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Department of Biochemistry, University of Wisconsin-Madison, Madison, WI 53706
2To whom correspondence should be addressed. E-mail: deluca{at}biochem.wisc.edu.
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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KEY WORDS: • vitamin D and reproduction • calcium and reproduction • vitamin D receptor • vitamin D deficiency • rats
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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). Depending on the gene, vitamin D can stimulate or inhibit gene transcription (1
).
Several lines of evidence suggest that vitamin D might have a direct role in female reproduction in rats. Most important are the nuclear localization of 1,25-(OH)2[3H]D3 and the presence of VDR in rat female reproductive organs such as uterus, oviduct, ovary, mammary gland and placenta as well as the pituitary gland and hypothalamus (2
,3
). Further, a hamster ovary cell line was shown to contain VDR (4
). Finally, the metabolism of vitamin D in female rats is altered during pregnancy and lactation. Serum concentrations of 1,25-(OH)2D3 increase two- to fourfold during pregnancy and lactation, whereas the plasma levels of 24,25-(OH)2D3, a catabolite, drop (5
–7
).
In 1979 Halloran and DeLuca (8
) demonstrated that vitamin D–deficient female rats could reproduce, lactate and maintain their offspring until weaning. However, vitamin D deficiency in female rats reduced mating efficiency, litter size and overall fertility. In addition, neonatal growth was retarded between d 6 and 15 postpartum when pups were nursed by a vitamin D–deficient dam (9
). The decrease in female reproductive efficiency was suggested to be a direct effect of vitamin D (10
). However, in those experiments it was not possible to normalize both serum calcium and serum phosphorus levels in vitamin D–deficient rats at the same time.
By feeding a high calcium, high phosphorus diet containing 20% lactose, serum calcium and phosphorus of vitamin D–deficient rats could be brought into the normal range of vitamin D–replete rats. Under these conditions, vitamin D–deficient female reproduction is completely normalized, demonstrating that vitamin D per se does not play a role therein.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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Female Holtzman weanling rats were obtained from Sprague-Dawley (Madison, WI) and fed one of the three diets: 1) Group 1, a 0.47% Ca+2 and 0.3% phosphorus (Pi) diet supplemented 3 times a week with 500 µg DL-
-tocopherol, 60 µg menadione, and 40 µg ß-carotene in 0.1 mL soybean oil (AEK); 2) Group 2, a 2.0% Ca+2, 1.25% Pi, 20% lactose diet + AEK; or 3) a 0.47% Ca+2 and 0.3% Pi diet + 1.875 µg cholecalciferol in addition to the AEK in 0.1 mL of soybean oil (Hunt Wesson oil, Fullerton, CA) given 3 times each week (ADEK). The basal diet was that described by Suda et al. (11
) and all additions including lactose were at the expense of the glucose monohydrate. The rats were housed in hanging wire cages and maintained on a 12-h light:dark cycle. To prevent endogenous production of vitamin D by UV irradiation of the skin, the rats were housed under incandescent lighting. All experimental protocols were received and approved by the Research Animal Resource Center (University of Wisconsin, Madison, WI).
At 12 wk of age, blood was taken from the tail for measurement of serum calcium and phosphorus concentrations. The serum calcium level was used to assess vitamin D depletion. Daily vaginal smears were performed to monitor estrus cycle for a period of 10 d (at least 2 estrus cycles).
Serum calcium and phosphorus.
Blood samples were obtained from the tail vein during the experiment or by decapitation. Whole blood was centrifuged at 1100 x g for 15 min at 25°C to yield serum. Serum Ca+2 concentration was determined using a 3110 atomic absorption spectrometer (Perkin Elmer, Norwalk, CT) on serum diluted at 1:40 with 1 g/L LaCl3 (12
). Serum phosphorus levels were determined by the method of Itaya et al. (13
).
Serum 25-hydroxyvitamin D3 (25-OH-D3).
The vitamin D metabolite, 25-OH-D3, was extracted from serum using a method described by Reinholz and DeLuca (14
). The levels of 25-OH-D3 in serum were quantitated using a modified version of a competitive ligand-binding assay as previously described by Shepard and DeLuca (15
). To separate protein-bound ligand from free ligand, a 50% (v/v) suspension of hydroxyapatite resin (Bio-Rad, Hercules, CA) in 50 mmol/L of Tris-HCl (pH 7.4) and 5 mmol/L EDTA (TE) was added and allowed to stand for 15 min. The resin was washed three times with TE + 0.5% Triton X-100. The washed collected resin was placed in Biosafe scintillation cocktail (Mount Prospect, IL) and counted in a Packard ß-counter (Packard Instrument, Meriden, CT).
Mating and fertility success.
At 13 wk of age, vitamin D–deficient female rats were mated with vitamin D–sufficient male rats and the offspring produced were used in the following study. The female offspring were weaned to the same diet as their dam (Group 1, n = 17, Group 2, n = 17, and vitamin D–replete n = 16). At 12 wk of age, the rats were tail bled and daily vaginal smears were performed for the next 10 d. At 13 wk of age, two female rats were mated with a vitamin D–sufficient male rat for an interval of 10 d or until the females were sperm positive. At this point, the female rat was indicated sperm positive, and was moved into a cage containing wood shavings.
Pregnant rats were monitored daily for signs of abortion. At parturition and once each week through the lactation period, the number of pups in each litter was counted. At 21 d (weaning age), the final pup number in each litter was recorded. After weaning, the dams were tail bled to determine serum calcium and phosphorus levels.
To evaluate the effect of vitamin D deficiency on female reproduction, the mating and fertility ratios were used as previously described by Halloran and DeLuca (9
). The mating ratio is defined as the total number of females becoming pregnant (i.e., sperm positive) divided by the total animal days mated (i.e., the summation number of females times number of days each was exposed to the male). With a normal estrus cycle of 4–5 d, the mating ratio should range from 0.2 to 0.25. The fertility ratio is defined as the total number of females becoming pregnant and giving birth to live, healthy litters divided by the total animal days mated. The fertility ratio attempts to encompass both mating and parturition. If there are no complications during pregnancy, the fertility ratio should equal the mating ratio.
Statistical methods.
SAS version 8 (SAS Institute, Cary, NC) was used to analyze the data. Serum calcium and phosphorus levels were analyzed using a one-way ANOVA, as were pup number, number of mating days and percentage of pups lost. Comparisons of the least-square means were used to determine the significance between treatment groups. Fisher’s exact test was used to analyze the differences in the fertility success. Differences with P < 0.05 were considered significant.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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Before mating, the vitamin D–deficient rats fed a 20% lactose diet (Group 2) had slightly lower than normal serum phosphorus levels but the serum calcium levels were normal (P < 0.02) (Table 1
). Vitamin D–deficient rats fed the 0.47% calcium diet (Group 1) had significantly lower serum calcium levels than vitamin D–deficient rats fed the 20% lactose diet (P < 0.001) and the vitamin D–replete rats (P < 0.001)(Table 1)
. Vitamin D–deficient rats fed the 0.47% calcium, 0.3% Pi diet had higher than normal serum phosphorus levels relative to vitamin D–replete rats and higher serum phosphorus levels than vitamin D-deficient rats fed the 20% lactose diet (P < 0.001). After mating, vitamin D–deficient rats fed the 0.47% calcium diet still had lower serum calcium level than vitamin D–replete rats and vitamin D–deficient rats fed the 20% lactose diet (P < 0.001). Serum phosphorus levels of the vitamin D–deficient rats fed the 0.47% calcium diet had decreased to levels not different from those in vitamin D–deficient rats fed the 20% lactose diet or in the vitamin D–replete rats.
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Mating and fertility success.
The vitamin D–replete females had a mating ratio of 0.25, whereas the vitamin D–deficient females fed the 0.47% calcium diet had a mating ratio of 0.22 and vitamin D–deficient females fed the 20% lactose diet had a mating ratio of 0.27 (Table 2
). This indicates that, on average, all groups become sperm positive within at least 5 d in the presence of the male. Based on daily vaginal smears, the majority of the vitamin D–deficient dams regardless of diet had a normal 4- to 5-d estrus cycle (data not shown) (10
).
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. Interestingly, the vitamin D–deficient rats fed the 20% lactose diet had a fertility ratio of 0.26, virtually identical to the fertility ratio observed in vitamin D–replete rats. Litter size and lactation.
Litters from hypocalcemic, vitamin D–deficient dams (Group 1) were 40% smaller than litters from vitamin D–replete dams (P < 0.001). The litter size was normalized when vitamin D–deficient dams were fed the high calcium, high phosphorus, 20% lactose diet (Table 3
).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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75% due to a decrease in mating success and complications during pregnancy (9
). To determine whether the decrease in reproductive capacity is a direct effect of vitamin D or an indirect effect via hypocalcemia and/or hypophosphatemia, vitamin D–deficient dams were produced with serum calcium and phosphorus concentrations in the normal range. These rats were as efficient in reproduction as vitamin D–replete rats. This result appears to contradict previous conclusions (10
). However, in the previous study, the investigators were unable to normalize both serum calcium and phosphorus levels at the same time in vitamin D–deficient rats. Thus, combining both the current results and the results previously reported by Kwiecinski et al. (10
), we conclude that hypocalcemia and hypophosphatemia interfere with reproductive capacity.
A decrease in litter size by 40% in the hypocalcemic vitamin D–deficient dams was consistent with the previous observation by Halloran and DeLuca (9
). With the adjustment of serum calcium and serum phosphorus levels in vitamin D–deficient dams through dietary means, the pup number increased to 12.6 ± 1.67, similar to the mean pup number from vitamin D–replete dams. This strongly suggests that the decreased litter size in vitamin D deficiency is due to hypocalcemia, not to a lack of vitamin D.
Vitamin D regulates both intestinal calcium and phosphorus absorption to produce normal serum calcium and phosphorus levels (16
). In the absence of vitamin D, passive intestinal absorption of calcium and phosphorus can occur in the presence of high dietary levels (16
). Previously published research indicates that to normalize serum calcium and phosphorus levels of vitamin D–deficient rats, 20% lactose is required (17
,18
); a high calcium and high phosphorus diet alone is not sufficient. Lactose has been shown to increase calcium and phosphorus absorption in the ileum section of the small intestine; however, the exact mechanism is unclear (19
,20
). Regardless of the role of lactose in intestinal absorption, our data certainly show that normal serum calcium and phosphorus levels result in normal reproduction in the absence of vitamin D. The absence of vitamin D in these rats was verified by measurement of the blood form of vitamin D, i.e., 25-OH-D3. Clearly, lactose itself has no direct effect on reproduction because vitamin D-replete rats have identical reproductive values as the lactose-fed rats.
The present findings support the conclusions reached with the VDR-null mutant mice previously reported by our group (21
). The VDR-null mutant mice are unable to reproduce unless they are provided with a high calcium and high phosphorus diet (21
). Both deficiency of the vitamin and the absence of the VDR had the same effect on female reproduction. Thus, it seems clear that the defect in female reproduction in either the vitamin D–deficient mice or the VDR-null mutant mice is the result of hypocalcemia and/or hypophosphatemia.
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FOOTNOTES |
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3 Abbreviations used: ADEK, 40 µg ß-carotene, 1.875 µg cholecalciferol, 500 µg -tocopherol and 60 µg menadione; AEK, 40 µg ß-carotene, 500 µg -tocopherol and 60 µg menadione in 0.1 mL soybean oil; Pi, phosphorus; VDR, vitamin D receptor; 1,25-(OH)2D3, 1,25-dihydroxycholecalciferol; 25-OH-D3, 25-hydroxyvitamin D3.
Manuscript received 23 August 2001. Initial review completed 17 September 2001. Revision accepted 21 April 2002.
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LITERATURE CITED |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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1. Jones, G., Strugnell, S. A. & DeLuca, H. F. (1998) Current understanding of the molecular actions of vitamin D. Physiol. Rev. 78:1193-1231.
2. Stumpf, W., Sar, M., Reid, F., Tanaka, Y. & DeLuca, H. (1979) Target cells for 1,25-dihydroxyvitamin D3 in intestinal tract, stomach, kidney, skin, pituitary, and parathyroid. Science (Wash., DC) 206:1188-1190.
3. Stumpf, W. E. & Denny, M. E. (1989) Vitamin D (soltriol), light, and reproduction. Am. J. Obstet. Gynecol. 161:1375-1384.[Medline]
4. Dokoh, S., Donaldson, C., Marion, S., Pike, W. & Haussler, M. (1983) The ovary: a target organ for 1,25-dihydroxyvitamin D3. Endocrinology 112:200-206.
5. Boass, A., Toverud, S. U., McCain, T., Pike, J. W. & Haussler, M. (1977) Elevated serum levels of 1, 25-dihydroxycholecalciferol in lactating rats. Nature (Lond.) 267:630-632.[Medline]
6. Halloran, B. P., Barthell, E. N. & DeLuca, H. F. (1979) Vitamin D metabolism during pregnancy and lactation in the rat. Proc. Natl. Acad. Sci. USA 76:5549-5553.
7. Pike, J. W., Parker, J. B., Haussler, M. R., Boass, A. & Toverud, S. (1979) Dynamic changes in circulating 1,25-dihydroxyvitamin D during reproduction in rats. Science (Wash., DC) 204:1427-1429.
8. Halloran, B. P. & DeLuca, H. F. (1979) Vitamin D deficiency and reproduction in rats. Science (Wash., DC) 204:73-74.
9. Halloran, B. P. & DeLuca, H. F. (1980) Effect of vitamin D deficiency on fertility and reproductive capacity in the female rat. J. Nutr. 110:1573-1580.
10. Kwiecinski, G. G., Petrie, G. I. & DeLuca, H. F. (1989) 1,25-Dihydroxyvitamin D3 restores fertility of vitamin D-deficient female rats. Am. J. Physiol. 256:E483-E487.
11. Suda, T., DeLuca, H. F. & Tanaka, Y. (1970) Biological activity of 25-hydroxyergocalciferol in rats. J. Nutr. 100:1049-1052.
12. Bowers, G. & Rains, T. (1988) Measurement of total calcium in biological fluids: flame atomic absorption spectrometry. Riordan, J. Vallee, B. eds. Methods in Enzymology 1988:302-319 Academic Press San Diego, CA. .
13. Itaya, K. & Ui, M. (1966) A new micromethod for the colorimetric determination of inorganic phosphate. Clin. Chim. Acta 14:361-366.[Medline]
14. Reinholz, G. & DeLuca, H. (1998) Inhibition of 25-hydroxyvitamin D3 production by 1,25-dehydroxyvitamin D3 in rats. Arch. Biochem. Biophys. 355:77-83.[Medline]
15. Shepard, R. & DeLuca, H. (1980) Determination of vitamin D and its metabolites in plasma. Methods in Enzymology 1980:393-413 Academic Press New York, NY. .
16. Wasserman, R. (1997) Vitamin D and the intestinal absorption of calcium and phosphorus. Feldman, D. Glorieux, F. H. Pike, J. W. eds. Vitamin D 1997:259-273 Academic Press New York, NY. .
17. Kollenkirchen, U., Walter, M. & Fox, J. (1991) Plasma Ca influences vitamin D metabolite levels as rats develop vitamin D deficiency. Am. J. Physiol. 23:E447-E452.
18. Kollenkirchen, U., Fox, J. & Walters, M. (1991) Normocalcemia without hyperparathyroidism in vitamin D-deficient rats. J. Bone Miner. Res. 6:273-278.[Medline]
19. Debiec, H. & Lorenc, R. (1988) Influence of lactose on phosphate metabolism in rats. Br. J. Nutr. 59:87-92.[Medline]
20. Dupuis, Y. & Fournier, P. (1962) Lactose and the absorption of calcium and strontium. Wasserman, H. eds. The Transfer of Calcium and Strontium Across Biological Membranes 1962:277-293 Academic Press New York, NY. .
21. Johnson, L. & DeLuca, H. (2001) Vitamin D receptor null mutant mice fed high levels of calcium are fertile. J. Nutr. 131:1787-1791.
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