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Articles

Vitamin D Receptor Null Mutant Mice Fed High Levels of Calcium Are Fertile¹

Department of Biochemistry, University of Wisconsin-Madison, Madison, WI 53706

²To whom correspondence should be addressed. E-mail: deluca{at}biochem.wisc.edu

	ABSTRACT

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Vitamin D receptor (VDR) null mutant mice provide a model to investigate the possible effect of vitamin D on female reproduction. Infertility in these mice has been reported but it is uncertain whether the infertility results from a lack of VDR or from the hypocalcemia that results from a lack of VDR. VDR null mutant mice and wild-type controls were fed a nonpurified, high calcium or medium calcium diet, plus a diet containing lactose and their reproductive efficiency was examined. VDR null mutant mice fed a nonpurified diet were hypocalcemic and were found to be largely infertile with 14% fertility, while the fertility percentage of normocalcemic VDR null mutant mice and wild-type mice was between 86% and 100%. A high calcium or medium calcium diet maintained 100% fertility in the VDR knockout mice; removal of the lactose from this diet did not diminish reproductive capability. Reproductive capacity of VDR null mutant mice was analyzed when they were fed purified diets containing 0.02–2% calcium. Mutant mice fed a low calcium diet (0.47%) had a lower reproductive efficiency than VDR null mutant mice fed a diet that resulted in normal serum calcium concentrations. Thus, high dietary calcium levels are required for normal reproduction in VDR null mutant female mice. It seems that the defect in reproduction reported previously for VDR null mutant mice is not the lack of a direct effect of 1,25-dihydroxycholecalciferol on reproductive function but is the result of hypocalcemia.

KEY WORDS: • fertility • reproduction • vitamin D receptor • vitamin D and reproduction • mice

	INTRODUCTION

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The biological activity of vitamin D is mediated by the vitamin D receptor (VDR)³ , which is a member of the steroid/thyroid superfamily of receptors. 1,25-Dihydroxycholecalciferol (1,25(OH)₂-D₃), the hormonally active form of cholecalciferol (D₃), is the ligand for the VDR. Once the ligand is bound, the VDR forms a heterodimer with the retinoid X receptor (RXR) that binds to responsive elements located in promoters of target genes (1) .

Vitamin D plays a role in calcium and phosphate homeostasis (1) . Previous reports have suggested that vitamin D also is important in female reproduction of rats (2 ,3) . However, female reproduction is not absolutely dependent on vitamin D (4) . Vitamin D did improve mating success and did increase litter size. Vitamin D deficiency also impairs neonatal growth and causes an increase in pregnancy complications. Vitamin D deficiency in female rats led to an overall reduction in fertility of 75%. The reduction resulted from a 50% decrease in mating efficiency and complications during pregnancy. In addition, litter sizes of vitamin D-deficient rats are 30% smaller than are those of vitamin D-sufficient rats (2) . In support of the idea that vitamin D potentially plays a direct role in female reproduction, the VDR has been found in the nuclei of reproductive tissues (5) and a hamster ovarian cell line (6) .

The observed decrease in fertility has been postulated to result from vitamin D deficiency rather than from the hypocalcemia associated with vitamin D deficiency (3) . Compared with vitamin D-sufficient female rats, fertility in vitamin D-deficient female rats was reduced by 67% (vitamin D-deficient diet, hypocalcemic and normophosphatemic), 100% (vitamin D-deficient diet, normocalcemic and hypophosphatemic), and 84% (vitamin D-deficient diet, normocalcemic and normophosphatemic). Regardless of Ca⁺² and inorganic phosphate (P_i) levels in the diet and, thus, the plasma, vitamin D deficiency led to a reduction in fertility. Reproductive capacity was restored in vitamin D-deficient rats within 3 wk by either D₃ or 1,25(OH)₂-D₃ (3) .

A new tool to investigate a possible role of vitamin D in reproduction was recently provided by two groups in the development of VDR null mutant mice (7 ,8) . Yoshizawa and colleagues (8) have reported that these female mice are unable to reproduce and that the defect was presumably due to inadequate uterine development. Dr. S. Kato kindly provided breeding stock of these mice that allowed additional study. These VDR null mutant mice were generated by a targeted disruption in exon 2, which includes the transcriptional start site and the first Zn⁺² finger of the VDR. The neomycin cassette insertion abolishes the activity of vitamin D by eliminating the translation of a functional VDR protein.

The VDR null mutant mice, after weaning, exhibit low serum calcium and phosphate levels, alopecia and bone formation similar to vitamin D-dependent rickets Type II in humans (8) . Infertility in the VDR null mutant mice has been investigated further by studying the role of vitamin D in the regulation of estrogen synthesis in the gonads, specifically aromatase enzyme activity and expression in the ovaries, testes and epididymis (9) . In the null mutant mice, gene expression was low when compared with wild-type mice and the aromatase activity was 24%, 58% and 35% of the wild-type values in the ovary, testis and epididymis, respectively. With dietary calcium supplementation (20% lactose), there was an increase in aromatase activity in the ovary to 60% of wild-type level. The authors conclude that estrogen biosynthesis is regulated by calcium, but vitamin D may play an additional role (9) . However, the authors did not directly examine fertility of these mice on the various diets.

The purpose of these experiments was to test whether the infertility in the VDR null mutant mice was a direct result of the nonfunctional VDR or a secondary result of the hypocalcemia. We now report that these VDR null mutant female mice are fully able to reproduce, despite a proven absence of a functional VDR; however, fertility in these mice is dependent on high dietary calcium levels.

	MATERIALS AND METHODS

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Ligand binding assay.

A modified nuclear extract of kidney and liver tissue was prepared as described previously (10) . A ligand-binding assay was conducted on nuclear extracts based on a procedure of Wecksler and Norman (11) . Dilutions of the extract were incubated overnight at 4°C with 2 nmol/L 1,25(OH)₂-[³H]D₃ [160 Ci/mmol 1,25(OH)₂-D₃] with and without 200 nmol/L 1,25(OH)₂-D₃. The next day, a 50% (v/v) suspension of hydroxyapatite resin in 50 mmol/L of Tris-Cl (pH 7.4), 5 mmol/L of EDTA and 5 mmol/L of dithiothreitol was mixed with the extract for 15 min. The resin was washed three times with TED + 0.5% Triton X-100. The washed collected resin was placed in a Biosafe scintillation cocktail (Mount Prospect, IL) and counted in a Beckman ß-counter (Palo Alto, CA). Protein concentration of nuclear extracts was determined using a Bradford Assay (Bio-Rad, Hercules, CA) using bovine serum albumin as the standard.

RNase protection assay.

Total RNA was isolated from mouse kidney and liver as described previously (12) . A polymerase chain reaction-generated DNA fragment encoding the exon 2 of the rat VDR was cloned into Bluescript vector (Stragene, La Jolla, CA). The Bluescript vector clone was used to generate an RNA probe using a commercially available in vitro transcription kit (Ambion, Austin, TX) and the probe was labeled with [³²P] uridine triphosphate. An RNase protection assay kit (Ambion) was used according to manufacturer’s instructions. A 250-base pair mouse ß-actin probe was used as a control. Ten micrograms or 30 µg of total RNA from VDR heterozygotes and null mutant mice was hybridized with the exon 2/VDR probe or the ß-actin probe. A 5% acrylamide · (8 mol/L urea) · (0.09 mol/L Tris borate, 0.002 mol/L EDTA) gel was run to separate the protected fragment. The gel was dried and exposed to a phosphorimager screen for 18 h.

Animal maintenance.

A breeding colony was established from two pairs of mice (a generous gift from Dr. Shigeaki Kato, Institute of Molecular and Cellular Biosciences, University of Tokyo, Tokyo, Japan) that were heterozygous for the VDR (8) . The mice were housed in a 12-h light:12-h dark cycle. They were maintained in cages with wooden shavings and consumed distilled water and nonpurified or purified diet ab libitum. All experimental protocols were received and approved by the Research Animal Resources Center (University of Wisconsin-Madison, Madison, WI).

Serum calcium analysis.

Blood was obtained by nicking the tail and serum was prepared by centrifugation. Serum was diluted 1:40 with 0.1 g/L of LaCl₂ and calcium levels were determined using atomic absorption spectroscopy (Perkin-Elmer, Norfolk, CT).

Reproductive studies.

Wild-type and VDR ablated mice were fed one of four diets: 1) nonpurified diet (5015 Purina Chow, Richmond, IN) containing 0.8% calcium, 0.5% phosphorus and 82.5 ng of vitamin D/g of diet, 2) 10% lactose (13 ,14) , 100 g/kg lactose, 1.2% calcium and 0.7% phosphorus diet supplemented with 75 ng of D₃/g of diet, 3) high calcium diet, 1.2% calcium, 0.7% phosphorus diet supplemented with 75 ng of D₃/g of diet (in the absence of lactose) or 4) medium calcium diet, 0.8% calcium, 0.5% phosphorus diet supplemented with 75 ng of D₃/g of diet (in the absence of lactose). In all cases, lactose was added at the expense of the cerelose (glucose monohydrate) in the diet described by Suda et al. (14) . Serum calcium levels were monitored at breeding age (7 wk). VDR null mutants and wild-type littermates were paired at 7 wk of age and allowed to mate. The time between potential mating and parturition was monitored. The fertility frequency was followed for 3 mo. The number of pups produced was counted the morning after parturition.

The effect of dietary calcium on fertility.

Both VDR ablated and wild-type mice were weaned onto the diet described by Suda et al. (14) that varied in calcium percentages: 1) 0.02%, 2) 0.47%, 3) 0.87%, 4) 1.2% and 5) 2%. All experimental diets were supplemented with 75 ng of D₃/g of diet. Serum calcium levels were determined as discussed in the serum calcium analysis section. At 7–8 wk of age, VDR null mutants and wild-type mice were paired and allowed to mate. The time between potential mating and parturition was monitored.

Statistical analysis.

SAS version 8 (SAS Institute, Cary, NC) was used to analyze statistical significance of the treatment groups. The data on serum calcium levels were analyzed using a mixed procedure (Proc GLM). A comparison of the least square means was used to determine significance between treatment groups. A Fisher’s exact test was used to analyze the significance of the fertility percentages. Differences with P < 0.05 value were considered significant.

	RESULTS

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Verification of VDR disruption.

Two lines of evidence were used to verify that the VDR was disrupted. The absence or presence of a functional VDR protein was determined using a ligand-binding assay (Fig. 1 ; 11 ). Kidney nuclear extracts of the null mutant mice (89.8 ± 55.4 fmol/mg) had significantly reduced 1,25(OH)₂-D₃ binding activity compared with extracts either from wild-type (573.9 ± 6.77 fmol/mg) or heterozygous mice (414.2 ± 50.24 fmol/mg). Liver extracts, a vitamin D unresponsive tissue, were used as a negative control in the ligand-binding assay for each genotype (< 24 fmol/mg).

View larger version (28K): [in this window] [in a new window]   Figure 1. Ligand-binding assay—verification of the VDR gene disruption in mouse kidneys from VDR null mutant mice. Modified nuclear extracts of kidney and liver from wild-type homozygous (+/+), heterozygous (±) and VDR null mutant (-/-) mice were incubated with 2 nmol/L 1,25(OH)2-[3H]D3 and 200 nmol/L 1,25(OH)2-D3 overnight at 4°C. Values represent a mean (± range) of the assay performed in duplicate at three dilutions. Two mice of each genotype were used to make the modified nuclear extracts.

Fertility of the VDR null mutant mice.

The VDR null mutant mice have previously been reported to be infertile (8) . In contrast, VDR null mutants originally obtained from Yoshizawa et al. (8) reproduced in our laboratory as efficiently as wild type but this ability depended on diet. Interestingly, VDR null mutant mice, when maintained on a nonpurified diet from weaning (21 d of age), were mostly infertile (14% fertile) compared with wild-type mice with a 86% fertility (Table 1 ). Both the VDR null mutant mice and wild-type mice were fertile when fed the 10% lactose diet (100% fertility). Importantly, even in the absence of lactose in the high calcium diet, the mice absent VDR could reproduce as well as wild-type mice (100% fertility; Table 1 ).

View larger version (60K): [in this window] [in a new window]   Figure 2. The absence of VDR does not alter the time of mating and gestation in VDR null mutant mice. At 7 wk of age, wild-type mice (WT) and VDR null mutant mice (KO) were combined in breeding pairs. They consumed one of four diets: 1) nonpurified, 2) 10% lactose, 3) high calcium or 4) medium calcium. The values represent the mean ± standard deviation (n = 4–7) number of days between potential mating and parturition during a 3-mo period. Genotypes did not differ; *P > 0.1.

Litter size of VDR null mutant mice.

In vitamin D-deficient rats, the litter size is 30% smaller than in vitamin D-sufficient rats (2 ,4) . The average litter sizes for the VDR null mutant mouse breeders consuming the lactose or high calcium diets were 4.2 ± 1.10 pups and 3.5 ± 1.29 pups, respectively, which were not significantly different from the litter sizes of wild-type breeders fed 10% lactose or high calcium diets (3.9 ± 2.67 pups and 5.29 ± 2.13 pups, respectively; both P > 0.1; Fig. 3 ). Thus, a 30% decrease in litter size as occurred in litters from vitamin D-deficient rats was not observed in the VDR null mutant mice.

View larger version (49K): [in this window] [in a new window]   Figure 3. VDR null mutant mice have litters similar in number to wild-type mice. At 7 of wk age, wild-type mice (WT) and VDR null mutant (KO) mice were combined in breeding pairs. They consumed one of the following diets: 10% lactose or high calcium. The litter size of the mice fed the nonpurified diet was excluded because only one VDR null mutant mouse was fertile. The values represent the mean (± standard deviation) number of pups per litter (n = 4–7 mice per group) observed the morning after parturition during 3 mo. Genotypes did not differ; *P > 0.1.

Initial experiments indicated that dietary calcium affected serum calcium levels in the VDR null mutant mice (Table 2) . Mice were then fed diets differing in calcium concentration and their serum calcium levels were monitored. VDR-ablated mice fed a diet containing 0.47% calcium had a significantly lower serum calcium level than wild-type mice (P < 0.001). With higher dietary calcium concentration (1.2% Ca⁺² and 2% Ca⁺²), VDR null mutant mice maintained serum calcium levels similar to serum calcium levels in wild-type mice (Table 3 ; P > 0.1). VDR null mutant mice fed a diet deficient in calcium (0.02% Ca⁺²) were extremely hypocalcemic and they died at 6–7 wk of age (Table 3) .

View larger version (52K): [in this window] [in a new window]   Figure 4. The time length of mating and gestation of fertile VDR null mutant mice was not affected by dietary calcium. At weaning, the mice consumed a base diet with various calcium percentages: 1) 0.02%, 2) 0.47%, 3) 0.87%, 4) 1.2% or 5) 2%. At 7 wk of age, wild-type mice and VDR null mutant mice were combined in breeding pairs. The values represent the mean ± standard deviation (n = 4–12) number of days between potential mating and parturition for 3 mo. Genotypes did not differ; *P > 0.1.

	DISCUSSION

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Dietary calcium levels had a marked effect on serum calcium concentrations of the VDR null mutant mice but not the wild-type mice. VDR-ablated mice that consumed a diet containing 0.47% calcium were hypocalcemic, and in these mice, reproduction efficiency was diminished by 30% compared with wild-type mice. VDR-ablated mice fed diets that could normalize serum calcium had 100% fertility, similar or higher than fertility percentage observed in wild-type mice. Thus, there seems to be a correlation between serum calcium levels and reproductive performance. Interestingly, VDR null mutant mice that did conceive in all cases had similar time length of mating and gestation as wild-type mice. Therefore, it seems that the defect in reproduction previously reported for these VDR null mutant mice is caused by hypocalcemia that resulted from the disruption of the function of vitamin D in calcium metabolism rather than the absence of the VDR itself.

Thus, in our hands, vitamin D null mutant mice do not fully illustrate the defect in female reproduction found with vitamin D-deficient rats. Vitamin D-deficient rats apparently have a 75% decrease in fertility because of a 50% decrease in mating efficiency and an overall increase in pregnancy complications. Based on these results, it was indeed unexpected that VDR null mutant mice were found capable of reproduction. Therefore, this represents a major departure in the two models to study the role of vitamin D in this function. The decrease in fertility due to deficiency of vitamin D was found in rats, whereas the present studies were carried out in mice. It is possible that an important species difference and it is indeed possible that vitamin D-deficient female mice will be fully capable of reproduction. In contrast, it is possible that vitamin D could have a function in female reproduction that is not mediated by the VDR. Still another possibility is that another, yet unknown, VDR functions in reproduction. These different possibilities warrant additional investigation but, at the present time, it is unknown why there is a discrepancy between the vitamin D-deficiency studies in rats and the current studies in the VDR null mutant mice.

	FOOTNOTES

 
¹ Supported by funds from the National Foundation for Cancer Research and the Wisconsin Alumni Research Foundation.

³ Abbreviations used: VDR, vitamin D receptor; 1,25(OH)₂-D₃, 1,25-dihydroxycholecalciferol; D₃, cholecalciferol; RXR, retinoid X receptor; P_i, inorganic phosphate.

Manuscript received December 18, 2000. Initial review completed January 24, 2001. Revision accepted March 23, 2001.

	REFERENCES

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