Ovulation-Associated Increase in Intestinal Calcium Absorption during the Rat Estrous Cycle is Blunted by Ovariectomy

BIOLOGY OF REPRODUCTION 49, 544-548 (1993) Ovulation-Associated Increase in Intestinal Calcium Absorption during the Rat Estrous Cycle Is Blunted by Ovariectomy ROBERT BROMMAGE,1 CLAUDINE BINACUA, and ANNE-LISE CARRIE Nestle Research Centre, Vers-chez-les-Blanc, 1000 Lausanne 26, Switzerland ABSTRACT INTRODUCTION Cessation of ovarian function, whether due to natural menopause or to surgical ovariectomy, leads to a loss of bone mass and is a contributing factor to osteoporosis. Alterations in Ca metabolism following menopause or ovariectomy include decreased intestinal Ca absorption [1, 2] possibly resulting from a lower responsiveness [3] to calcitriol (1,25-dihydroxyvitamin D3), increased urinary Ca excretion [4], a transient increase in bone remodeling lasting several years [5], and a lower "set-point" for bone mass [6]. Most of these changes are thought to result from low estrogen levels, but a lack of progesterone may also be a contributing factor [7]. Since normal ovarian function produces cyclical changes in the circulating levels of the sex hormones, the beneficial effects of ovarian activity on bone mass and Ca metabolism might vary during the menstrual cycle. These effects could be mediated by estrogen and progesterone directly or through alterations in serum levels of parathyroid hormone (PTH), calcitonin, and calcitriol, the three major Ca-regulating hormones. Evidence supporting cyclical changes in Ca metabolism during the menstrual cycle includes observations of variations in the serum levels of ionized Ca [8], PTH [9], calcitriol [10-12], and osteocalcin [13], a presumed marker of osteoblastic, and hence bone-forming, activity. In each instance, serum concentrations were highest around the time of ovulation. However, these changes have not been consistently observed, as other investigators have found no influence of the menstrual cycle on serum levels of total Ca [3, 14,15], PTH [8, 13, 14], calcitriol [13-16], or osteocalcin [17]. Possible variations in intestinal Ca absorption, total urinary Ca excretion, and bone resorption during the menstrual cycle have not yet been examined. One report has indicated that the fasting urinary Ca/creatinine ratio remains unchanged during the menstrual cycle [11]. Ovariectomized rats also lose bone and have a transient increase in skeletal remodeling [18]. During the ovulatory phase of the estrous cycle in young, rapidly growing rats, the longitudinal growth rate of the proximal tibia is at its lowest [19], but bone creatine kinase activity is highest [20]. Serum levels of PTH have been reported to remain unchanged during the rat estrous cycle [21, 22], but serum calcitonin levels are lowest near ovulation [21, 22]. We have recently shown that intestinal Ca absorption is highest around the time of ovulation in normally cycling rats at 12, 32, and 50 wk of age [23]. The purpose of the present study was to further characterize the variations in intestinal Ca absorption during the rat estrous cycle by examining rats fed diets containing both low and high contents of Ca. Furthermore, normally cycling rats were compared to ovariectomized rats to determine the influence of cessation of ovarian activity on intestinal Ca absorption. MATERIALS AND METHODS Female rats of the Fischer 344 strain were purchased from IFFA-CREDO (Les Oncins, France) at 4 wk of age and fed a modified AIN-76A pelleted diet formulated to contain 0.5% Ca (as CaCO 3), 0.5% P, and 1 u/g vitamin D3. At 12 wk of age, intestinal Ca absorption was measured during the estrous cycle; these data have been published previously [23]. The dietary Ca content was reduced to 0.2% at 17 wk of age. Ovariectomy was performed on several rats at 29 wk of age, and at the same time the dietary Ca content was increased to 1.4% for all of the rats. Intestinal Ca absorption was then measured at 32 wk of age, after which the dietary Ca content was reduced to 0.11% and intestinal Ca absorption was determined again at 36 wk of age. The rats continued to receive the 0.11% Ca diet and were killed over Accepted April 19, 1993. Received January 24, 1992. 'Correspondence and current address: Celtrix Pharmaceuticals, 3055 Patrick Henry Drive, Santa Clara, CA 95054-1815. FAX: (408) 450-4700. 544 Intestinal calcium (Ca) absorption was measured under noninvasive conditions in both normally cycling and ovariectomized 47 47 rats by determining the decrease in Ca/ Sc ratio between diet and feces. In 32-wk-old rats fed a 1.4% Ca diet, both fractional and total intestinal Ca absorption varied during the estrous cycle (p < 0.03), being highest during estrus and lowest during the second day of diestrus. Similarly, in 36-wk-old rats fed a 0.11% Ca diet, both fractional and total intestinal Ca absorption varied during the estrous cycle (p < 0.001), being highest during estrus and lowest during the first day of diestrus. In both studies, Ca absorption in ovariectomized rats was identical to the mean values for all of the cycling rats. Serum zinc (Zn) levels, but not those of Ca, phosphorus (P), and magnesium (Mg), varied during the estrous cycle when measured in 37-wk-old rats fed the 0.11% Ca diet. INTESTINAL CA ABSORPTION DURING ESTROUS CYCLE AND O O X FIG. 1. Body weights of rats throughout the experiments. The hatched bars indicate dietary Ca contents during the different periods of the experiment. OOX indicates the time of ovariectomy and the symbols #1 and #2 indicate the weeks when Ca absorption was measured. 6 days during Week 37 in order to obtain blood from the abdominal aorta under ethrane anesthesia for measurements of serum electrolytes. On the day animals were killed, food was removed by 1000 h and the blood was obtained between 1600 h and 1715 h. The stages of the estrous cycle were determined from vaginal smears taken each morning during Weeks 29 through 37 (except for a 4-day period starting 5 days after Ca absorption was measured during Week 32). The smears of the ovariectomized rats consistently showed the cell morphology typical of diestrus. For measurements of Ca absorption (which were made during the night), the stage of the cycle was defined from the smear obtained the following morning. For the serum electrolyte data (obtained in the afternoon), the stage of the cycle was defined from the smear obtained the same day. Intestinal Ca absorption was determined from the decrease in the ratio of 4 7 ~toa 4 7 in~ feces ~ relative to the diet by the method of McCredie et al. [24], modified to permit inclusion of the isotopes in the diet instead of administration by stomach gavage. The gamma-emitting 4 7 ~(t112 a = 4.53 days and energies of 490,810, and 1290 kev) decays ~ ~= 3.43 days and energy of to the gamma-emitting 4 7 (tl/g 160 kev), and the two isotopes were counted simultaneously by appropriate discrimination of their gamma energies. We have confirmed (unpublished data) previous observations [24] that Sc is not absorbed by the intestine. Thus, with the use of 4 7 as (...truncated)