Consequences of vitamin D deficiency or overdosage on follicular development and steroidogenesis in Normo and hypo calcemic mouse models

Abstract

Vitamin D deficiency (VDD) is a widespread situation, linked to patients' dietary habits and/or geographical origins. On the other hand, hypervitaminosis D (VDO) is also a worldwide problem, mainly associated with uncontrolled self-administration. In this study, we investigated the effects of VDD and VDO on sex steroid production and ovarian histology in mice. In addition to addressing the rarely explored situation of VDO, the originality of our approach is to disconnect VDD/VDO situations from the well-known calciotrophic effect of vitamin D (VitD). Our data indicate that VDD led to a significant decrease in serum LH and FSH levels, independently of serum calcium levels. VDD was also associated with increased testosterone and reduced oestradiol levels. VDO animals showed increased LH and reduced testosterone levels. Hormonal changes in the VDO animal groups were correlated with a lower accumulation of transcripts of steroidogenic genes such as CYP11A1 and 3ß-HSD, whereas these transcripts were higher in the VDD groups. CYP19A1 transcripts were lower in VDD animals than in controls. This study highlights the complex interaction between vitamin D status, the regulation of reproductive hormones and, consequently, reproductive performance. It underlines the need for caution when oral vitamin D supplementation is chosen as a therapeutic action to boost female reproductive performance, as VDO can be as detrimental as VDD.

Keywords: Liver enzyme; Ovary; Reproduction; Sex steroids; Vitamin D deficiency; Vitamin D overdose.

Fig. 1

Experimental design. Black circles and black Square: paricalcitol and Zoledronic acid injections, respectively. Black Rectangle: Calcitriol gavage. In this experiment, 195 NMRI female mice were utilized. Prior to the allocation of groups, 5 mice were randomly chosen for the assessment of blood 25-hydroxyvitamin D2 and calcium levels. The remaining 190 mice were subsequently divided into 5 groups (n = 38 each): (1) control group, (2) vitamin D overdose (VDO) and normal calcium (VitD⁺Ca), these mice were treated with vitamin D daily with low phosphorus and calcium diet, (3) vitamin D and calcium deficient (VitD⁻Ca⁻), these mice were treated with paricalcitol and normal phosphorus and calcium diet, (4) vitamin D deficient and normal- calcium (VitD⁻Ca), these mice were treated with paricalcitol and high phosphorus and calcium diet, (5) vitamin D normal and calcium deficient (VitD Ca⁻), these mice were treated zoledronic acid with low phosphorus and calcium diet.

Fig. 2

Assessment of serum levels of 25-hydroxyvitamin D2 (A), calcium (B), Alanine transaminase (ALT) (C) and Aspartate aminotransferase (AST) (D) content at day 72 in the different groups of mice. ^{b c d} indicates significant differences groups with ^a control group. Statistical differences between groups were obtained by a one-way ANOVA test (P < 0.01, P < 0.001, P < 0.0001).

Fig. 3

Assessment of serum levels of Luteinizing Hormone (LH) (A), Follicle-stimulating hormone (FSH) (B), Estradiol (C), Progesterone (D) and Testosterone (E), in the different groups of mice at day 72. ^{b c d} indicates significant differences groups with ^a control group. Statistical differences between groups obtained by one-way ANOVA test (P < 0.01, P < 0.001, P < 0.0001).

Fig. 4

Distributions of different types of follicles per ovary, primary(A), primordial (B), secondary (C) tertiary (D)and Graafian (E) follicles. ^{b c d} indicates significant differences groups with ^a control group. Statistical differences between groups were obtained by a one-way ANOVA test (P < 0.01, P < 0.001, P < 0.0001).

Fig. 5

Distributions of different types of follicles per ovary, primary(A), primordial (B), secondary (C) tertiary ()Dand Graafian (E) follicles. ^{b c d} indicates significant differences groups with ^a control group. Statistical differences between groups were obtained by a one-way ANOVA test (P < 0.01, P < 0.001, P < 0.0001).

Fig. 6

Cross-sections of ovarian tissue from different groups; the cross-section from the control group represented the normal distribution of follicles at different stages along with distributed corpus luteums from the previous cycles in the cortex area. See the expanded follicular atresia in the sections from the experimental groups at various stages of the folliculogenesis. The intact follicles contain normal oocytes (NO), with homogenous cytoplasm without cytoplasmic vacuolation (CV), well-formed theca layers (black arrow), and normal integrity of granulosa cells (NGC) with no granulosa cell dissociation (GCD) and antrum formation (*), no hyalinization (HCG), normal cumulus oophorus integrity (NCOI) with no apoptotic granulosa cells (AGC) when compared to the atretic follicles (H&E staining, the photomicrographs with higher magnifications: 400× microscopic and 2.4× optical magnification).

Fig. 7

Real-time PCR assessment of Granulosa cells CYP11A1(A) 3β-HSD (B), CYP19A1(C) in the different groups of mice at 72 days. ^{b c d} indicates significant differences groups with ^a control group. Statistical differences between groups obtained by one-way ANOVA test (P < 0.01, P < 0.001, P < 0.0001).