REVERBA couples the circadian clock to Leydig cell steroidogenesis.

The involvement of the molecular clock in regulating cell physiological processes on a specific time scale is a recognized concept, yet its specific impact on optimizing androgen production in Leydig cells has been unclear. This study aimed to confirm the role of the REVERBA (NR1D1) gene in controlling the transcription of key genes related to Leydig cell steroid production. We investigated daily variations by collecting Leydig cells from rats at various times within a 24-h period. Chromatin immunoprecipitation study showed a time-dependent pattern for genes linked to steroid production (Nur77, Star, Cyp11a1, and Cyp17a1), which closely matched the 24-h REVERBA levels in Leydig cells, peaking between zeitgeber time (ZT) 7-11. To understand the physiological significance of REVERBA's interaction with promoters of steroidogenesis-related genes, Leydig cells from rats at two different times (ZT7 and ZT16; chosen based on REVERBA expression levels), were treated with either an agonist (GSK4112) or an antagonist (SR8278). The results revealed that the REVERBA agonist stimulated gene transcription, while the antagonist inhibited it, but only when REVERBA was sufficiently present, indicating a reliance on REVERBA's circadian fluctuation. Moreover, this REVERBA-dependent stimulation had a clear impact on testosterone production in the culture medium, underscoring REVERBA's involvement in the circadian regulation of testosterone. This study indicates that REVERBA, in addition to being a core component of the cellular clock, plays a key role in regulating androgen production in Leydig cells by influencing the transcription of critical steroidogenesis-related genes.

Growing Up Under Constant Light: A Challenge to the Endocrine Function of the Leydig Cells.

The factors influencing Leydig cell maturity and the acquisition of functional capacity are incompletely defined. Here we analyzed the constant light (LL) influence on Leydig cells' endocrine function during reproductive maturation. Rats were exposed to LL from P21 to P90. Data were collected at juvenile (P35), peri/pubertal (P42, P49), and adult (P90) stages of life. The results proved the effect of LL on rats' physiology by changing of bimodal voluntary activity pattern into free-running. Additionally, the peripheral clock in Leydig cells changed in LL condition, indicating disturbed rhythm: the positive element (Bmal1) increased in pre-/pubertal but decreased in the adult period, while negative elements (Per2 and Reverba) were increased. The effects of LL were most prominent in puberty: pituitary genes encoding gonadotropic hormones (Cga, Lhb, Fshb) decreased; serum corticosterone increased, while serum androgens and mass of testicular and sex accessory organs reduced; markers of Leydig cells maturity/differentiation (Insl3, Lhcgr) and steroidogenesis-related genes (Scarb1, Star, Cyp11a1, Cyp17a1) decreased; the steroidogenic and energetic capacity of the Leydig cell mitochondria decreased; the mtDNA copy number reduced, and mitochondrial dynamics markers changed: fusion decreased (Opa1 and Mfn2), and mitophagy increased (Pink1). In adults, the negative effect of LL on mitochondrial function and steroidogenic capacity persists in adult Leydig cells while other parameters reached control values. Altogether, the results indicate that LL slows down Leydig cells' maturation by reducing the endocrine and energy capacity of cells leading to the delay of reproductive development.

Dependence of Leydig Cell's Mitochondrial Physiology on Luteinizing Hormone Signaling.

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.