Stimulation of pregnenolone metabolism and aromatase activity by luteinizing hormone in mouse uterus

Abstract

In vitro metabolism of pregnenolone (P 5 ) as well as production of 17b-estradiol (E 2 ) were studied in uteri of untreated and luteinizing hormone (LH)-treated mice that had been ovariectomized (OVX) at late-diestrus stage. In the uteri of untreated mice, [ 3 H]pregnenolone was shown to be metabolized to D 5-components such as 17a-hydroxypregnenolone (17a-P 5 ) and dehydroepiandrosterone (DHEA), whereas LH treatment resulted in significant increases in the formation of progesterone (P 4 ), 17a-hydroxyprogesterone (17a-P 4 ), androstenedione (AD) and testosterone (T). This was assessed by thinlayer chromatography (TLC) and high-performance liquid chromatography (HPLC). The content and release of P 4 was shown to be stimulated by LH. Trilostane, an inhibitor of 3b-hydroxysteroid dehydrogenase (3b-HSD), inhibited LH-induced P 4 synthesis and its release in a dose-dependent manner. A considerable increase in [ -3-oxosteroids. In vitro effects of LH on the synthesis and metabolism of P 4 , as well as on the stimulation of aromatase activity, were more pronounced in the uterine tissue of LH-primed OVX mice. Thus the results of the present study indicate that, under specific conditions, the uterus of the mouse behaves like steroidogenic tissue. Its prompt response to LH reveals the probable physiological relevance of the existence of LH receptors of high binding affinity in the uterine tissue of the mouse, as reported earlier.

Expression of LH receptor in nonpregnant mouse endometrium: LH induction of 3β-HSD and de novo synthesis of progesterone

“…The reported rise of uterine LHR/HCG receptor at metestrus in rats after ovulation (Bonnamy et al 1990) may be due to modulation of P 4 content (Bonnamy et al 1989). Our previous study also showed LH-induced increases in P 4 synthesis in mouse uterus at metestrus (Debnath et al 2000). Thus, the maximum expression of LHR in mouse endometrium at metestrus in this study corroborates the earlier findings with rat uterus, indicating that this rise was due to post-ovulatory production of P 4 in mouse endometrium.…”

“…Activity of 3b-HSD enzyme has been described in the endometrium of several species, either by conversion study during follicular (Sweat & Bryson 1969) and luteal phase (Henricks & Tindall 1971) or histochemically during maternal recognition of pregnancy (Flood & Ghazi 1981). Our previous study also reported LH-induced stimulation of 3b-HSD enzyme activity in mouse uterus (Debnath et al 2000). In this study, there is a clear indication for the stimulation of 3b-hsd mRNA expression and its enzyme activity leading to increased de novo P 4 synthesis by nonpregnant mouse endometrium after LH stimulation particularly at metestrus.…”

“…A role for MAPK/ERK signaling in regulating gonadotropin-induced steroidogenesis in mammalian and avian granulosa cells (Moore et al 2001, Seger et al 2001, Dewi et al 2002, Cottom et al 2003, Su et al 2006, Woods & Johnson 2007 and in rat Leydig cells (Martinelle et al 2004) has also been reported. Although LH-induced P 4 synthesis by the mouse/rat uteri has been documented earlier (Bonnamy et al 1989, Mukherjee et al 1994, Debnath et al 2000, little is known about its mechanism of action. To our knowledge, demonstration of MAPK signaling in LH-induced P 4 synthesis in mouse endometrium may be the first one in any non-gonadal tissues of mammals.…”

“…02% NaN 3 , 1% Triton X-100, 100 mg/ml phenylmethylsulphonyl fluoride, and 1 mg/ml aprotinin). 3b-HSD enzyme activity was measured following the procedure described previously (Debnath et al 2000). In brief, endometrial tissues (50 mg each) were incubated with or without LH (100 ng/ml) or trilostane (a specific inhibitor of 3b-HSD) in the presence of [ 3 H]pregnenolone (2!10 6 c.p.m., 138 pmol) and NAD (300 mM) in 1 ml SF-DMEM with constant shaking for 8 h. Inhibitor was added 1 h before LH addition.…”

“…The methods of extraction and estimation of labeled steroids for determination of 3b-HSD activity and estimation of P 4 from incubation media by RIA were similar to those described previously (Debnath et al 2000). The sensitivity of the RIA was 12 pg/incubation and the intra-and interassay coefficients of variation were 7 and 9% respectively.…”