Vitamin D Regulates Anti-Mullerian Hormone Expression in Granulosa Cells of the Hen1

Abstract

Anti-Mullerian hormone (AMH) is involved in the regression of the Mullerian ducts in mammalian and avian male embryos as well as the right oviduct in avian female embryos. AMH is expressed by granulosa cells of adult hens and mammals and is thought to be involved in the recruitment of follicles from the primordial pool as well as in regulating follicle-stimulating hormone (FSH) sensitivity. We have shown that AMH expression by the granulosa layer of hens is high in the small follicles but decreased in the larger hierarchical follicles. The decline in expression of AMH with increasing follicle size is associated with an increase in expression of the receptor for FSH (FSHR) in the granulosa layer, although the mechanism is not known. In this study, we tested whether vitamin D (1,25-dihydroxyvitamin D3) regulates expression of AMH mRNA in granulosa cells of the hen. Granulosa cell layers were removed from follicles 3-5 mm and 6-8 mm in size, dispersed, and cultured for 24 h in Medium 199 + 5% fetal bovine serum (n = 7). The medium was removed and replaced with Medium 199 + 0.1% bovine serum albumin and vitamin D (at doses of 0, 10, and 100 nM) and cultured for 24 h. Cells were harvested and RNA was extracted for use in quantitative PCR. Parallel 96-well plates were set up to examine cell proliferation. AMH and FSHR mRNA expressions were evaluated, and all values were standardized to 18S reactions. There was a significant (P < 0.05) dose-related decrease in the expression of AMH mRNA in granulosa cells of 3- to 5-mm and 6- to 8-mm follicles in response to vitamin D. Additionally, FSHR mRNA and cell proliferation were significantly (P < 0.05) increased by vitamin D in both groups. Western blot analysis for the vitamin D receptor (VDR) showed doublet bands at the expected sizes (58 and 60 kDa) in protein isolated from the chicken granulosa layer. Immunohistochemistry was used to identify VDR within the follicle, and it predominantly localized to the nucleus of granulosa cells. VDR mRNA expression in the granulosa layer, relative to follicle development, was increased (n = 4; P < 0.05) with follicle development, with greatest expression in the F1 follicle. There was no evidence for expression (mRNA or protein) of the calcium-binding protein, calbindin (CALB1), in the ovary or granulosa layer. Overall, these results suggest that vitamin D regulates AMH expression, and thereby may influence follicle selection in the hen.

Direct vitamin D3 actions on rhesus macaque follicles in three-dimensional culture: assessment of follicle survival, growth, steroid, and antimüllerian hormone production

“…High-dose VD3 appeared to have a greater impact than low-dose VD3 on promoting later folliculogenesis by improving small antral follicle growth. The data were consistent with findings in hens in which a significant enhancement of cell proliferation was observed for non-luteinized granulosa cells incubated with VD3 (17). A previous study indicated that a relative high-dose VD3 not only down-regulated AMH receptor II gene expression, but also significantly reduced the phosphorylation and nuclear localization of SMAD 1/5/8, which are critical for AMH signaling, in cultured granulosa cells collected from IVF patients (20).…”

“…Previous studies suggested that FSH was a survival factor for preantral follicles developed in vitro in both nonhuman primates and humans (15, 16). Although not demonstrated in primate species, FSH receptor mRNA expression was up-regulated in non-luteinized granulosa cells in hens following VD3 treatment in culture (17). Therefore, exogenous VD3 may increase FSH sensitivity in macaque follicles, and thereby promote follicle survival in vitro .…”

Molecular actions of vitamin D in reproductive cell biology

“…VitD increased AMH mRNA in prostate cancer cells were a VDRE site was detected (Malloy et al 2009). Conversely, VitD decreased AMH mRNA in primary granulosa cells from hens (Wojtusik & Johnson 2011). In a study with primary human granulosa cells, 1α,25(OH) 2 D decreased AMH receptor II and FSH receptor mRNA expression, while promoting 3β-HSD expression and activity leading to increased progesterone secretion (Merhi et al 2014) (Fig.…”

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

“…Thus, the observation that many small follicles grow in PCOS may be explained by excess testosterone. Alternatively, 1,25D 3 might inhibit the testosterone-stimulated early stages of follicular growth because 1,25D 3 is known to inhibit the proliferation and induce the differentiation of a variety of cells [48], and VDR is expressed in the granulosa cells of largest follicle [49]. It is possible that 1,25D 3 paired with testosterone preferentially improves the growth of larger follicles.…”