Stimulation of androgen production by d-aspartate through the enhancement of StAR, P450scc and 3β-HSD mRNA levels in vivo rat testis and in culture of immature rat Leydig cells

• d-Asp is endogenously present in vivo adult rat testis and in vitro immature rat primary LCs.
• d-Asp enhances StAR, P450scc and 3β-HSD mRNA levels and androgen production in vivo adult testis.
• Immature rat LCs specifically takes up d-Asp after its in vitro incubation.
• d-Asp directly acts on StAR, P450scc and 3β-HSD mRNA levels in vitro immature rat LCs.
• d-Asp directly enhances the production of androgens in vitro immature rat LCs.

Previous studies have shown a role of d-aspartic acid (d-Asp) in testicular steroidogenesis. Here, we evaluated the effects of d-Asp on androgen production and on expression levels of mRNAs encoding specific steroidogenic key molecules. d-Asp was endogenously present in adult rat testis and its content paralleled to serum luteinizing hormone (LH) and, local and circulating androstenedione and testosterone levels. In vivod-Asp administration induced serum LH release, causing an indirect increase of androstenedione and testosterone levels by enhancing steroidogenic acute regulatory protein (StAR), cytochrome P450 cholesterol side-chain cleavage enzyme (P450scc) and 3β-hydroxysteroid dehydrogenase/D5-D4 isomerases (3β-HSD) mRNA levels. The direct endocrine role of d-Asp was evaluated using cultured immature Leydig cells (ILCs) obtained from 35 days old rats. Cytoplasm and nucleus of ILCs localized d-Asp, while StAR marked the cytoplasm only. After 12 h from d-Asp in vitro administration, ILCs resulted intensely d-Asp stained, and StaR protein level, evaluated by Western blotting, significantly increased. After 24 h, significant androstenedione and testosterone syntheses were induced. At molecular level, d-Asp administration significantly increased StAR, P450scc and 3β-HSD mRNAs at 2, 4 and 12 h, respectively. The temporal shift on relative mRNA expression levels indicated that d-Asp exerted its physiological role through sequential gene cascade activation of those molecules implicated in the synthesis of androgens. Conclusively, our findings demonstrated that d-Asp is a local messenger in testis and give a contribution in understanding the complexity of local endocrine regulation as well as the molecular events leading the acquisition to a steroidogenic competence by ILCs.

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