Vitamin D3 Regulates Follicular Development and Intrafollicular Vitamin D Biosynthesis and Signaling in the Primate Ovary

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.

Keywords: 1α; 1α-hydroxylase; 25-dihydroxyvitamin D3; 25-hydroxylase; 25-hydroxyvitamin D3; rhesus macaque; vitamin D receptor.

FIGURE 1

The expression of vitamin D receptor (VDR) in rhesus macaque follicles developed in vivo. Immunohistochemistry was performed without (A) or with (B,C) counterstain hematoxylin to allow for visualization of the VDR nuclear and cytoplasmic staining. Mouse non-immune IgG was used as the negative control (C insert). PM, primordial follicle; PR, primary follicle; SD, secondary follicle; SA, small antral follicle; LA, large antral follicle. Scale bar = 100 μm for (A,C), and 50 μm for (B).

FIGURE 2

The growth, as well as mRNA levels of cytochrome P450 family 2 subfamily R polypeptide 1 (CYP2R1) and vitamin D receptor (VDR), of rhesus macaque follicles developed in vitro. A representative isolated secondary follicle (A) survived the first 2 weeks of culture under control conditions (B), and then grew to the small antral stage at week 5 (C). The mRNA levels of CYP2R1 and VDR in preantral (week 2) and antral (week 5) follicles developed in vitro under control conditions were determined by real-time PCR (4 pools from 8 animals, with follicles from 2 animals per pool) (D). Mitochondrial ribosomal protein S10 (MRPS10) served as the internal control. Following recombinant human chorionic gonadotropin exposure, a metaphase II oocyte was harvested from in vitro-developed antral follicles treated with 1α,25-dihydroxyvitamin D3 during culture weeks 3–5 (E). ^∗Significant difference between preantral and antral follicles, P < 0.05. Data are presented as the mean ± SEM. Scale bar = 100 μm for follicles and 25 μm for the oocyte.

FIGURE 3

Effects of 1α,25-dihydroxyvitamin D3 (VD3) supplementation during weeks 0–2 (preantral stage) on the development and function of rhesus macaque preantral follicles developed in vitro. Follicle survival was calculated as the percentage of surviving preantral follicles relative to the total follicles cultured (n = 6 animals per group) (A). Follicle growth was determined by measuring follicle diameters (16–17 follicles assessed per group) (A). Anti-Müllerian hormone (AMH) concentrations in the culture media were measured by ELISA (16–17 follicles assessed per group) (B). AMH mRNA levels in cultured follicles were determined by real-time PCR (4 pools from 8 animals, with follicles from 2 animals per pool) (B). The mRNA levels of cytochrome P450 family 2 subfamily R polypeptide 1 (CYP2R1) and vitamin D receptor (VDR) were determined by real-time PCR (4 pools from 8 animals, with follicles from 2 animals per pool) (C). Mitochondrial ribosomal protein S10 (MRPS10) served as the internal control. ^∗Significant difference between experimental groups, P < 0.05. Data are presented as the mean ± SEM.

FIGURE 4

Effects of 1α,25-dihydroxyvitamin D3 (VD3) supplementation during weeks 3–5 (antral stage) on the development and function of rhesus macaque antral follicles developed in vitro. Follicle survival was calculated as the percentage of surviving antral follicles relative to the total follicles cultured (n = 6 animals per group) (A). Follicle growth was determined by measuring follicle diameters (12–19 follicles assessed per group) (A). Estradiol and progesterone concentrations in the culture media were measured by the electrochemiluminescence immunoassay (12–19 follicles assessed per group) (B). Anti-Müllerian hormone (AMH) concentrations in the culture media were measured by ELISA (12–19 follicles assessed per group) (C). AMH mRNA levels in cultured follicles were determined by real-time PCR (4 pools from 8 animals, with follicles from 2 animals per pool) (C). The mRNA levels of cytochrome P450 family 2 subfamily R polypeptide 1 (CYP2R1) (D), cytochrome P450 family 27 subfamily B polypeptide 1 (CYP27B1) (E), and vitamin D receptor (VDR) (F) were determined by real-time PCR (4 pools from 8 animals, with follicles from 2 animals per pool). Mitochondrial ribosomal protein S10 (MRPS10) served as the internal control. ^∗Significant difference between experimental groups, P < 0.05. Data are presented as the mean ± SEM.