Abstract
There is an increasing recognition that vitamin D plays important roles in female reproduction. Recent studies demonstrated that 1α,25-dihydroxyvitamin D3 (VD3), the biologically active form of vitamin D, improved ovarian follicle survival and growth in vitro. Therefore, we investigated the direct effects of VD3 at the specific preantral and antral stages of follicular development, and tested the hypothesis that vitamin D receptor (VDR) and enzymes critical for vitamin D biosynthesis are expressed in the primate ovary. Fourteen adult rhesus macaques provided ovarian tissue. Secondary and antral follicles were isolated for PCR analysis on VDR, vitamin D3 25-hydroxylase, and 25-hydroxyvitamin D3-1α-hydroxylase. VDR protein localization was determined by immunohistochemistry on ovarian sections. Isolated secondary follicles were cultured under conditions of control and VD3 supplementation during the preantral or antral stage. Follicle survival, growth, steroid and anti-Müllerian hormone (AMH) production, as well as oocyte maturation were evaluated. In vivo- and in vitro-developed follicles were also assessed for genes that are critical for vitamin D biosynthesis and signaling, gonadotropin signaling, steroid and paracrine factor production, and oocyte quality. The mRNA encoding VDR, 25-hydroxylase, and 1α-hydroxylase was detectable in in vivo- and in vitro-developed preantral and antral follicles. The 25-hydroxylase was elevated in cultured follicles relative to in vivo-developed follicles, which further increased following VD3 exposure. VD3 treatment increased 1α-hydroxylase in in vitro-developed antral follicles. The absence of VD3 during culture decreased VDR expression in in vitro-developed antral follicles, which was restored to levels comparable to those of in vivo-developed antral follicles by VD3 supplementation. Positive immunostaining for VDR was detected in the nucleus and cytoplasm of granulosa cells and oocytes. While only survival was improved in preantral follicles treated with VD3, VD3 supplementation promoted both survival and growth of antral follicles with increased estradiol and AMH production, as well as oocyte maturation. Thus, Vitamin D biosynthesis and signaling systems are expressed in primate ovarian follicles. Our findings support a role for VD3 in regulating follicular development in a stage-dependent manner, as well as the intrafollicular vitamin D biosynthesis and signaling, directly in the ovary.
Highlights
Vitamin D3 is produced in the skin or obtained from diet
Follicular Vitamin D Biosynthesis Enzyme and vitamin D receptor (VDR) Expression in vivo and in vitro The mRNA encoding CYP2R1, CYP27B1, and VDR was present in all pooled samples of secondary and small antral follicles isolated from ovaries of 8 macaques
While mRNA levels of CYP27B1 were comparable between the control and VD3-treated preantral follicles (CYP27B1/Mitochondrial ribosomal protein S10 (MRPS10) = 0.01 ± 0.01 versus 0.1 ± 0.1), mRNA levels of CYP2R1 and VDR increased (P < 0.05) in preantral follicles treated with VD3 (Figure 3C)
Summary
Vitamin D3 (cholecalciferol) is produced in the skin or obtained from diet. Vitamin D3 25-hydroxylase encoded by cytochrome P450 family 2 subfamily R polypeptide 1 (CYP2R1), converts vitamin D3 to 25-hydroxyvitamin D3 (calcifediol). Through actions of 25-hydroxyvitamin D3-1α-hydroxylase, encoded by cytochrome P450 family 27 subfamily B polypeptide 1 (CYP27B1), 25-hydroxyvitamin D3 is converted to 1α,25dihydroxyvitamin D3 (VD3; calcitriol) (Bikle, 2014). The kidney is the primary source of circulating VD3, other organs, including the ovary, express 1α-hydroxylase, which could catalyze VD3 biosynthesis (Bikle, 2014; Xu et al, 2016c) (RNA-Seq of macaca mulatta: adult female ovarian follicle; accession number: SRP044327). VDR is expressed in various organs, including the hypothalamus, pituitary, ovary, oviduct, uterus and placenta (Stumpf and Denny, 1989; Xu et al, 2016c) (RNA-Seq of macaca mulatta: adult female ovarian follicle; accession number: SRP044327), which could alter their function through VDR activation by circulating or locally synthesized VD3. Ovarian parameters need to be evaluated in the presence of physiological levels of VD3, especially under non-pathological conditions, to assess the direct impact of VD3 on processes critical for the development of follicles yielding oocytes that undergo fertilization and subsequent embryonic development
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