Estrogen receptor-alpha gene deficiency enhances androgen biosynthesis in the mouse Leydig cell

Abstract

Leydig cells, which produce the primary male steroid hormone testosterone (T), express the two estrogen receptor (ER) subtypes, ERalpha and ERbeta, and have the capacity to convert testosterone to the natural estrogen 17beta-estradiol. Thus, Leydig cells are subject to estrogen action. The development of transgenic mice that are homozygous for targeted deletion of genes encoding the ER subtypes provides an opportunity to examine the role of estrogen in Leydig cell function. In this study androgen biosynthesis was analyzed in Leydig cells from mice that were homozygous for targeted deletion of the ERalpha gene (alphaERKO). T production by alphaERKO Leydig cells was 2-fold higher than that in wild-type (WT) cells. Serum T levels were accordingly higher in alphaERKO compared with WT mice (5.1 +/- 1.1 vs. 2.2 +/- 0.4 ng/ml; P </= 0.01) as were serum LH levels (1.31 +/- 0.3 vs. 0.45 +/- 0.08 ng/ml; P </= 0.01). Mice that were treated with the pure antiestrogen ICI 182,780 at 100 micro g/kg.d for 7 d, effectively abrogating ER-mediated activity, also had 2-fold elevations in the serum levels of LH (1.15 +/- 0.3 vs. 0.45 +/- 0.2 ng/ml) and T (4.3 +/- 1.1 vs. 2.2 +/- 0.2 ng/ml; P </= 0.01). Increased androgen biosynthesis by alphaERKO Leydig cells was associated with higher steroidogenic enzyme activity, especially of cytochrome P450 17alpha-hydroxylase/17-20 lyase (P450(17alpha)) and 17beta-hydroxysteroid dehydrogenase (17beta-HSD), as measured by conversion of radiolabeled steroid substrates to T or its precursors. The largest increases in enzymatic activity were observed for P450(17alpha) (423 +/- 45 pmol/min.10(6) cells in alphaERKO Leydig cells vs. 295 +/- 27 pmol/min.10(6) cells in WT cells; P < 0.01). Consistent with steroidogenic enzyme activity, the testis of alphaERKO mice expressed higher steady state mRNA levels for steroidogenic acute regulatory protein and two enzymes involved in androgen biosynthesis, P450(17alpha) and 17beta-HSD type III, as determined by semiquantitative RT-PCR. Compared with the controls, higher steady state mRNA levels for steroidogenic acute regulatory protein and P450(17alpha) were also measured in the testis of ICI 182,780-treated mice. In a second set of experiments estrogen administration reduced serum LH and T levels in WT controls, whereas alphaERKO mice were unaffected. Although exposure of WT and alphaERKO Leydig cells to estrogen in vitro did not affect androgen biosynthesis, incubation with ICI 182,780 reduced T production by WT, but not alphaERKO, Leydig cells. These observations indicate that abrogation of the ERalpha gene by targeted deletion or treatment with an antiestrogen increases Leydig cell steroidogenesis in association with elevations in the serum levels of LH, which presumably is the result of estrogen insensitivity at the level of the hypothalamus and/or pituitary gonadotropes. Furthermore, the decrease in T production by WT Leydig cells and not alphaERKO Leydig cells occasioned by incubation with ICI 182,780 suggests that of the ER subtypes, ERalpha has a regulatory role in Leydig cell steroidogenic function.