D-Aspartic acid stimulates steroidogenesis through the delay of LH receptor internalization in a mammalian Leydig cell line

J Endocrinol Invest. 2016 Feb;39(2):207-13. doi: 10.1007/s40618-015-0333-4. Epub 2015 Jun 28.


Purpose: Recent experimental evidence on non-mammalian animal models showed that D-Aspartic acid (d-Asp) administration increases testosterone levels through upregulation of StAR in Leydig cells. In this study, we aimed to investigate in vitro the signaling pathway associated with d-Asp stimulation in MA-10 murine Leydig cells.

Methods: MA-10 cells were stimulated with different concentrations of d-Asp, in presence or absence of hCG. Then total testosterone (T) levels in the culture medium were evaluated by electrochemiluminescence immunoassay, and StAR and LHR protein expressions were quantified by the means of Western blotting. LHR cellular localization after hormonal stimulation was assessed by immunofluorescence.

Results: Stimulation with the sole d-Asp did not induce any relevant increase of T release from cultured cells. On the other hand, stimulation with hCG induced significant increase of T (P = 0.045). Concomitant stimulation with hCG and d-Asp, at the concentration of 0.1 and 1 nM, induced additional and significant increase of released T (P = 0.03 and P = 0.04, respectively). StAR protein levels increased after concomitant stimulation with hCG and d-Asp 0.1 nM, compared with stimulation with the sole hCG (P = 0.02), whereas no variation in LHR protein expression was observed. Finally, d-Asp attenuated displacement of LHR staining, from cell membrane to cytoplasm, subsequent to hCG stimulation.

Conclusions: In this study, we confirmed a steroidogenic role for d-Asp, in concert with hCG, on murine Leydig cells, which is mediated by an increase in StAR protein levels. In addition, we showed that the possible mechanism subtending the effect of d-Asp could rely on the modulation of LHR exposure on the cell membrane.

Keywords: D-Aspartic acid; LH receptor internalization; Leydig cells; Steroidogenesis; Testosterone.

Publication types

  • Comparative Study

MeSH terms

  • Animals
  • Cell Line
  • Cell Membrane / drug effects*
  • Cell Membrane / metabolism
  • Chorionic Gonadotropin / agonists*
  • Chorionic Gonadotropin / pharmacology
  • Clone Cells
  • D-Aspartic Acid / pharmacology*
  • Humans
  • Kinetics
  • Leydig Cells / cytology
  • Leydig Cells / drug effects*
  • Leydig Cells / metabolism
  • Male
  • Membrane Transport Modulators / pharmacology*
  • Mice
  • Microscopy, Fluorescence
  • Microscopy, Video
  • Phosphoproteins / metabolism
  • Protein Transport / drug effects
  • Receptors, LH / antagonists & inhibitors*
  • Receptors, LH / metabolism
  • Reproductive Control Agents / agonists*
  • Reproductive Control Agents / pharmacology
  • Signal Transduction / drug effects
  • Testosterone / metabolism


  • Chorionic Gonadotropin
  • Membrane Transport Modulators
  • Phosphoproteins
  • Receptors, LH
  • Reproductive Control Agents
  • steroidogenic acute regulatory protein
  • Testosterone
  • D-Aspartic Acid