1,25-Dihydroxyvitamin D3 increases testosterone-induced 17beta-estradiol secretion and reverses testosterone-reduced connexin 43 in rat granulosa cells.

Ching-Tien Lee,Ming-I Hsu,Jiz-Yuh Wang,Kuang-Yi Chou

doi:10.1186/1477-7827-12-90

Abstract

BackgroundAromatase converts testosterone into 17beta-estradiol in granulosa cells, and the converted 17beta-estradiol contributes to follicular maturation. Additionally, excessive testosterone inhibits aromatase activity, which can lead to concerns regarding polycystic ovary syndrome (PCOS). Generally, 1,25-dihydroxyvitamin D3 (1,25D3) supplements help to improve the symptoms of PCOS patients who exhibit low blood levels of 1,25D3. Therefore, this study investigated the interaction effects of 1,25D3 and testosterone on estrogenesis and intercellular connections in rat granulosa cells.MethodsPrimary cultures of granulosa cells were treated with testosterone or testosterone plus 1,25D3, or pre-treated with a calcium channel blocker or calcium chelator. Cell lysates were subjected to western blot analysis to determine protein and phosphorylation levels, and 17beta-estradiol secretion was examined using a radioimmunoassay technique. Cell viability was evaluated by MTT reduction assay. Connexin 43 (Cx43) mRNA and protein expression levels were assessed by qRT-PCR, western blot, and immunocytochemistry.ResultsTestosterone treatment (0.1 and 1 microg/mL) increased aromatase expression and 17beta-estradiol secretion, and the addition of 1,25D3 attenuated testosterone (1 microg/mL)-induced aromatase expression but improved testosterone-induced 17beta-estradiol secretion. Furthermore, testosterone-induced aromatase phosphotyrosine levels increased at 10 min, 30 min and 1 h, whereas 1,25D3 increased the longevity of the testosterone effect to 6 h and 24 h. Within 18–24 h of treatment, 1,25D3 markedly enhanced testosterone-induced 17beta-estradiol secretion. Additionally, pre-treatment with a calcium channel blocker nifedipine or an intracellular calcium chelator BAPTA-AM reduced 1,25D3 and testosterone-induced 17beta-estradiol secretion. Groups that underwent testosterone treatment exhibited significantly increased estradiol receptor beta expression levels, which were not affected by 1,25D3. Neither testosterone nor 1,25D3 altered 1,25D3 receptor expression. Finally, at high doses of testosterone, Cx43 protein expression was decreased in granulosa cells, and this effect was reversed by co-treatment with 1,25D3.ConclusionsThese data suggest that 1,25D3 potentially increases testosterone-induced 17beta-estradiol secretion by regulating aromatase phosphotyrosine levels, and calcium increase is involved in both 1,25D3 and testosterone-induced 17beta-estradiol secretion. 1,25D3 reverses the inhibitory effect of testosterone on Cx43 expression in granulosa cells.

Highlights

Aromatase converts testosterone into 17beta-estradiol in granulosa cells, and the converted 17beta-estradiol contributes to follicular maturation
Biosynthesis of 17β-estradiol from androgen precursors is catalysed by the enzyme aromatase, which is expressed from the cyp19 gene and exists in the ovaries, placenta, testes, breasts, brain, fat, liver and muscles [1]. 17βEstradiol production and follicular development are controlled by the expression level and activity of aromatase [2]
Circulating 17β-estradiol levels are low in VDR-null mutant mice and do not rise after calcium supplementation [16]. These results indicate that 1,25D3 plays a role in estrogenesis that is only partially mediated by extracellular calcium homeostasis

Summary

Introduction

Aromatase converts testosterone into 17beta-estradiol in granulosa cells, and the converted 17beta-estradiol contributes to follicular maturation. Excessive testosterone inhibits aromatase activity, which can lead to concerns regarding polycystic ovary syndrome (PCOS). Aromatase mRNA expression levels in granulosa cells from dominant follicles and 17β-estradiol levels in the follicular fluid are significantly increased [3,4]. Androgens have been shown to stimulate the growth of small antral follicles and inhibit apoptosis of preovulatory follicles in primate ovaries [6,7], whereas AR-knockout mice exhibit greatly increased apoptosis of granulosa cells in preovulatory follicles [8]. Excessive testosterone is involved in polycystic ovary syndrome (PCOS), which is the most common endocrine disorder in females and is associated with arrested follicular development and the failure to select a dominant follicle [11]. A daily 1,25D3 supplement enhances the intestinal absorption of calcium and alleviates both PCOS symptoms and gonadal dysfunction in 1,25D3 receptor (VDR)-null mutant mice [14]

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