity and functionality. Additionally, two markers of Leydig cell maturation, which encode enzymes involved in steroid metabolism, exhibited distinct expression patterns. Leydig cells from juvenile rats (35 pnd) showed high levels of steroid 5α-reductase 1 (Srd5a1), responsible for the synthesis of 5α-reduced androgens, but this expression decreased with maturation to adult Leydig cells (Fig. 3G). In contrast, the transcription of 3α-hydroxysteroid hydrogenase (Akr1c14), an enzyme responsible for converting 3-ketosteroids into 3α-hydroxysteroids, decreased as Leydig cells matured (Fig. 3H).

Under constant darkness (DD), the expression of gonadotropin subunit genes (Cga, Lhb, Fshb) was elevated during the juvenile and peripubertal stages (pnd 35 and 42), but significantly reduced in adulthood (pnd 90), indicating a dysregulated temporal pattern of gonadotropin gene activation (Fig. 3A-C). This persistent suppression indicates impaired activation of pituitary gonadotropin synthesis during postnatal development. Similarly, Gnrhr expression (Fig. 3D) showed only a modest increase at pnd 42 and was significantly reduced compared to LD levels by adulthood, suggesting diminished GnRH responsiveness in the pituitary under DD conditions.

In the Leydig cells, Lhcgr (Fig. 3E), Insl3 (Fig. 3F), and Srd5a (Fig. 3G) expression levels in DD rats remain largely unchanged or lower across time points compared to LD. Notably, Akr1c14 (Fig. 3H) is elevated at pnd 42 under DD, but declines thereafter, possibly reflecting a transient or compensatory response during maturation.

To further characterize Leydig cell maturation, the expression of genes related to steroidogenesis (Fig. 4A), mitochondrial dynamics (Fig. 4B), and circadian regulation (Fig. 4C) was analyzed, as these pathways are essential for supporting their differentiation and function.

Principal component analysis (PCA) assessed coordinated transcriptional changes during Leydig cell maturation. While the analysis revealed clear clustering of steroidogenesis-related genes according to developmental stage, Leydig cells from peripubertal rats (pnd 42) did not form a strong gene cluster, suggesting limited or transitional transcriptional activation at this time point (Fig. 4A). Only a few genes, including the negative steroidogenic regulator Gata4 and the enzyme Hsd17b4, were associated with this stage, possibly reflecting an early shift toward steroidogenic differentiation rather than full functional maturation. In contrast, adult Leydig cells (pnd 90) clustered with genes such as Scarb1 and Star (involved in cholesterol uptake and mitochondrial import), Cyp11a1 (catalyzing the conversion of cholesterol to pregnenolone), Cyp17a1 (responsible for converting progesterone to androstenedione), and transcription factors Sf1, Dax1, and Creb1a, which are established positive regulators of steroidogenesis. This transcriptional profile reflects the establishment of a mature and fully competent steroidogenic phenotype in adult Leydig cells. Notably, Hsd3b1/2 and Nur77 clustered with the pnd 35 group, indicating their involvement in the early activation of the steroidogenic program. These genes likely represent initial functional and regulatory responses in progenitor Leydig cells, preceding the robust expression of steroidogenic genes characteristic of fully differentiated adult cells, such as Star, Cyp17a1, and Sf1.

Similarly, PCA of mitochondria-related genes (Fig. 4B) revealed distinct developmental clustering. At pnd 42, Leydig cells formed a separate cluster associated with genes such as Tfam, Nrf1, Cytc, and Cox4i2, which are involved in mitochondrial biogenesis and respiratory function. This pattern suggests metabolic upregulation during the peripubertal period to meet the increased energy demands associated with steroidogenic activation. Genes related to mitochondrial fusion (Opa1, Mfn1) and fission (Drp1, Fis1) also exhibited coordinated expression, indicating active mitochondrial remodeling during this transitional phase. By pnd 90, the clustering pattern shifted again, now associated with Prkn and Pink1 (key regulators of mitophagy) and Ppargc1a, a master regulator of mitochondrial biogenesis. This transition is consistent with the stabilization and refinement of mitochondrial function in adult Leydig cells. Overall, the PCA highlights mitochondrial remodeling as a critical component of Leydig cell maturation, supporting the energetic and biosynthetic requirements of steroidogenesis in adults.

PCA of core clock genes (Fig. 4C) revealed the strongest transcriptional activity during the peripubertal transition. Most clock genes, including positive Clock, and negative feedback loop regulators Cry1, Cry2, Reverba, Reverbb, Per1 and Rora, cluster closely at that period, suggesting synchronized upregulation. In contrast, 90 pnd form a more dispersed cluster, indicating more stabilized expression of clock genes in adult Leydig cells. Notably, Bmal1 and Per2 appear to diverge from the main cluster, possibly due to unique regulatory dynamics or phase shifts in their expression relative to other clock components.

Exposure to DD significantly altered the expression of genes involved in steroidogenesis (Fig. 5), highlighting a sustained disruption of Leydig cell function across critical developmental stages. Specifically, Scarb1 and Star, key genes for cholesterol uptake and mitochondrial import, were downregulated under DD, with Scarb1 affected in both progenitor and adult Leydig cells, and Star showing reduced expression at all examined time points. Among steroidogenic enzymes, the expression of Hsd3b1/2, and Cyp17a1 was significantly decreased in adult Leydig cells, whereas Hsd17b4 expression remained unchanged. Transcriptional activators such as Sf1 and Dax1 showed reduced expression in adult Leydig cells, while Creb1a was selectively downregulated in progenitor stages. In contrast, the repressive factor Gata4 was upregulated in immature Leydig cells, suggesting a potential role in suppressing the steroidogenic gene program under DD conditions. Additionally, Arr19, a marker associated with androgen regulation, was also inhibited in progenitor cells, further indicating compromised early Leydig cell differentiation.

Steroidogenesis in Leydig cells initiates within the mitochondria, which must maintain both mitochondrial membrane potential (Δψm) and efficient ATP production to support this process. The bioenergetic demands of steroid hormone synthesis are closely linked to mitochondrial dynamics, making their regulation essential for sustaining steroidogenic efficiency. Given the critical role of mitochondria in Leydig cell function and maturation, mitochondrial membrane potential, content, and ATP levels were assessed as key indicators of mitochondrial health and steroidogenic capacity.

Leydig cell maturation is accompanied by an increase in Δψm (Fig. 6A) and ATP content (Fig. 6D), while mitochondrial quantity remains unchanged (Fig. 6B). This pattern indicates that in LD, Leydig cells undergo dynamic mitochondrial activation characterized by increased polarization and a boost in energy output, without a need for large changes in mitochondrial quantity.

In contrast, Leydig cells developing under DD show a disrupted mitochondrial profile. Δψm is elevated at all developmental stages compared to LD (Fig. 6A), indicating sustained mitochondrial hyperpolarization. However, this does not correlate with improved function. Despite the high Δψm, mitochondrial content progressively declines, with a significant reduction by adulthood (Fig. 6B). Most notably, ATP production remains consistently low across all time points under DD (Fig. 6D), revealing a clear disconnect between Δψm and bioenergetic output. This suggests a functional uncoupling of the electron transport chain and ATP synthase activity, possibly due to impaired oxidative phosphorylation or mitochondrial stress.

Since mitochondrial function is closely linked to testosterone production in Leydig cells, the correlation analysis was performed between circulating testosterone levels and markers of mitochondrial function (Fig. 6C). Data are presented as a heatmap of correlation coefficients comparing testosterone (T) with TMRE fluorescence (Δψm) and mitochondrial content (MitoTracker), under both LD and DD conditions. In LD animals, positive correlation was observed between testosterone levels and a key indicator of mitochondrial function, Δψm (Fig. 6C). This suggests that under normal photoperiodic conditions, efficient mitochondrial activity supports androgen synthesis in Leydig cells. In contrast, under DD, this correlation was reversed (Fig. 6C). Notably, both Δψm and mitochondrial content showed negative correlations with testosterone levels in DD animals, indicating a decoupling of mitochondrial function from steroidogenic output. These findings imply that without environmental light cues, mitochondrial performance becomes dysregulated, contributing to reduced testosterone synthesis and impaired Leydig cell function.

Furthermore, under LD conditions, the expression of canonical mitochondrial biogenesis genes and genes regulating mitochondrial function, Ppargc1a (Fig. 6E), Tfam (Fig. 6F), Nrf1 (Fig. 6G), and Cox4i2 (Fig. 6I), remained relatively stable, showing no significant transcriptional increases throughout development. This aligns with data indicating that mitochondrial content does not increase with age, suggesting that mitochondrial biogenesis is not the primary regulatory mechanism supporting Leydig cell maturation in this context. Despite the stable mitochondrial quantity, mitochondrial functional capacity seems to increase, as evidenced by the age-dependent rise in Cytc (Fig. 6H), a component of the electron transport chain critical for oxidative phosphorylation. This trend suggests enhanced mitochondrial efficiency or respiratory activity rather than an expansion in mitochondrial mass. However, DD did not significantly alter the expression of these genes (Fig. 6E-I), indicating that the fundamental transcriptional programming of mitochondrial function was preserved.

Additionally, the developmentally regulated expressions of mitochondrial dynamics genes, including Opa1 (Fig. 6J), Mfn1 (Fig. 6K), Mfn2 (Fig. 6M), and Fis1 (Fig. 6N), as well as Drp1 (Fig. 6L), remained stable without a significant increase in LD. In contrast, DD stimulated the expression of fusion-related genes Opa1 (Fig. 6J) and Mfn1 (Fig. 6K), as well as the fission gene Drp1 (Fig. 6L) during the peripubertal period, indicating enhanced mitochondrial remodeling activity in response to altered environmental cues during this sensitive developmental window. Similarly, the mitophagy-related gene Pink1 (Fig. 6O) showed a developmental increase under LD conditions, while Prkn (Fig. 6P) remained largely unchanged. Under DD, Pink1 expression was notably elevated during the peripubertal period, suggesting enhanced mitochondrial quality control activity, whereas Prkn remained unresponsive.

Adult Leydig cells exhibit circadian regulation, with a rhythmic expression of transcription factors, steroidogenic enzymes, and testosterone production, indicating that steroidogenesis is temporally organized. These cells also rhythmically express core clock genes, aligning endocrine function with the internal circadian timing system. However, the developmental trajectory and expression dynamics of clock genes in Leydig cells under constant conditions, particularly DD, remain poorly understood.

To investigate the developmental regulation of circadian genes in Leydig cells, the transcriptional activity of core clock components was analyzed in cells isolated from 35-, 42-, and 90-day-old rats (Fig. 7). Under LD conditions, the core clock genes Clock, Per2, Cry1, and Cry2 showed a modest but consistent increase in expression by pnd 90, indicating the progressive establishment of circadian regulation during Leydig cell maturation. The accessory loop gene Reverba also demonstrated gradual developmental upregulation, suggesting a role in fine-tuning the temporal coordination of steroidogenic and metabolic functions. In contrast, the expression of Bmal1, Reverbb, and Rorb remained relatively stable across all time points while Per1 decreased around puberty (Fig. 7).

Notably, DD triggered significant upregulation of Clock, Bmal1, Per2, Reverba, Reverbb, and Rorb during the peripubertal period, indicating a phase of heightened circadian gene activation without light cues. Additionally, the expression of Per1, Per2, and Cry1 was reduced at pnd 35 but increased at later stages, Per1 at both pnd 42 and 90, and Per2 specifically at pnd 42, indicating that the absence of environmental light cues may delay or disrupt the typical developmental trajectory of circadian gene expression in Leydig cells (Fig. 7).

The findings of this study suggest that disruption of circadian rhythms interferes with the proper maturation of Leydig cells, resulting in diminished circulating testosterone levels. Given the central role of testosterone in regulating spermatogenesis, this decline may negatively affect male reproductive capacity.

Spermatogenesis is a tightly regulated process that depends on the coordinated activation of testis-specific genes involved in germ cell differentiation and chromatin remodeling. To assess how DD affects this process, the expression of critical markers of sperm maturation in the seminiferous tubules, Tnp1 and Prm2, was examined. Under LD conditions, Tnp1 expression, a marker of round spermatids, increased progressively with age, consistent with normal spermatogenic maturation (Fig. 8A). In contrast, rats exposed to DD exhibited reduced Tnp1 transcription during the early stages of Leydig cell development, suggesting impaired or delayed initiation of spermatid differentiation. By adulthood, Tnp1 expression in DD animals approached control levels, indicating a potential recovery or catch-up effect, albeit with a delayed onset of spermatogenic progression (Fig. 8A).

Likewise, Prm2, a marker of condensed and elongated spermatids, showed a clear age-dependent increase under LD conditions, reaching its peak in adulthood (Fig. 8B). In contrast, DD-exposed animals exhibited reduced Prm2 expression during the early developmental stage, suggesting a potential impairment in the transition from round to elongated spermatids (Fig. 8B). This observation aligned with histological analysis of seminiferous tubules at stage VII of spermatogenesis, an androgen-dependent phase marked by the transition from round to elongated spermatids. This stage is highly sensitive to androgen signaling and is often used as a histological marker for Leydig cell function and endocrine regulation of spermatogenesis. In LD animals, a high number of elongated spermatids was observed at pnd 42, reflecting normal progression of spermatogenesis and coinciding with elevated serum androgen levels (Figs. 2A and 8C and D). In contrast, rats raised under DD exhibited a reduction in elongated spermatids during the same peripubertal period (pnd 42), indicating impaired androgen-dependent germ cell differentiation (Fig. 8C, D).

These findings are supported by histological analysis, which revealed delayed spermatogenesis in DD-exposed rats, as evidenced by the absence of spermatozoa in the seminiferous tubules during the peripubertal stage (pnd 42; Fig. 8D, right panel), in contrast to the presence of mature germ cells observed under LD conditions (Fig. 8D, left panel).

The number of elongated spermatids in the testes of adult rats was also analyzed, revealing a significant reduction in animals maintained under DD conditions (Fig. 8E, F).

Additionally, in the epididymis of adult DD rats lower number of spermatozoa was found compared to LD conditions (Fig. 8G).

Moreover, spermatozoa isolated from the DD rats were less functional than those in the LD rats, as evidenced by a significant reduction in the progesterone-induced acrosome reaction (Fig. 8H).