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

Reproductive Biology and Endocrinology

Ming-I Hsu³  Kuang-Yi Chou²  Jiz-Yuh Wang⁴  Ching-Tien Lee¹ 

[1] Department of Nursing, Hsin Sheng College of Medical Care and Management, Taoyuan, Taiwan;General Education Center, National Taipei University of Nursing and Health Sciences, Taipei, Taiwan;Department of Obstetrics and Gynecology, Wan Fang Hospital, Taipei Medical University, Taipei, Taiwan;Department of Neurology, School of Medicine, College of Medicine, Kaohsiung Medical University, Kaohsiung, Taiwan

关键词: Granulosa cells;    Calcium;    Polycystic ovary syndrome;    1,25-dihydroxyvitamin D3;    Testosterone;    Connexin 43;    Estrogenesis;    17beta-estradiol;   

Others  :  1139769
DOI  :  10.1186/1477-7827-12-90

 received in 2014-05-08, accepted in 2014-09-13,  发布年份 2014

【 摘 要 】

Background

Aromatase 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.

Methods

Primary 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.

Results

Testosterone 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.

Conclusions

These 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.

【 授权许可】

   
2014 Lee et al.; licensee BioMed Central Ltd.

【 图 表 】

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