Vitamin D in Follicular Development and Oocyte Maturation

Abstract

Vitamin D (VD) is a secosteroid hormone synthesized predominantly in the skin upon ultraviolet light exposure, which can also be obtained from dietary sources. In target cells, the bioactive VD binds to specific VD receptor to regulate downstream transcription of genes that are involved in a wide range of cellular processes. There is increasing recognition that the proper physiological levels of VD are critical for optimizing reproductive potential in women. The direct VD action in the ovary was first suggested in the 1980s. Since then, research has attempted to determine the role of VD in follicular development and oocyte maturation in animal models and clinical settings. However, data published to date are inconclusive due to the complexity in VD metabolism and the fact that VD actions are pervasive in regulating physiological functions in various systems, including the reproductive, endocrine and nervous systems that control reproduction. This review summaries in vitro, in vivo, and clinical evidence regarding VD metabolism and signaling in the ovary, as well as VD-regulated or VD-associated ovarian follicular development, steroidogenic function, and oocyte maturation. It is suggested that adequate animal models are needed for well-controlled studies to unravel molecular mechanisms of VD action in the ovary. For clinical studies, follicular development and function may be evaluated more effectively in a relatively homogeneous patient population under a well-controlled experimental design. A comprehensive understanding of VD-regulated folliculogenesis and oogenesis will provide critical insight into the impact of VD in female reproductive health.

Introduction

Vitamin D (VD) is a secosteroid hormone synthesized from cholesterol predominantly in the skin upon ultraviolet light exposure, with an additional ~10% being derived from dietary sources (Wagner et al. 2012). In addition to the well-known function of VD in regulating calcium absorption during osteogenesis, VD affects a wide range of cellular processes, including cell proliferation and differentiation, as well as apoptosis and inflammation (Verstuyf et al. 2010). There is increasing recognition that VD plays a crucial role in female reproduction. The proper physiological levels of VD are considered critical for optimizing reproductive potential in women (Franasiak et al. 2017). For women of reproductive age, serum 25-hydroxyvitamin D3 (calcifediol) levels of 12.0–20.0 ng/ml and <12.0 ng/ml are defined as VD insufficiency and deficiency, respectively (Zhao et al. 2012). Notably, VD insufficiency and deficiency has been a global health problem for the past few decades. People with increased skin melanin pigmentation, obesity or abstinence from direct sun exposure are especially at high risk (Hossein-nezhad & Holick 2013). Therefore, studies are conducted to unravel VD actions in reproductive organs in order to provide insight into benefits of VD in female reproductive health for clinical interventions.

Generally, vitamin D3 (cholecalciferol; animal source) and D2 (ergocalciferol; plant source) originated from the skin and diet are first converted to 25-hydroxyvitamin D3 in the liver via the action of vitamin D3 25-hydroxylase encoded by cytochrome P450 family 2 subfamily R polypeptide 1 (CYP2R1). The precursor is then converted to biologically active 1α,25-dihydroxyvitamin D3 (calcitriol; VD3) in the kidney by cytochrome P450 family 27 subfamily B polypeptide 1 (CYP27B1) encoded 25-hydroxyvitamin D3–1α-hydroxylase (Bikle 2014). All forms of VD metabolites exist in the circulation. In target cells, the bioactive VD3 binds to specific VD receptor (VDR), a DNA-binding transcription factor which is a member of the nuclear hormone receptor superfamily. VD3-VDR binding fastens the association between VDR and its heterodimeric co-receptor, retinoid X receptor (RXR). This active signal transduction complex recognizes vitamin D responsive elements (VDREs) in the DNA sequence and regulates downstream gene transcription (Haussler et al. 2011).

The potential of direct VD action in the ovary was first suggested by a study in hens in the 1980s (Dokoh et al. 1983). As the female gonad, the ovary is responsible for producing steroid hormones to maintain endocrine function and generating competent oocytes for fertilization. Follicles are basic functional units in the ovary, each containing an oocyte surrounded by follicular cells. Ovarian folliculogenesis consists of primordial (dormant) follicle activation, preantral (primary and secondary) follicle growth, and antral (gonadotropin-responsive) follicle maturation to generate the preovulatory (Graafian) follicle. This dynamic follicular development process, along with the growth and maturation of enclosed oocyte, is controlled by local factors in combination with or mediating the actions of gonadotropic hormones (Gougeon 1996). Studies have attempted to determine the role of VD in folliculogenesis and oogenesis in animal models and clinical settings (Irani & Merhi 2014). However, data published to date are inconclusive due to the complexity in VD metabolism and the fact that VD actions are pervasive in regulating physiological functions in various systems, including the reproductive, endocrine and nervous systems that control reproduction (Stumpf & Denny 1989). The following sections summarize evidence regarding VD metabolism and signaling in the ovary, as well as VD-regulated or VD-associated ovarian follicular development, steroidogenic function, and oocyte maturation.

VD and Its Signaling in the Ovary

VD sources for the ovarian follicles

Vitamin D3 25-hydroxylase and 25-hydroxyvitamin D3–1α-hydroxylase are recognized as the hepatic and renal enzyme, respectively, because the circulating VD metabolites (25-hydroxyvitamin D3 and VD3) are predominantly synthesized in the liver and the kidney (Bikle 2014). However, research demonstrated that VD metabolism could also take place locally in the ovary. As summarized in Table 1, CYP2R1 mRNA encoding vitamin D3 25-hydroxylase was detected in human ovarian tissue using RT-PCR (Bièche et al. 2007). CYP27B1 mRNA and 25-hydroxyvitamin D3–1α-hydroxylase protein expression was identified by RT-PCR and Western blot, respectively, in human ovarian tissue and granulosa cells (GCs) from women receiving in vitro fertilization (IVF) treatment (Fischer et al. 2009). Consistently, both CYP2R1 and CYP27B1 were expressed by nonhuman primate (rhesus macaque) preantral and antral follicles, as determined by RNA sequencing and real-time PCR (Xu et al. 2016b; Xu et al. 2018). Therefore, follicular VD can be systemic and intraovarian origin.

Table 1.

Vitamin D metabolic enzyme and receptor expression in the ovary.

Protein	Gene	Species	Materials	Methods	mRNA	Protein	References
Vitamin D3 25-hydroxylase	CYP2R1	Human	Ovarian tissue	RT-PCR	Yes	NA	Bièche et al. (2007)
		Rhesus macaque	Isolated follicles	RNAseq, RT-PCR	Yes	NA	Xu et al. (2016), (2018)
25-hydroxyvitamin D3–1α-hydroxylase	CYP27B1	Human	Ovarian tissue, GCs	RT-PCR, WB	Yes	Yes	Fischer et al. 2009
		Rhesus macaque	Isolated follicles	RNAseq, RT-PCR	Yes	NA	Xu et al. (2016b), (2018)
Vitamin D receptor	VDR	Fish	In vivo	Autoradiograph	NA	Yes	Bidman et al. (1997)
		Hen	Ovarian tissue	Chromatography, sucrose density gradient analysis, saturation analysis	NA	Yes	Dokoh et al. (1983)
		Hen	Isolated follicles, GCs	Real-time PCR, WB, IHC	Yes	Yes	Wojtusik & Johnson (2012)
		Rat	Ovary	IHC	NA	Yes	Johnson et al. (1996)
		Goat	Ovary, GCs	Real-time PCR, WB, IHC	Yes	Yes	Yao et al. (2017)
		Pig	GCs	WB	NA	Yes	Herian et al. (2018)
		Rhesus macaque	Ovary, isolated follicles	Real-time PCR, IHC	Yes	Yes	Xu et al. (2018)
		Human	GCs	RT-PCR	Yes	NA	Parikh et al. (2010)
		Human	GCs	Real-time PCR	Yes	NA	Thill et al. (2009)

CYP2R1, cytochrome P450 family 2 subfamily R polypeptide 1; CYP27B1, cytochrome P450 family 27 subfamily B polypeptide 1; GC, granulosa cell; IHC, immunohistochemistry; RNAseq, RNA sequencing; WB, Western blot; NA, not assessed.

A study in patients undergoing ovarian stimulation first showed that both VD metabolites, 25-hydroxyvitamin D3 and VD3, were present in the follicular fluid (Potashnik et al. 1992). Although levels of VD metabolites in the follicular fluid were relatively low, they correlated positively with those in the serum. Interestingly, when rhesus macaque antral follicles developed in vitro in VD-depleted conditions, follicular CYP2R1 mRNA levels were increased compared with size-matched in vivo-developed antral follicles (Xu et al. 2018). It appears that the reduced or loss of systemic VD supply could induce follicular VD biosynthesis for intraovarian VD production.

VD signaling in the ovarian follicles

The specific binding of VD3 to VDR in the ovary was demonstrated in hens by DNA cellulose chromatography, sucrose density gradient analysis, and saturation analysis (Dokoh et al. 1983), and later in fish (Xiphophorus helleri) by autoradiography (Bidman et al. 1997) (Table 1). The follicular expression of VDR was subsequently identified in various mammalian species (Table 1). VDR protein was localized predominantly in the oocyte of follicles at the early developmental stages, especially the primordial and primary follicles, as shown by immunohistochemistry staining in the caprine and rhesus macaque ovaries (Yao et al. 2017; Xu et al. 2018), though oocyte-specific staining could be further validated by including negative control tissue sections incubated with primary antibodies preabsorbed with blocking peptides. When follicle growth progresses to the preantral and antral stages, VDR expression becomes evident in GCs. VDR mRNA was detected in caprine and human GCs by RT-PCR and real-time PCR (Yao et al. 2017; Parikh et al. 2010; Thill et al. 2009). GC VDR immunostaining was localized in the ovary of rats, goats and rhesus macaques (Johnson et al. 1996; Yao et al. 2017; Xu et al. 2018). VDR protein was also detected by Western blot in GCs from caprine and porcine ovaries (Yao et al. 2017; Herian et al. 2018). The levels of VDR mRNA and protein expression appeared to correlate positively with antral follicle sizes (Wojtusik & Johnson 2012; Yao et al. 2017). These data suggest that VD has the potential to act directly on the oocyte and GCs to maintain or alter the quiescent state of primordial follicles, and to regulate growth and maturation of growing follicles.

Recent in vitro evidence demonstrated that the VD ligand availability could directly impact follicular VDR expression. VD3 supplementation increased levels of VDR mRNA and protein in cultured caprine GCs (Yao et al. 2017). When isolated rhesus macaque follicles were cultured in the absence of VD, follicular VDR mRNA levels decreased compared with size-matched in vivo-developed follicles. With VD3 supplementation, VDR mRNA expression in cultured follicles was restored to levels observed in follicles developed in vivo (Xu et al. 2018). VD3-enhanced VDR protein expression was also reported recently in cultured human ovarian surface epithelial cells (Pejovic et al. 2020). Although data in follicular cells are absent, research in mouse pre-osteoblastic cells and human lymphoblastoid cell lines indicated that the number of VDRE occupied by the VD3-VDR-RXR complex were dynamically controlled by the VD ligand availability (Meyer et al. 2010; Ramagopalan et al. 2010). The VD3-VDR-RXR complex binding to VDRE was enhanced by VD3, which was associated with increased VDR occupancy and gene expression. Thus, VD could generate a positive feedback on VDR expression to enhance its endocrine and paracrine signaling in the ovary.

VD in Follicular Development and Oocyte Maturation

VD-regulated follicular function in animal models

Because VDR is expressed in various organs, including the hypothalamus, pituitary, ovary, oviduct, uterus and placenta (Stumpf & Denny 1989), it is challenging to delineate the direct VD action on the ovarian follicles in vivo. In vitro studies are also limited due to the lack of adequate models to mimic the dynamic follicular development process.

In vivo studies

To date, a few studies have investigated VD in regulating ovarian function in vivo using animal models (Table 2). An early study reported that hens fed with a VD-deficient diet with calcium supplementation ceased laying and had decreased ovarian weight and plasma estradiol (E2)/progesterone (P) levels compared with those on a control diet (Ruschkowski & Hart 1992). Consistently, although calcium was supplemented for proper calcium homeostasis, mice fed with a VD-deficient diet exhibited arrested follicular development and prolonged estrous cycles, with fewer mature oocytes retrieved following gonadotropin stimulation (Dicken et al. 2012). In addition, VDR-null mice showed ovarian insufficiency with impaired follicular development (Kinuta et al. 2000). E2 biosynthesis was disrupted due to decreased gene expression and activity of aromatase, which could not be fully recovered to the levels of control mice by calcium supplementation. Collectively, these data suggest an essential role of VD for proper follicular development and oocyte maturation, which is independent of calcium action. However, alterations in metabolism and function of organs other than the ovary, which compromise the overall health, cannot be ruled out in VD-deficient animals (Hadjadj et al. 2018). Recently, studies were conducted in polycystic ovary syndrome (PCOS) rat models to determine VD effects in the ovary. VD supplementation in the diet improved follicle viability and growth, as well as follicular E2 and P production (Behmanesh et al. 2019). Follicle morphology and follicular cell ultrastructure (e.g., cell junctions, endoplasmic reticulum, and lipid droplets), as well as serum testosterone levels, were ameliorated by VD3 injection (Kuyucu et al. 2020). The evidence supports trophic actions of VD on follicular health and function. However, it is difficult to elucidate the direct VD effect on follicles and enclosed oocytes in these in vivo studies because of the systemic effects generated by global VD manipulations, including altered gonadotropin levels (Behmanesh et al. 2019; Kuyucu et al. 2020).

Table 2.

Vitamin D (VD)-regulated follicular function in animal models.

Study setting	Species	Model	Outcomes	References
In vivo	Hen	VD-deficient diet	Laying ceased, ↓ovarian weight, ↓plasma E2 and P	Ruschkowski & Hart (1992)
	Mouse	VD-deficient diet	Arrested follicular development, prolonged estrous cycles, ↓mature oocyte number	Dicken et al. (2012)
	Mouse	VDR-knockout	Impaired follicular development, ↓aromatase expression and E2 production	Kinuta et al. (2000)
	Rat	PCOS+VD supplementation	Improved follicle viability and growth, ↑serum E2 and P	Behmanesh et al. (2019)
	Rat	PCOS+VD supplementation	Improved follicle morphology and follicular cell ultrastructure, ↓serum testosterone	Kuyucu et al. (2020)
In vitro with VD3 supplementation	Hen	GC culture	↑Cell proliferation	Wojtusik & Johnson (2012)
	Goat	GC culture	↑Cell proliferation, ↑E2 and P production, ↑STAR and HSD3B mRNA levels	Yao et al. (2017)
	Pig	GC culture	↑E2 production, ↑HSD17B1 mRNA levels	Hong et al. (2017)
	Rhesus macaque	Follicle culture	↑Preantral follicle survival, ↑antral follicle growth, improved E2 production and oocyte maturation	Xu et al. (2016a), (2018)

↓, decrease; ↑, increase; E2, estradiol; GC, granulosa cell; HSD17B1, 17β-Hydroxysteroid dehydrogenase 1; HSD3B, 3β-hydroxysteroid dehydrogenase; P, progesterone; PCOS, polycystic ovary syndrome; STAR, steroidogenic acute regulatory protein; VD3, 1α,25-dihydroxyvitamin D3; VDR, VD receptor.

In vitro studies

In vitro studies have attempted to assess the direct VD effects using cultured GCs and isolated follicles (Table 2). VD3 supplementation in the culture media promoted GC proliferation in hens and goats, as assessed by the MTT assay and flow-cytometric cell cycle analysis (Wojtusik & Johnson 2012; Yao et al. 2017). E2 and/or P production by cultured porcine and caprine GCs increased after VD3 treatment, which could be due to elevated expression of steroidogenic enzymes, including 3β-hydroxysteroid dehydrogenase and 17β-hydroxysteroid dehydrogenase 1, as determined by real-time PCR (Hong et al. 2017; Yao et al. 2017). While GC culture is a relevant model for cellular function of large antral follicles from which cells are collected, follicle culture approach offers an opportunity to study follicular activity at the preantral and small antral stages with intact oocyte-follicular cell interactions. Data from cultured rhesus macaque follicles revealed dose- and stage-dependent VD impact on follicular development and function. Low-dose (25 pg/ml) VD3 increased preantral follicle survival, whereas high-dose (100 pg/ml) VD3 enhanced antral follicle growth (Xu et al. 2016a). The VD3 doses bordered on the reference interval of systemic VD3 levels in humans (19.9–79.3 pg/ml; labcorp.com/tests/081091/calcitriol-1–25-di-oh-vitamin-d; Pasquali et al. 2015). VD3 supplementation also promoted E2 production and oocyte maturation of antral follicles developed in vitro (Xu et al. 2016a; 2018). The beneficial effects exerted by VD may be due to its anti-apoptotic and antioxidant actions, as well as by altering cell cycle activities (Yao et al. 2017). Thus, detailed studies are warranted to further understand the underlying mechanisms of VD in regulating folliculogenesis and oogenesis at specific follicular developmental stages.

VD-regulated follicular function in humans

Although research to assess VD effects on the ovary in women without reproductive disorders is limited, clinical studies examined the association between VD levels and ovarian function in patients with a history of infertility. Most of these studies were performed in the context of PCOS or assisted reproductive technology treatment, and the precursor form of VD (25-hydroxyvitamin D3) was often the VD metabolite analyzed in the blood or follicular fluid.

Clinical trials

Primordial follicles represent the dormant follicle pool in the ovary, i.e., the “ovarian reserve” (Wallace & Kelsey 2010). Many clinical studies did not identify any correlations between serum VD levels and ovarian reserve parameters (Table 3). A prospective cross-sectional study and a retrospective cohort study reported that serum VD levels were not associated with ovarian reserve in women who received infertility treatment (Drakopoulos et al. 2017; Shapiro et al. 2018). When serum VD3 levels were analyzed retrospectively in oocyte donors, a high prevalence of VD insufficiency did not impair ovarian reserve (Fabris et al. 2017). However, an observational prospective study suggested that severely low systemic VD levels might be associated with reduced ovarian reserve in VD-deficient patients (Arefi et al. 2018). An inverse relationship between serum VD levels and ovarian reserve was also identified in patients with uterine fibroids (Jukic et al. 2015). The caveat of current clinical research is that only indirect markers of ovarian reserve were evaluated, i.e., follicle-stimulating hormone (FSH) and anti-Müllerian hormone (AMH) levels and antral follicle count, which represent the growing follicle pool, particularly antral follicles, instead of the dormant primordial follicles. Direct assessment on the number and quality of primordial follicles are needed to determine the true ovarian reserve associated with VD status, which is challenging in human studies.

Table 3.

Vitamin D (VD)-regulated follicular function in humans.

Study setting/Patients		VD status or treatment	Outcomes	References
Clinical trial
	PCOS	VD deficiency	↓Ovulation by induction	Pal et al. (2016)
	PCOS	Calcitriol supplementation	↑Spontaneous ovulation	Bonakdaran et al. (2012)
	PCOS	Ergocalciferol and calcium supplementation	Resumption of normal menstrual cycles	Thys-Jacobs et al. (1999)
	IVF	VD deficiency	Serum VD levels were not associated with serum AMH concentrations or AFC	Drakopoulos et al. (2017)
	IVF	VD deficiency	Serum VD levels were not associated with serum AMH or FSH concentrations	Shapiro et al. (2018)
	IVF	VD deficiency	Serum VD levels correlated positively with AFC	Arefi et al. (2018)
	IVF	Normal	High serum VD levels was associated with increased fertilization rates	Abadia et al. (2016)
	IVF	VD deficiency and insufficiency	High serum VD levels was associated with increased fertilization rates	Liu et al. (2019)
	IVF	VD deficiency and insufficiency	High serum VD levels was associated with increased number of oocytes retrieved and fertilization rates	Wu et al. (2018)
	IVF	VDR polymorphism (Taql)	↓AFC	Reginatto et al. (2018)
	IVF	Normal	Low FF VD levels was associated with high AFC and serum E2 concentrations	Antunes et al. (2018)
	IVF	VD deficiency	Low FF VD levels was associated with high fertilization rates	Ciepiela et al. (2018)
	IVF	VD deficiency and insufficiency	Serum VD levels was not associated with fertilization rates	Firouzabadi et al. (2014)
	IVF	VD deficiency and insufficiency	Serum VD levels was not associated with the number of oocytes retrieved, fertilization rates, or embryo quality	Rudick et al. (2012)
	Uterine fibroid	VD deficiency and insufficiency	Serum VD levels correlated negatively with urinary FSH concentrations	Jukic et al. (2015)
	Oocyte donor	VD deficiency and insufficiency	Serum VD levels were not associated with serum AMH concentrations	Fabris et al. (2017)
	Oocyte donor	VD deficiency and insufficiency	Serum VD levels was not associated with the number of oocytes retrieved, fertilization rates, or embryo quality	Banker et al. (2017)
	Healthy	VD deficiency and insufficiency	Serum VD levels correlated negatively with follicular phase and menstrual cycle lengths	Jukic et al. (2015)
GC culture
	IVF	VD3 supplementation	↑P production, ↑HSD3B mRNA levels	Merhi et al. (2014)
	IVF	VD3 supplementation	↓AGE adverse effects on steroidogenesis	Merhi et al. (2018)
	IVF	VD3 supplementation	↑E2 and P production, ↑insulin inhibitory effect on IGFBP1 production	Parikh et al. (2010)

↑, increase; ↓, decrease; AFC, antral follicle count; AGE, advanced glycation end products; AMH, anti-Müllerian hormone; E2, estradiol; FF, follicular fluid; FSH, follicle-stimulating hormone; GC, granulosa cell; HSD3B, 3β-hydroxysteroid dehydrogenase; IGFBP1, insulin-like growth factor-binding protein 1; IVF, in vitro fertilization; P, progesterone; PCOS, polycystic ovary syndrome; VDR, VD receptor; VD3, 1α,25-dihydroxyvitamin D3.

To date, clinical data on VD-associated follicular growth and oocyte maturation yielded equivocal or inconsistent outcomes (Table 3). A number of studies supported the trophic role of VD in promoting follicular development and function, as well as oocyte maturation and competence, during the spontaneous menstrual cycle and IVF cycle. VD deficiency was associated with prolonged follicular phase and overall menstrual cycle length in reproductive-aged women in a prospective cohort study (Jukic et al. 2019). Secondary analysis of a multicenter randomized controlled trial suggested that VD-deficient patients with PCOS were less likely to ovulate following ovulation induction compared with those with sufficient circulating levels of VD (Pal et al. 2016). Calcitriol (VD3) supplementation improved spontaneous ovulation of patients with PCOS in a randomized placebo-controlled trial (Bonakdaran et al. 2012). PCOS patients with documented chronic anovulation resumed normal menstrual cycles after ergocalciferol (vitamin D2) repletion along with calcium therapy (Thys-Jacobs et al. 1999). Serum VD concentrations correlated positively with the number of mature oocytes retrieved and oocyte fertilization rates in patients undergoing the IVF cycle in prospective studies, which was suggested to involve anti‐inflammatory effects of VD (Abadia et al. 2016; Liu et al. 2019; Wu et al. 2018). A VDR polymorphism (Taql) resulted in decreased antral follicle counts in patients receiving ovarian stimulation (Reginatto et al. 2018). However, contradictory findings were reported in studies with the similar IVF setting, wherein high follicular fluid VD levels were associated with low antral follicle count, serum E2 concentrations, and oocyte fertilization rates (Antunes et al. 2018; Ciepiela et al. 2018). In addition, some studies did not identify a significant relationship between serum or follicular fluid VD levels and the IVF outcomes, such as the number of oocytes retrieved, oocyte fertilization rates, and embryo quality in either patients receiving infertility treatment or oocyte donors (Firouzabadi et al. 2014; Rudick et al. 2012; Banker et al. 2017). The controversial results are likely due to the heterogeneity of patient background and medical treatment protocols, as well as the fact that, in most cases, only the precursor form of VD (25-hydroxyvitamin D3) was analyzed. There is a lack of information as to levels of bioactive VD3 in the follicular fluid or follicular and oocyte VDR expression leading to downstream actions via ligand-receptor binding.

In vitro studies

Similar to animal studies, in vitro experiments have been conducted mainly in cultured GCs (mural or cumulus GCs) from large antral follicles in patients undergoing IVF, yielding consistent results in terms of VD-enhanced GC function in steroid production (Table 3). VD3 supplementation stimulated E2 and/or P production directly by promoting the expression of steroidogenic enzymes, e.g., 3β-hydroxysteroid dehydrogenase (Merhi et al. 2014), as determined by real-time PCR, or indirectly by altering the actions of steroidogenic-related factors, e.g., advanced glycation end products and insulin-like growth factor (Merhi et al. 2018; Parikh et al. 2010). Data suggest that VD supports ovarian steroidogenesis, which is essential for follicular development and oocyte maturation. However, data on steroidogenic enzyme protein expression and activity are limited. VD effects has not been studied in intact human preantral for small antral follicles in vitro. Additional investigations are needed to evaluate the relationship between VD metabolic/signaling components and follicular function in humans.

Species Differences in VD-regulated Follicular Signaling Pathways

While many VD-regulated molecular and cellular processes during follicular development and oocyte maturation are similar in animal models and humans, species differences are observed involving key regulatory factors and signaling pathways. For example, studies using GC culture yielded inconclusive results on VD-regulated expression of FSH receptor (FSHR) and AMH signaling pathway components. While VD3 supplementation increased FSHR mRNA levels in hens (Wojtusik & Johnson 2012), it did not alter FSHR expression in pigs and reduced the mRNA and/or protein expression of FSHR in goats and humans (Hong et al. 2017; Yao et al. 2017; Merhi et al. 2014). Although VD3 supplementation decreased AMH mRNA levels in hens (Wojtusik & Johnson 2012), the mRNA and/or protein expression of AMH ligand was not affected by VD3 treatment in pigs, goats or humans (Hong et al. 2017; Yao et al. 2017; Merhi et al. 2014). Instead, VD3 supplementation led to reduced AMH-specific receptor expression in goats and humans (Yao et al. 2017; Merhi et al. 2014). It is consistent in terms of the stimulatory effects of VD on E2 and P production in cultured GCs. However, the alterations in steroidogenic enzyme expression patterns by VD3 supplementation vary between different species. For example, the mRNA levels of steroidogenic acute regulatory protein (STAR) and aromatase (CYP19A1) increased in goats and pigs, respectively, but remained unchanged in humans after VD3 treatment (Hong et al. 2017; Yao et al. 2017; Merhi et al. 2014). It appears that VD actions on regulating follicular cell function could be species-specific.

Involvement of VD in Regulating Mitochondrial Biogenesis and Function

Mitochondria participate in various cellular processes, including adenosine triphosphate (ATP) synthesis, calcium signaling, reactive oxygen species production, and apoptosis, which have critical impact on follicular development and oocyte maturation/competence (May-Panloup et al. 2016). Recently, emerging studies intend to investigate VD actions on mitochondrial biogenesis and function, particularly in cultured rodent GCs. When rat GCs were treated with VD3, mitochondrial biogenesis-related protein expression was increased, which in turn promoted cellular oxygen consumption and ATP production (Lee et al. 2018). VD3 supplementation in the culture media increased mitochondrial DNA copy number, biogenesis and membrane integrity, as well as upregulated gene expression of antioxidant and anti-apoptotic factors, in GCs from a PCOS mouse model (Safaei et al., 2020a; 2020b). Although data are not available for VD-regulated mitochondrial biogenesis and function in the oocyte, these data provide a foundation for studies in vivo and in other animal models or humans to further elucidate VD-regulated mitochondrial activity during folliculogenesis and oogenesis.

Conclusions and future perspectives

Although VD metabolism and signaling pathway are well-known, their existence and importance during folliculogenesis and oogenesis remain obscure. Sufficient VD action appears to be important for the coordinated development of ovarian follicles with improved ovarian steroidogenic function and oocyte maturation. However, it has been challenging to study the direct VD effects in the ovary due to the fact that actions of VD as an endocrine and paracrine factor are highly context-dependent (Stumpf & Denny 1989; Xu et al. 2018). Species and follicular developmental stages, as well as VD metabolite forms, doses and treatment interval, need to be carefully considered during the experimental design. Therefore, adequate animal models are needed for performing well-controlled in vivo and in vitro studies to increase our knowledge surrounding the dynamics of follicular VD metabolites and expression of VDR, VD-regulated follicular development and oocyte maturation, as well as the underlying molecular and cellular mechanisms of VD action in the ovary.

In clinical studies, the correlation between VD levels in the circulation and the follicle or oocyte parameters may vary significantly among patients who are different in VD metabolism and signaling due to diverse physiological and pathological conditions. Follicular development and function may be evaluated more effectively in a relatively homogeneous patient population under a well-controlled experimental design. Although there is general consensus that the total precursor form of VD (25-hydroxyvitamin D3) is the biochemical marker for nutritional VD status, the value may not reflect the bioavailable levels of VD in the ovary. VD is transported to target tissues by binding to high-affinity carrier proteins in the circulation, such as VD binding protein (Bikle et al. 2017). Because VD binding protein polymorphisms, which vary in humans with different ethnic background, affect the binding affinity to VD (Constans et al. 1985; Arnaud and Constans 1993), VD status may be better assessed by calculating bioavailable VD based on levels of total VD and carrier proteins (Fabris et al. 2014). In addition, it is known that serum VD levels vary significantly throughout the year (Tjellesen & Christiansen 1983). Therefore, correction for seasonal variation during data analysis may enhance the validity of study results (Hartwell et al. 1990). Follicular fluid levels of the bioactive form VD3 and the inactive VD metabolite (24,25-dihydroxyvitamin D3), as well as the expression of follicular VDR, may provide further information on VD-exerted biological activities in the ovary (Franasiak et al. 2017). Thus, the association between VD status in patients and their ovarian-related reproductive outcomes warrants further investigations.

In summary, collective evidence suggests a potential role of VD in ovarian follicular development and function from primordial follicle activation to ovulation to generate mature oocytes. VD effects on follicular cells and the oocyte could be directly via regulating its downstream factors or indirectly via mediating gonadotropin actions. A comprehensive understanding of VD-regulated folliculogenesis and oogenesis will provide critical insight into the impact of VD in female reproductive health. Importantly, VD supplementation may have potential therapeutic benefits on improving endocrine function and fertility in women.