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

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

Research output: Contribution to journalArticle

12 Citations (Scopus)

Abstract

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.

Original languageEnglish
Article number90
JournalReproductive Biology and Endocrinology
Volume12
Issue number1
DOIs
Publication statusPublished - Sep 20 2014

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Connexin 43
Calcitriol
Granulosa Cells
Testosterone
Estradiol
Aromatase
Phosphotyrosine
Polycystic Ovary Syndrome
Calcium Channel Blockers
Western Blotting
Estradiol Receptors
Therapeutics
Calcitriol Receptors
Proteins

Keywords

  • 1,25-dihydroxyvitamin D3
  • 17beta-estradiol
  • Calcium
  • Connexin 43
  • Estrogenesis
  • Granulosa cells
  • Polycystic ovary syndrome
  • Testosterone

ASJC Scopus subject areas

  • Developmental Biology
  • Endocrinology
  • Reproductive Medicine
  • Medicine(all)

Cite this

1,25-Dihydroxyvitamin D3 increases testosterone-induced 17beta-estradiol secretion and reverses testosterone-reduced connexin 43 in rat granulosa cells. / Lee, Ching Tien; Wang, Jiz Yuh; Chou, Kuang Yi; Hsu, Ming I.

In: Reproductive Biology and Endocrinology, Vol. 12, No. 1, 90, 20.09.2014.

Research output: Contribution to journalArticle

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abstract = "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.",
keywords = "1,25-dihydroxyvitamin D3, 17beta-estradiol, Calcium, Connexin 43, Estrogenesis, Granulosa cells, Polycystic ovary syndrome, Testosterone",
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T1 - 1,25-Dihydroxyvitamin D3 increases testosterone-induced 17beta-estradiol secretion and reverses testosterone-reduced connexin 43 in rat granulosa cells

AU - Lee, Ching Tien

AU - Wang, Jiz Yuh

AU - Chou, Kuang Yi

AU - Hsu, Ming I.

PY - 2014/9/20

Y1 - 2014/9/20

N2 - 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.

AB - 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.

KW - 1,25-dihydroxyvitamin D3

KW - 17beta-estradiol

KW - Calcium

KW - Connexin 43

KW - Estrogenesis

KW - Granulosa cells

KW - Polycystic ovary syndrome

KW - Testosterone

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U2 - 10.1186/1477-7827-12-90

DO - 10.1186/1477-7827-12-90

M3 - Article

VL - 12

JO - Reproductive Biology and Endocrinology

JF - Reproductive Biology and Endocrinology

SN - 1477-7827

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M1 - 90

ER -