Effect of Vitamin D3 on Biosynthesis of Estrogen in Porcine Granulosa Cells via Modulation of Steroidogenic Enzymes

Abstract

Vitamin D3 is a fat-soluble secosteroid responsible for enhancing intestinal absorption of calcium, iron, and other materials. Vitamin D3 deficiency, therefore, can cause health problems such as metabolic diseases, and bone disorder. Female sex hormones including estrogen and progesterone are biosynthesized mainly in the granulosa cells of ovary. In this study, we isolated granulosa cells from porcine ovary and cultured for the experiments. In order to examine the effect of vitamin D3 on the ovarian granulosa cells, the mRNA and protein levels of genes were analyzed by real-time PCR and Western blot assay. The production of estrogen from the granulosa cells was also measured by the ELISA assay. Genes associated with follicle growth were not significantly altered by vitamin D3. However, it increases expression of genes involved in the estrogen-biosynthesis. Further, estrogen concentrations in porcine granulosa cell-cultured media increased in response to vitamin D3. These results showed that vitamin D3 is a powerful regulator of sex steroid hormone production in porcine granulosa cells, suggesting that vitamin D deficiency may result in inappropriate sexual development of industrial animals and eventually economic loss.

Keywords: Estrogen; Follicle growth; Granulosa cell; Steroidogenesis; Vitamin D3.

Fig. 1

The mRNA expression of folliculogenic enzymes in granulosa cells treated with VD3. AMH, FSHR, and Foxl2 expression levels were analyzed by real-time PCR. Gene expression levels were normalized to the level of β-actin. Data are expressed as the mean ± SD.

Fig. 2

The mRNA expression of E2 biosynthesis-related enzymes in granulosa cells treated with VD3. CYP17A1, HSD17B1, and CYP19A1 expression levels were analyzed by real-time PCR. Gene expression levels were normalized to the level of β-actin. Data are expressed as the mean ± SD. *P< 0.05 compared to control group.

Fig. 3

The protein expression of E2 biosynthesis-related proteins in granulosa cells treated with VD3. CYP17A1 and CYP19A1 levels were analyzed by Western blot assay. Protein levels were normalized by β-actin. Data are expressed as the mean ± SD.

Fig. 4

Effects of VD3 on E2 secretion in granulosa cells. After treatment of granulosa cells with VD3, medium was harvested. E2 concentration in cell growth medium was analyzed by ELISA assay. NC, negative control, media without granulosa cells; Con, control media with granulosa cells. Data are expressed as the mean ± SD. *P< 0.05 compared to control group.