Peripubertal vitamin D(3) deficiency delays puberty and disrupts the estrous cycle in adult female mice

Abstract

The mechanism(s) by which vitamin D(3) regulates female reproduction is minimally understood. We tested the hypothesis that peripubertal vitamin D(3) deficiency disrupts hypothalamic-pituitary-ovarian physiology. To test this hypothesis, we used wild-type mice and Cyp27b1 (the rate-limiting enzyme in the synthesis of 1,25-dihydroxyvitamin D(3)) null mice to study the effect of vitamin D(3) deficiency on puberty and reproductive physiology. At the time of weaning, mice were randomized to a vitamin D(3)-replete or -deficient diet supplemented with calcium. We assessed the age of vaginal opening and first estrus (puberty markers), gonadotropin levels, ovarian histology, ovarian responsiveness to exogenous gonadotropins, and estrous cyclicity. Peripubertal vitamin D(3) deficiency significantly delayed vaginal opening without affecting the number of GnRH-immunopositive neurons or estradiol-negative feedback on gonadotropin levels during diestrus. Young adult females maintained on a vitamin D(3)-deficient diet after puberty had arrested follicular development and prolonged estrous cycles characterized by extended periods of diestrus. Ovaries of vitamin D(3)-deficient Cyp27b1 null mice responded to exogenous gonadotropins and deposited significantly more oocytes into the oviducts than mice maintained on a vitamin D(3)-replete diet. Estrous cycles were restored when vitamin D(3)-deficient Cyp27b1 null young adult females were transferred to a vitamin D(3)-replete diet. This study is the first to demonstrate that peripubertal vitamin D(3) sufficiency is important for an appropriately timed pubertal transition and maintenance of normal female reproductive physiology. These data suggest vitamin D(3) is a key regulator of neuroendocrine and ovarian physiology.

FIG. 1.

Timeline of experimental events. All experiments were carried out with mice born from heterozygote matings in which the dams received a Vit D₃+ or Vit D₃− diet. WT and Cyp27b1 null mice female pups were weaned at 21 days and randomized to either a Vit D₃+ or a Vit D₃− diet supplemented with calcium gluconate. WT (+/+) mice that received a Vit D₃+ diet throughout gestation, lactation, and weaning served as controls. All mice were inspected daily for vaginal opening. After vaginal opening, vaginal lavage was performed daily at 1500 h for a minimum of 7 wk, and estrous cycle length and estrous stage frequency were determined with vaginal smears.

FIG. 2.

Peripubertal 1,25-(OH)₂ vitamin D₃ deficiency delays puberty. A) Age at vaginal opening (VO) in WT (Cyp27b1^+/+) mice fed a Vit D₃+ diet throughout pregnancy (in utero) and lactation (perinatal period) and peripubertal transition and in Cyp27b1 null female mice subjected to a Vit D₃− diet in utero and perinatal period and peripubertal transition, or in utero and perinatal period, or only during the peripubertal transition. B) Age at first estrus in WT mice fed a D+ diet in utero and perinatal period and peripubertal transition and in Cyp27b1 null female mice subjected to D− diet in utero and perinatal period and peripubertal transition or in utero and perinatal period, or only during the peripubertal transition. C) Number of days between VO and first estrus in WT mice fed a D+ diet in utero and perinatal period and peripubertal transition and in Cyp27b1 null female mice subjected to D− diet in utero and perinatal period and peripubertal transition, or in utero and perinatal period, or only during the peripubertal transition (n = 8–15); ^aP < 0.0001 vs. WT; ^bP < 0.0001 vs. in utero and lactation; ^cP < 0.05 vs. WT; ^dP < 0.01 vs. WT.

FIG. 3.

Mice fed a vitamin D₃-deficient diet during the prepubertal period have a delayed pubertal onset but normal developmental growth curves. A) Age at vaginal opening. B) Age at first estrus. C) Days between vaginal opening and first estrus. D) Developmental growth curves of mice. KO denotes Cyp27b1 null mice. WT denotes Cyp27b1^+/+ mice. Vit D₃+ or D+ denotes mice fed a Vit D₃-sufficient diet before and after weaning. Vit D₃− or D− denotes mice supplemented with calcium gluconate and fed a Vit D₃-deficient diet after weaning (n = 8–15). ^aP < 0.01 vs. Vit D₃+; ^bP < 0.0001 vs. Vit D₃+

FIG. 4.

Vitamin D₃ deficiency extends the estrous cycle by increasing time spent in diestrus. A) Average cycle length in WT and Cyp27b1 null mice fed a Vit D₃+ or Vit D₃− diet during the peripubertal period. Average percentage of time spent in diestrus I/II (B), proestrus (C), and estrus (D) in WT and Cyp27b1 null mice fed a Vit D₃+ diet or Vit D₃− diet during the prepubertal period and into early adulthood (n = 8–15); ^aP < 0.05 vs. Vit D₃+ diet; ^bP < 0.001 vs. Vit D₃+ diet. E) Representative estrous cycle of WT mouse fed Vit D₃+ diet throughout gestation and lactation and after weaning. F) Representative estrous cycle of WT mouse fed Vit D₃+ diet throughout gestation and lactation and weaned onto a Vit D₃− diet. G) Representative estrous cycle of Cyp27b1 mouse fed Vit D₃+ diet throughout gestation and lactation and after weaning. H) Representative estrous cycle of Cyp27b1 mouse fed Vit D₃+ diet throughout gestation and lactation and weaned onto a Vit D₃− diet. P, proestrus; E, estrus; D1, diestrus 1; D2, diestrus 2.

FIG. 5.

Vitamin D₃ deficiency reversibly disrupts the estrous cycle. Average percentage of time spent in diestrus I (A), diestrus II (B), proestrus (C), and estrus (D) when Vit D₃− Cyp27b1 mice (KO [D-]) were switched to a Vit D₃+ (D+) diet. WT (Vit D₃+) mice described in Figure 4 were included for comparison. ^aP < 0.03 vs. WT and KO (D+) mice; n = 6. E) Representative estrous cycle of Cyp27b1 mouse fed Vit D₃+ diet throughout gestation and lactation and weaned onto a Vit D₃− diet. F) Representative estrous cycle of the same Cyp27b1 mouse transferred to a Vit D₃+ diet for 4 wk. P, proestrus; E, estrus; D1, diestrus 1; D2, diestrus 2. (n = 6).

FIG. 6.

Diet-induced vitamin D₃ deficiency is associated with a robust response to superovulation with exogenous gonadotropins. Representative photomicrograph (original magnification ×40) of WT (top) and Cyp27b1 null mice fed a Vit D₃-deficient diet during the peripubertal period (bottom), injected with saline or superovulated with eCG plus hCG. A) WT mice injected with saline. B) Cyp27b1 null mice fed a Vit D₃− diet, injected with saline. C) WT mice fed a Vit D₃+ diet, injected with eCG and hCG. D) Cyp27b1 null mice fed a Vit D₃− diet, injected with eCG and hCG. E) Number of oocytes deposited into the oviduct of WT and CYP27b1 null (KO) mice fed a Vit D₃+ diet and Cyp27b1 null mice fed a Vit D₃− diet after superovulation with eCG and hCG. AF, antral follicle; CL, corpus luteum; PA, early preantral; 2°, secondary. ^aP = 0.02 vs. WT and KO mice fed a Vit D₃+ diet during the peripubertal transition (n = 4–6).

FIG. 7.

Effects of vitamin D₃ on serum gonadotropin levels during mice in diestrus, VDR expression in GT1-7, and density of hypothalamic GnRH neurons. A) Serum FSH in reproduction-aged WT and Cyp27b1 null mice fed a vitamin D₃-sufficient diet (KO [D+]) or a D₃-deficient diet (KO [D−]) (n = 4–12). B) Serum LH in reproduction-aged WT and Cyp27b1 null mice fed a vitamin D₃-sufficient diet (KO [D+]) or D₃-deficient diet (KO [D−]) (n = 4–12). C) Western blot showing VDR in cell lysates from GT1-7 neurons and kidney. The positive control is WT mouse kidney, and negative control is GT1-7 and kidney lysate without primary VDR antibody. D) Representative sections of single-label immunohistochemistry (original magnification ×40) showing GnRH neurons (brown cytoplasm) in WT Vit D₃+ and Cyp27b1 null Vit D₃− mice. Arrows indicate GnRH immunoreactive neurons (n = 4). E) Average number of GnRH neuron numbers per hypothalamic section (means ± SEM) in WT Vit D₃+ and Cyp27b1 null Vit D₃− mice. Hypothalamic sections reviewed corresponded to plates 25–32 of the Paxinos and Watson mouse atlas [49] and the hypothalamic region between the organum vasculosum of lamina terminalis and the medial POA (n = 4).