Progesterone Action in a Murine Leydig Tumor Cell Line (mLTC-1), Possibly through a Nonclassical Receptor Type¹

Abstract

In a recent report we demonstrated that a high (micromolar) concentration of progesterone (P) specifically down-regulates LH receptor (R) expression and function in murine Leydig tumor cells. The aim of the present study was to characterize further the putative novel R, mediating these P effects in the murine Leydig tumor cell line, mLTC-1. The binding of [3H]P to these cells revealed a high (Kd, approximately 9.3 nmol/liter) and a low affinity (Kd, approximately 284 nmol/liter) component, and the binding displayed with specificity (P > dehydroepiandrosterone > 17-OHP). The binding was apparently different from that of the classical nuclear PR in the following ways. 1) The P/glucocorticoid antagonist RU 486 did not compete with [3H]P binding to the mLTC-1 cells. 2) No expression of the classical PR messenger RNA was detected, despite clear P binding to these cells, by Northern hybridization or RT-PCR. 3) An antibody against the C-terminal end of the classical PR (alpha c-262) revealed in mLTC-1 cells several molecular size protein bands between 45-57 kDa on Western hybridization, whereas these immunoreactive proteins were faintly recognized by another antibody (alpha-PR) directed toward the NH2-terminal region of the classical PR. The sizes of the immunoreactive molecules were relatively similar to those detected using the same antibodies in human sperm lysates, but were at variance with the classical PR (120, 94, and 60 kDa), detected with these antibodies in human uterus. The immunoreactive proteins bound peroxidase-labeled-P, which could be displaced in the presence of a 10-fold excess of free P. 4) An immediate increase in the intracellular free calcium level was observed after P treatment in cultured mLTC-1 cells, whereas it also increased the 45Ca2+ entry within 15 min in these cells. 5) Increasing doses of P (0.1-10 micromol/liter) demonstrated significant inhibition of LH receptor messenger RNA levels in a dose-dependent manner in mLTC-1 cells. In conclusion, a nonclassical PR is expressed and functional in these cells, and it is clearly distinct from the classical nuclear PR. It is apparent that recently reported inhibitory effects of P on LH receptor gene expression and function are mediated through this novel type PR in mouse Leydig cells.

Non-Genomic Action of Androgens is Mediated by Rapid Phosphorylation and Regulation of Androgen Receptor Trafficking

“…Binding of estradiol causes additional ER translocation to the membrane [17-19]. The progesterone receptor is also reportedly localized in the plasma membrane [20, 21]. We conducted experiments to test this hypothesis in murine testicular Sertoli cell line TM4 cells.…”

Mifepristone Treatment Promotes Testicular Leydig Cell Tumor Progression in Transgenic Mice

“…P4 has been shown to be produced and involved in the regulation of LC and LCT function and proliferation [24,30], suggesting that LCTs could be susceptible to antiprogestin treatment. MF has been shown to inhibit in vitro the growth of cancer cells of reproductive and non-reproductive origin, independently of their PGR expression status [8].…”

“…In mouse LCTs (mLTC-1) cell line, P4 significantly inhibited luteinizing hormone receptor (LHR) expression and function. Presumably it was through their membrane PR (mPR), as mLTC-1 cells did not express classical PGRs [24,30]. Interestingly, the disruption of α and β PGR isoforms did not affect male fertility [31,32], which may suggest a non-classical P4 pathway activation in LCs.…”

Development of an in vitro reproductive screening assay for novel pharmaceutical compounds

“…The steroidogenic pathway starts with cholesterol and finishes with steroid hormones [19,20]. In male Leydig cells, progesterone is converted into 17‐OH progesterone, which in turn is converted into testosterone [18,20,21].…”

“…The MLTC‐1 cell line has been used in several studies to analyse the mechanism of progesterone signalling pathways and the importance of Ca 2+ ‐dependent pathways in the regulation of steroidogenesis but has not been previously used to assess the reproductive toxicity of novel compounds [20,21].…”