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

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Abstract

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.

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References

  1. Hamase K, Morikawa A, Zaitsu K (2002) d-Amino acids in mammals and their diagnostic value. J Chromatogr B Analyt Technol Biomed Life Sci 781(1–2):73–91

    Article  PubMed  CAS  Google Scholar 

  2. Hamase K, Homma H, Takigawa Y, Fukushima T, Santa T, Imai K (1997) Regional distribution and postnatal changes of d-amino acids in rat brain. Biochim Biophys Acta 1334(2–3):214–222

    Article  PubMed  CAS  Google Scholar 

  3. Long Z, Homma H, Lee JA et al (1998) Biosynthesis of d-aspartate in mammalian cells. FEBS Lett 434:231–235

    Article  PubMed  CAS  Google Scholar 

  4. Long Z, Lee JA, Okamoto T, Nimura N, Imai K, Homma H (2000) d-Aspartate in a prolactin-secreting clonal strain of rat pituitary tumor cells (GH(3)). Biochem Biophys Res Commun 276:1143–1147

    Article  PubMed  CAS  Google Scholar 

  5. Sakai K, Homma H, Lee JA et al (1998) Localization of d-aspartic acid in elongate spermatids in rat testis. Arch Biochem Biophys 351(1):96–105

    Article  PubMed  CAS  Google Scholar 

  6. D’Aniello G, Ronsini S, Guida F, Spinelli P, D’Aniello A (2005) Occurrence of d-aspartic acid in human seminal plasma and spermatozoa: possible role in reproduction. Fertil Steril 84(5):1444–1449

    Article  PubMed  Google Scholar 

  7. Burrone L, Raucci F, Di Fiore MM (2012) Steroidogenic gene expression following d-aspartate treatment in frog testis. Gen Comp Endocrinol 175(1):109–117

    Article  PubMed  CAS  Google Scholar 

  8. Furuchi T, Suzuki T, Sekine M, Katane M, Homma H (2009) Apoptotic inducers activate the release of d-aspartate through a hypotonic stimulus-triggered mechanism in PC12 cells. Arch Biochem Biophys 490(2):118–128

    Article  PubMed  CAS  Google Scholar 

  9. Katane M, Homma H (2011) D-Aspartate–an important bioactive substance in mammals: a review from an analytical and biological point of view. J Chromatogr B Analyt Technol Biomed Life Sci 879(29):3108–3121

    Article  PubMed  CAS  Google Scholar 

  10. Cooke BA, Choi MC, Dirami G, Lopez-Ruiz MP, West AP (1992) Control of steroidogenesis in Leydig cells. J Steroid Biochem Mol Biol 43(5):445–449

    Article  PubMed  CAS  Google Scholar 

  11. Ascoli M (1981) Characterization of several clonal lines of cultured Leydig tumor cells: gonadotropin receptors and steroidogenic responses. Endocrinology 108:88–95

    Article  PubMed  CAS  Google Scholar 

  12. Clark BJ, Wells J, King SR, Stocco DM (1994) The purification, cloning, and expression of a novel luteinizing hormone-induced mitochondrial protein in MA-10 mouse Leydig tumor cells. Characterization of the steroidogenic acute regulatory protein (StAR). J Biol Chem 269:28314–28322

    PubMed  CAS  Google Scholar 

  13. Nagata Y, Homma H, Matsumoto M, Imai K (1999) Stimulation of steroidogenic acute regulatory protein (StAR) gene expression by d-aspartate in rat Leydig cells. FEBS Lett 454:317–320

    Article  PubMed  CAS  Google Scholar 

  14. Furuchi T, Homma H (2005) Free d-aspartate in mammals. Biol Pharm Bull 28:1566–1570

    Article  PubMed  CAS  Google Scholar 

  15. Topo E, Soricelli A, D’Aniello A, Ronsini S, D’Aniello G (2009) The role and molecular mechanism of d-aspartic acid in the release and synthesis of LH and testosterone in humans and rats. Reprod Biol Endocrinol 7:120–130

    Article  PubMed  PubMed Central  Google Scholar 

  16. Chandrashekar KN, Muralidhara (2008) Oxidative alterations induced by d-aspartic acid in prepubertal rat testis in vitro: a mechanistic study. Theriogenology 70(1):97–104

    Article  PubMed  CAS  Google Scholar 

  17. Tsuchiya M, Inoue K, Matsuda H et al (2003) Expression of steroidogenic acute regulatory protein (StAR) and LH receptor in MA-10 cells. Life Sci 73(22):2855–2863

    Article  PubMed  CAS  Google Scholar 

  18. Smrcka AV (2008) G protein βγ subunits: central mediators of G protein-coupled receptor signaling. Cell Mol Life Sci 65(14):2191–2214

    Article  PubMed  CAS  PubMed Central  Google Scholar 

  19. Wang H, Segaloff DL, Ascoli M (1991) Lutropin/choriogonadotropin down-regulates its receptor by both receptor-mediated endocytosis and a cAMP-dependent reduction in receptor mRNA. J Biol Chem 266:780–785

    PubMed  CAS  Google Scholar 

  20. Kanamarlapudi V, Thompson A, Kelly E, López Bernal A (2012) ARF6 activated by the LHCG receptor through the cytohesin family of guanine nucleotide exchange factors mediates the receptor internalization and signaling. J Biol Chem 287(24):20443–20455

    Article  PubMed  CAS  PubMed Central  Google Scholar 

  21. Hirakawa T, Galet C, Ascoli M (2002) MA-10 cells transfected with the human lutropin/choriogonadotropin receptor (hLHR): a novel experimental paradigm to study the functional properties of the hLHR. Endocrinology 143(3):1026–1035

    Article  PubMed  CAS  Google Scholar 

  22. Zhang Y, Cudmore RH, Lin DT, Linden DJ, Huganir RL (2015) Visualization of NMDA receptor-dependent AMPA receptor synaptic plasticity in vivo. Nat Neurosci. doi:10.1038/nn.3936. [Epub ahead of print]

  23. Santillo A, Falvo S, Chieffi P et al (2014) D-aspartate affects NMDA receptor-extracellular signal-regulated kinase pathway and upregulates androgen receptor expression in the rat testis. Theriogenology 81(5):744–751

    Article  PubMed  CAS  Google Scholar 

  24. D’Aniello A, Di Fiore MM, Fisher GH et al (2000) Occurrence of d-aspartic acid and N-methyl-d-aspartic acid in rat neuroendocrine tissues and their role in the modulation of luteinizing hormone and growth hormone release. FASEB J 14(5):699–714

    PubMed  Google Scholar 

  25. Gore AC (2001) Gonadotropin-releasing hormone neurons, NMDA receptors, and their regulation by steroid hormones across the reproductive life cycle. Brain Res Rev 37(1–3):235–248

    Article  PubMed  CAS  Google Scholar 

  26. Morris RG, Steele RJ, Bell JE, Martin SJ (2013) N -methyl-d-aspartate receptors, learning and memory: chronic intraventricular infusion of the NMDA receptor antagonist d-AP5 interacts directly with the neural mechanisms of spatial learning. Eur J Neurosci 37(5):700–717

    Article  PubMed  CAS  Google Scholar 

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Author notes

    Authors and Affiliations

    1. Department of Medicine, Section of Endocrinology and Centre for Human Reproduction Pathology, University of Padova, Via Giustiniani, 2, 35128, Padua, Italy

      A. Di Nisio, L. De Toni, M. S. Rocca, E. Speltra, A. Ferlin & C. Foresta

    2. Department of Molecular Medicine, University of Padova, Padua, Italy

      M. Ferigo

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    1. A. Di Nisio
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    2. L. De Toni
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    3. M. Ferigo
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    4. M. S. Rocca
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    Correspondence to C. Foresta.

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    A. Di Nisio and L. De Toni have equally contributed to the manuscript.

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    Cite this article

    Di Nisio, A., De Toni, L., Ferigo, M. et al. d-Aspartic acid stimulates steroidogenesis through the delay of LH receptor internalization in a mammalian Leydig cell line. J Endocrinol Invest 39, 207–213 (2016). https://doi.org/10.1007/s40618-015-0333-4

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    • DOI: https://doi.org/10.1007/s40618-015-0333-4

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