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Endocrinology, doi:10.1210/en.2003-0892
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Endocrinology Vol. 144, No. 12 5521-5529
Copyright © 2003 by The Endocrine Society

Low Estrogen and High Parathyroid Hormone-Related Peptide Levels Contribute to Accelerated Bone Resorption and Bone Loss in Lactating Mice

Joshua N. VanHouten and John J. Wysolmerski

Section of Endocrinology and Metabolism, Department of Internal Medicine, Yale University School of Medicine, New Haven, Connecticut 06520

Address all correspondence and requests for reprints to: John J. Wysolmerski, Section of Endocrinology and Metabolism, Department of Internal Medicine, FMP 102, 333 Cedar Street, New Haven, Connecticut 06520-8020. E-mail: john.wysolmerski{at}yale.edu.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Providing enough calcium for milk production stresses calcium homeostasis in lactating mammals. A universal response to these demands for calcium appears to be the mobilization of maternal skeletal reserves, and bone loss during lactation has been well documented. However, the regulation of calcium and skeletal metabolism during lactation remains enigmatic. Our study was designed to examine mineral and bone metabolism in lactating mice. We found that mice lose bone rapidly at all sites during lactation. Bone mineral density as determined by dual-energy x-ray absorptiometry was 20 to 30% lower at the spine, femur, and total body in lactating compared with either age-matched virgin or pregnant mice. The decrease in bone mineral density was accompanied by dramatic reductions in bone volume and changes in trabecular architecture. Bone loss was also accompanied by increases in bone turnover as determined by biochemical markers and histomorphometry. PTHrP levels were elevated during lactation and correlated positively with markers of bone resorption and negatively with bone mass at all sites. Estrogen levels were low during lactation and correlated negatively with bone resorption markers. Finally, estrogen and pamidronate treatment lowered rates of bone resorption to baseline virgin levels and mitigated, but did not prevent, bone loss. These data suggest that the combination of estrogen deficiency and elevations in circulating PTHrP during lactation act to stimulate bone resorption and promote bone loss.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
VERTEBRATE OFFSPRING DEPEND on their mothers for calcium to support the initial growth of their skeletons. In eutherian mammals, calcium is transferred from mother to fetus across the placenta during pregnancy, and after delivery it is supplied in milk (1, 2, 3, 4, 5, 6). In humans, approximately 300–400 mg of calcium are secreted into milk on a daily basis, and the average nursing woman transfers more than 80 g to her child in this manner over a 9-month period of lactation (4, 5, 6, 7). The need to supply calcium for milk production represents a major stress on maternal calcium and skeletal homeostasis. The maternal adaptations to the calcium demands of lactation have been reviewed recently in detail (1, 2, 3, 4, 5, 6). One adaptation shared by all mammals is the mobilization of skeletal calcium stores, resulting in bone loss (1, 4, 5).

Lactating women typically lose 6–10% of their bone mineral content at trabecular sites over 6 months of lactation, a rate of loss far exceeding that seen after menopause (4, 5, 6, 8, 9, 10, 11, 12, 13, 14, 15, 16). Rats, which typically nurse many more offspring than women, lose up to 35% of their bone mineral content over 21 d of lactation (5, 8, 17, 18, 19, 20, 21, 22, 23, 24). Although trabecular bone is most severely affected, bone loss also occurs in cortical bone. Not surprisingly, given the decline in bone mass, lactation is also associated with deterioration in the biomechanical strength of bone (8, 25). Perhaps the most remarkable aspect of the bone loss associated with lactation is its rapid and complete reversibility after weaning (4, 5, 6). Histomorphometric studies in rats have demonstrated an almost complete restoration of bone mass within 4 wk after the cessation of lactation, and measurements in women post lactation suggest that bone mineral density (BMD) is restored to baseline values within 6–12 months (1, 4, 5, 14, 16, 24, 25, 26, 27, 28). Furthermore, epidemiological data suggest that, in women, neither the duration nor the number of offspring nursed represents a risk for lower bone density or a higher risk of osteoporotic fracture later in life (4, 5, 10). Thus, lactation and weaning represent a cycle of reversible bone loss encompassing the most rapid periods of skeletal catabolism and anabolism known during adult life.

The mechanisms of bone loss during lactation are incompletely understood. Previous studies have indicated that lactation is a period of high bone turnover, so it has been suggested that bone loss is mediated by osteoclastic bone resorption (22, 25, 29, 30, 31, 32, 33). However, it is not clear what drives the elevated rate of bone resorption. PTH levels have been reported to rise during lactation in rats, but PTH levels fall in lactating humans. Furthermore, experiments in rats have demonstrated that bone loss occurs during lactation in the absence of either PTH or vitamin D (4, 5, 34, 35), and hypoparathyroid patients can lactate successfully (36). PTHrP levels have been reported to be elevated during lactation, and there is some data to suggest that PTHrP levels correlate with bone loss in nursing mothers (5, 11, 37, 38, 39). However, not all studies have supported a role for PTHrP in regulating calcium or bone metabolism during lactation (40). Finally, lactation in humans is a period of hyperprolactinemia and hypoestrogenemia. Both hormones have been shown to be active in bone (41, 42), and, given the parallels between postmenopausal bone loss and lactation-related bone loss, it has been widely assumed that estrogen deficiency plays an important role in triggering the bone resorption seen during lactation (4, 5, 6). However, the potential roles that either estrogen or prolactin may play in this process have not been formally examined (4).

In the past several years, the study of genetically altered mice has led to much new knowledge regarding the biology of bone (43). Similar animal models might also further our understanding of bone loss during lactation. However, to date, there have been no studies examining bone and/or mineral metabolism in lactating mice. Our aims in this study were to provide baseline data on calcium and skeletal metabolism during lactation in mice and to examine directly the role of estrogen deficiency and bone resorption in regulating bone loss in lactating mice. Our data demonstrate that mice, like other mammals that have been studied, lose bone rapidly during lactation. This is associated with increases in circulating levels of PTHrP and decreases in circulating estrogen levels. Bone turnover is increased during lactation, and suppressing bone resorption rates with either estrogen or bisphosphonate treatment mitigates, but does not prevent, bone loss during lactation.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Animals
Age-matched CD1 female mice were purchased from Charles River Laboratories (Wilmington, MA). All animals were 12–15 wk old at the time of analysis. Baseline measurements were performed on cohorts of virgin mice, pregnant mice at 18 d gestation, and lactating mice on 2, 5, 12, and 18 d postpartum. We also analyzed mice 7 d after forced weaning following 12 d of lactation. For all lactating mice, the litter size ranged from 12–16 pups.

Blood collection
Mice were anesthetized with methoxyflurane (Medical Developments Australia, Springvale, Victoria, Australia), and blood was collected by cardiac puncture into syringes containing 28 U heparin (Becton Dickinson, Franklin Lakes, NJ) and 50 µl of a protease inhibitor cocktail (Nichols Institute Diagnostics, San Juan Capistrano, CA). Blood was centrifuged at 750 x g for 10 min at 4 C, and plasma was separated, aliquoted, and stored at -70 C. Milk and urine were collected immediately before terminal bleeding.

Dual-energy x-ray absorptiometry (DEXA)
BMD measurements were done by DEXA using a Lunar PIXImus (LUNAR Corporation, Madison, WI) operated by the Yale Core Center for Musculoskeletal Disorders (YCCMD). Mice were anesthetized with 50 mg/kg ketamine (Ketaset III, Fort Dodge Animal Health, Fort Dodge, IA) and 10 mg/kg xylazine (AnaSed, Lloyd, Shenandoah, IA) by ip injection before analysis. For untreated mice, single DEXA measurements were obtained on individuals in each cohort. Lactating mice receiving injections of vehicle, estrogen, or pamidronate had serial measurements of BMD performed at d 4 and 12 postpartum. Total skeletal BMD, excluding the head, was recorded, and region-of-interest rectangles were drawn around the vertebrae and total femur to calculate the BMD in these subcompartments. The vertebral region of interest encompassed the entire thoracic spine and the lumbar spine to the pelvis.

Histomorphometry
Static and dynamic histomorphometric analyses of tibias were performed by the YCCMD using the Osteomeasure system (OsteoMetrics, Atlanta, GA). Bones were labeled by injecting mice ip with 1 mg mouse calcein (Macalister Bicknell, New Haven, CT) at 4 d and 1 d before terminal bleeds.

Treatments
All injections were begun on d 1 postpartum and continued until d 12 postpartum. One group of mice was injected ip with 25 µg 17-ß-estradiol (E2) daily. E2 was made as a 5 mg/ml solution in sterile PBS from a premixed water-soluble powder with 50 mg E2 per 950 mg 2-hydroxypropyl-ß-cyclodextrin (Sigma, St. Louis, MO). To determine the effectiveness of the E2 injections, uteri were removed and weighed at the time of death. In addition, circulating E2 concentrations were determined with an enzyme immunoassay (DSL10–39100, Diagnostic Systems Laboratories, Webster, TX). Vehicle-treated controls received 100-µl ip injections of sterile saline. Another group of mice was injected sc every third day with 0.15 mg/kg pamidronate disodium (Novartis Pharmaceuticals, East Hanover, NJ) in 0.9% sterile saline, whereas the corresponding vehicle control mice were injected sc with the saline alone.

Assays
PTH was measured in plasma using a two-site immunoradiometric assay for rat PTH (Immutopics International, San Clemente, CA). Plasma calcium was measured with an atomic absorptiometer (model 2380; Perkin-Elmer, Norwalk, CT) by the Clinical Chemistry laboratory at Yale-New Haven Hospital. PTHrP levels were measured in plasma containing protease inhibitor cocktail using an immunoradiometric assay specific for amino acids 1–74 (Nichols Institute Diagnostics). Osteocalcin concentrations were measured in plasma by the YCCMD with a RIA (44). Urinary collagen C-telopeptide (CTx) levels were determined with the RatLaps enzyme immunoassay (Nordic Bioscience Diagnostics, Atlanta, GA) and corrected for creatinine concentrations, measured by the picric acid method (Sigma).

Statistical analysis
When comparing three or more groups, we used one-way ANOVA with the Student-Neuman-Keuls posttest. To compare two groups, the unpaired, two-tailed Student’s t test was used. Pearson correlations were used to analyze the relationship between PTHrP or E2 and CTx or BMD in virgin, pregnant, lactating, and weaned animals. All statistical analyses were carried out using Graph Pad Prism 4.00 for Windows (GraphPad Software, San Diego, CA).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Bone mass declines during lactation
We measured BMD by DEXA in separate cohorts of age-matched CD1 mice on d 2, 5, 12, and 18 postpartum. We also measured BMD in mice 7 d post weaning after a 12-d period of lactation. These results were compared with measurements taken in nonlactating, virgin mice and with those taken at the end of pregnancy (d 18 of gestation). Figure 1Go shows the mean and individual BMD data for these mice at the spine, femur, and total body. We found that the mean BMD tended to be lower at all three sites at the end of pregnancy compared with virgin controls, although only the differences at the spine reached statistical significance (15% decline; P < 0.01). As expected, BMD progressively declined over the course of lactation. Bone loss appeared maximal at 12 d postpartum and did not significantly change between 12 and 18 d of lactation, at which time the pups were beginning to wean and consume solid food. On average, compared with virgin controls, BMD after 12 d of lactation was 33% lower at the spine, 22% lower at the femur, and 21% lower when the total body was measured. If the end of pregnancy is used as the frame of reference, BMD was 21% lower at the spine, 20% lower at the femur, and 16% lower for total body measurements. Within 1 wk of weaning, average BMD had increased at all three sites, although the difference between d 12 of lactation and 1 wk post weaning was only statistically significant at the spine (P < 0.001).



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  		FIG. 1. BMD at the spine (A), hip (B), and total body (C) of virgin, pregnant, lactating, and weaned mice as measured by DEXA. Each point represents the measurement for an individual mouse, and the horizontal lines represent the means for the group.

 
The changes in bone density were paralleled by similar changes in bone volume on histomorphometric analysis of bone sections through the proximal tibia (Table 1Go). There were significant declines in trabecular bone volume (BV/TV), trabecular number (TbN), and trabecular thickness (TbTh) at 12 d of lactation compared with either virgin or pregnant mice. One week after weaning, there were significant increases in BV/TV and TbTh, with a nonsignificant increase in TbN. These changes in histomorphometric parameters are reflected in the von Kossa-stained bone sections shown in Fig. 2Go. As one can see, there was a dramatic loss of trabeculi and a significant thinning of the metaphyseal cortex during lactation (Fig. 2CGo), followed by a partial recovery within 1 wk after weaning (Fig. 2DGo). These changes are consistent with a rapid and reversible loss of bone tissue during lactation.


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  		TABLE 1. Histomorphometric analysis of tibias from virgin, pregnant, lactating, and weaned mice, and lactating mice treated with estrogen or treated with pamidronate

 


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  		FIG. 2. Representative von Kossa-stained sections through the proximal tibias of virgin (A), pregnant (B), lactating (C), and weaned (D) mice. Note the dramatic dropout of trabecular elements in the tibia from the lactating mouse (C) and their rapid reappearance by 1 wk post weaning (D). Also shown are tibias from lactating mice treated with either estrogen (E) or pamidronate (F). Note the preservation of trabecular elements in the estrogen- and pamidronate-treated lactating mice (E and F) compared with the untreated lactating (C) mice.

 
Bone turnover is elevated during lactation
Figure 3AGo demonstrates the results of urine collagen C-telopeptide (CTx, RatLaps) measurements in mice at the various times during pregnancy, lactation and weaning described previously. We found that this biochemical marker of bone resorption was significantly elevated during lactation compared with virgin mice (P < 0.05 vs. L2, P < 0.01 vs. L12). At 1 wk after weaning, CTx levels had decreased back to baseline. Figure 3BGo demonstrates the results for circulating levels of osteocalcin, a biochemical marker of bone formation. In contrast to the results for urine CTx, osteocalcin levels did not rise, but rather fell during lactation. By 1 wk after the cessation of lactation, osteocalcin levels had increased toward baseline.



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  		FIG. 3. Biochemical markers of bone turnover in virgin, pregnant, lactating, and weaned mice. A, Results for urine collagen C-telopeptide measurements, an index of bone resorption. B, Results for osteocalcin measurements, a marker of osteoblast function. Rates of bone resorption are elevated during pregnancy and lactation and decline after weaning. Osteocalcin levels decline during pregnancy and lactation and increase toward baseline after weaning. Letters above the bars indicate statistically significant differences, such that bars with at least one letter in common are not significantly different (e.g. virgin "a" is not significantly different from pregnant "abc"), whereas bars with no letters in common are significantly different (e.g. virgin "a" is significantly different from lactating d 5 "bc").

 
In addition to measuring biochemical markers, we also examined bone turnover via static and dynamic bone histomorphometry. As shown in Table 1Go, in concert with the increase in bone resorption markers, there were increases in the number of osteoclasts per millimeter bone perimeter (OcN/BPm) and in the osteoclast surface [OcS/bone surface (BS)] in lactating mice compared with virgin mice. The numbers of osteoclasts were similar in pregnant compared with lactating animals. In contrast, despite the decrease in osteocalcin levels, there was no corresponding decrease in osteoblast numbers (ObN/BPm) or the osteoblast surface (ObS/BS) in lactating compared with virgin mice. Both parameters increased during lactation compared with virgin mice, although these changes did not reach statistical significance. Interestingly there was a drop in both osteoblast number and surface at the end of pregnancy compared with virgin mice. Thus, the transition between pregnancy and lactation was associated with a significant increase in ObN/BPm and ObS/BS. Bone formation rates as assessed by double calcein labeling reflected the changes in osteoblast numbers, being decreased at the end of pregnancy compared with virgins and being elevated during lactation compared with both virgin and pregnant mice. One week after weaning, ObN/BPm and ObS/BS as well as bone formation rates tended to be lower, but OcN/BPm and OcS/BS were unchanged when compared with lactation. Taken together, these data suggest lactation is a time of increased bone turnover.

Calcium metabolism during lactation
As shown in Fig. 4Go, plasma levels of total calcium were slightly higher during pregnancy and lactation than they were in virgin mice. Calcium levels declined to baseline after weaning. PTH levels were more variable than plasma calcium levels at all time points, and there were no differences between lactating, pregnant, or virgin mice. Although average PTH levels appeared to be slightly higher during late lactation, these changes were not statistically significant. In contrast, circulating PTHrP levels were significantly increased in lactating compared with either virgin or pregnant mice. Although PTHrP levels hovered near the threshold of sensitivity of our assay (0.3 pM) in virgin mice, lactating mice had mean levels of PTHrP around 1 pM, a more than 3-fold elevation. With the cessation of lactation after weaning, PTHrP levels again declined back to baseline. Given these elevations in PTHrP, we performed a univariate analysis to determine whether circulating PTHrP concentrations correlated with urine CTx levels, systemic calcium concentrations, or BMD. PTHrP levels correlated positively with urine CTx (P < 0.005; r2 = 0.50) and negatively with vertebral BMD (P < 0.01; r2 = 0.37), femur BMD (P < 0.05; r2 = 0.28), and total body BMD (P < 0.05; r2 = 0.26).



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  		FIG. 4. Some parameters of calcium metabolism during lactation. A, Plasma calcium levels, which are slightly higher during pregnancy and lactation than in virgins or after weaning. B, PTH levels, which are variable but unchanged during lactation. C, PTHrP levels, which increase significantly during lactation and then decline back to baseline after weaning. Letters above the bars indicate statistically significant differences, such that bars with at least one letter in common are not significantly different, whereas bars with no letters in common are significantly different.

 
Inhibiting bone resorption with estrogen or a bisphosphonate mitigates, but does not prevent, bone loss during lactation
To investigate what role estrogen deficiency has in mediating the bone loss seen during lactation, we treated lactating mice with injections of estrogen. To determine whether the estrogen treatment was effective, we first measured circulating levels of E2 and uterine weights in treated mice and controls. As one can see in Fig. 5AGo, E2 levels were lower at d 12 of lactation than they were in virgin, nonlactating mice (12.2 ± 3.7 vs. 21.5 ± 5.7 pg/ml, respectively), confirming that midlactation represents a time of relative estrogen deficiency in mice. Furthermore, estrogen levels correlated negatively with urine CTx levels (P < 0.05; r2 = 0.38) but not with BMD. Although vehicle treatment had no effect on circulating estrogen levels, estrogen treatment resulted in a tripling of circulating E2 concentrations. Reflecting these changes in systemic E2 levels, vehicle-treated mice had an average uterine weight of 140 ± 10 mg, which was 27% lower than the average weight in virgin mice, and estrogen-treated mice had an average uterine weight of 300 ± 10 mg, which was double the uterine weight of vehicle-treated mice (Fig. 5BGo).



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  		FIG. 5. A, Circulating E2 levels in virgin, lactating, and estrogen-treated mice. Note that estrogen levels are low during lactation. Treatment with vehicle had no effect, but estrogen treatment significantly increased circulating E2 levels. B, Uterine weights in virgin, lactating, and estrogen-treated lactating mice. Note that uterine weights reflect circulating E2 levels. In particular, the increase in uterine weights in the estrogen-treated mice demonstrates that the estrogen treatment regimen was biologically active. C, Response of urine CTx measurements in lactating mice to treatment with estrogen or pamidronate. Note that both estrogen and pamidronate prevent the increase in urine CTx normally observed during lactation. D, Response of circulating osteocalcin levels in lactating mice to treatment with estrogen or pamidronate. Osteocalcin levels decline further in response to treatment with antiresorptive agents. Letters above the bars indicate statistically significant differences, such that bars with at least one letter in common are not significantly different, whereas bars with no letters in common are significantly different.

 
Estrogen had no effect on plasma calcium, PTH, or PTHrP levels (Fig 6Go), but it had a significant effect on biochemical markers of bone turnover. As shown in Fig. 5CGo, estrogen treatment prevented the rise in urine CTx normally seen at d 12 of lactation. Similar to the results shown in Fig. 3Go, osteocalcin levels were lower in vehicle-treated, lactating mice than in virgin controls, and estrogen treatment led to a slight further decline (Fig. 5DGo). Bone histomorphometry (Table 1Go) confirmed that estrogen treatment effectively prevented the rise in bone resorption usually seen during lactation; both OcN/BPm and OcS/BS in estrogen-treated lactating mice were no different than in virgins. However, estrogen treatment did not lower ObN/BPm, ObS/BS, or bone formation rates. Estrogen treatment caused a significant decrease in plasma calcium levels compared with vehicle treatment, but calcium levels in the estrogen-treated lactating mice were still significantly higher than in virgin mice (Fig. 6AGo). Despite its significant effects on bone turnover and plasma calcium, estrogen had no effect on PTH or PTHrP levels (Fig. 6Go, B and C).



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  		FIG. 6. Response of circulating calcium (A), PTH (B), and PTHrP (C) levels to antiresorptive treatment in lactating mice. Estrogen, but not pamidronate, treatment significantly reduced plasma calcium levels, compared with vehicle treatment. Treatment with estrogen or pamidronate had little effect on PTH or PTHrP levels. Letters above the bars indicate statistically significant differences, such that bars with at least one letter in common are not significantly different, whereas bars with no letters in common are significantly different.

 
Figure 7Go shows the BMD results from vehicle vs. estrogen-treated mice. As shown, treatment with estrogen attenuated bone loss at all three sites. At the spine, bone loss was reduced from 14.8 to 6.9% (Fig. 7AGo); at the femur it was reduced from 12 to 4.5% (Fig. 7BGo); and for the total body it was reduced from 10.2 to 4.9% (Fig. 7CGo). As demonstrated in Fig. 2Go and Table 1Go, histologically, estrogen treatment preserved trabecular elements and preserved bone volume and trabecular thickness compared with untreated mice. Thus, estrogen effectively prevented the increase in bone resorption normally associated with lactation and preserved bone tissue. However, although estrogen treatment reduced the amount of bone loss by approximately 50–60%, a significant decline in BMD remained (Fig. 7Go).



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  		FIG. 7. Bone loss in lactating mice treated with either estrogen or pamidronate as assessed by serial DEXA measurements at the spine (A), femur (B), and total body (C). Note that at all three sites, treatment with estrogen or pamidronate reduced the amount of bone lost by approximately 50–60%. However, note that estrogen- and pamidronate-treated mice continued to lose 5–7% of their bone mass between d 4 and 12 of lactation. Letters above the bars indicate statistically significant differences, such that bars with at least one letter in common are not significantly different, whereas bars with no letters in common are significantly different.

 
To determine whether the stimulation of bone turnover and bone loss by estrogen deficiency mobilizes calcium to supply milk production, we measured milk calcium levels in d 12 lactating mice treated with vehicle or estrogen. Like plasma calcium levels, compared with vehicle treatment, milk calcium levels were significantly reduced by treatment with estrogen (Fig. 8Go).



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  		FIG. 8. Milk calcium concentrations decreased significantly in mice treated with estrogen, whereas the milk calcium levels of vehicle-treated and pamidronate-treated mice were not significantly different. Letters above the bars indicate statistically significant differences, such that bars with at least one letter in common are not significantly different, whereas bars with no letters in common are significantly different.

 
We also treated lactating mice with another potent antiresorptive agent, the bisphosphonate pamidronate. Mice received systemic injections of pamidronate or vehicle starting on d 1 of lactation, and BMD was measured on d 4 and 12. During lactation, pamidronate treatment, as opposed to estrogen treatment, had no effect on plasma calcium (Fig. 6AGo) or milk calcium (Fig. 8Go). Similar to the results with estrogen, pamidronate had no effect on PTH levels or PTHrP levels (Fig. 6Go, B and C). However, it completely prevented the rise in urine CTx normally seen during lactation (Fig. 5AGo). Osteocalcin levels were similar to the values in estrogen-treated mice (Fig. 5BGo). Like estrogen, treatment with pamidronate led to a significant decrease in osteoclast numbers and surfaces (Table 1Go). However, in contrast to estrogen, osteoblast numbers and surfaces were also reduced by pamidronate treatment (Table 1Go). These changes were associated with a significant reduction in bone formation rates (Table 1Go). Figure 7Go illustrates the changes in BMD in pamidronate-treated mice. Similar to the results with estrogen, bisphosphonate treatment mitigated but did not prevent bone loss. The degree of bone loss was reduced from 14.3 to 7.3% at the spine (Fig. 7AGo), from 13.5 to 7.1% at the femur (Fig. 7BGo), and from 13 to 8.3% for the total body (Fig. 7CGo). Histological examination of bone sections documented that pamidronate preserved trabeculi, and significantly increased trabecular number and decreased trabecular spacing (Fig. 2Go and Table 1Go). Thus, treatment with pamidronate reduced the rate of bone turnover during lactation, which in turn, reduced, but did not eliminate bone loss.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Providing calcium for milk production represents a major challenge to maternal calcium homeostasis. In response to this stress, nursing mothers mobilize skeletal calcium stores and consequently lose significant amounts of bone. Although this phenomenon is well documented, its regulation is incompletely understood. Our goal in this study was to examine calcium and skeletal metabolism in detail to further our understanding of the nature and the regulation of bone loss during lactation. Our data demonstrate that lactating mice, like humans and rats, rapidly lose significant amounts of bone. We found that lactation is a time of increased bone turnover, with elevations in both rates of bone formation and bone resorption. Circulating estrogen levels declined during lactation, and PTHrP levels were elevated. Finally, antiresorptive treatment with either estrogen or pamidronate lessened, but did not prevent, bone loss during lactation. Our data were generated in CD1 mice, the outbred nature of which makes these findings unlikely to be strain-specific.

The degree and rapidity of bone loss in lactating mice is remarkable. We found that mice had already lost some bone by the end of pregnancy, probably in response to the transplacental transfer of calcium that serves to mineralize the fetal skeleton. Bone loss during lactation was progressive until the time at which the pups began to wean to solid food and suckling intensity declined. We noted 20–30% reductions in BMD at the spine, hip, and total body in lactating compared with virgin mice. This is comparable to the 15–35% reductions in bone mass reported for lactating rats (5, 8, 17, 18, 19, 20, 21, 22, 23, 24). Histologically, there was a loss of trabecular elements and thinning of the metaphyseal cortex in the proximal tibias of lactating compared with virgin mice. The degree of bone loss in rodents far exceeds the average 6–8% reduction in trabecular bone mass noted in women (4, 5, 6). This is most likely due to the large number of suckling offspring in rodents compared with humans, as previous studies have suggested that the degree of bone loss during lactation is associated with the duration of lactation and the number of offspring nursed (5, 6, 8, 9, 10). The speed with which this bone loss is reversed is also remarkable. Within 1 wk of forced weaning, there were significant increases in spinal bone mass, and trabecular elements reappeared in the proximal tibias. Thus, although bone is lost throughout the skeleton, the most radical changes (both catabolic and anabolic) occur in trabecular bone.

We found that lactating mice have increased bone turnover. Rates of bone resorption as assessed by urine CTx measurements were elevated at the end of pregnancy and remained so throughout lactation, until after weaning. Bone histomorphometry revealed that the numbers of osteoclasts and the osteoclastic surface were elevated at the end of pregnancy and during midlactation, compared with virgin mice. Interestingly, these histomorphometric parameters remained elevated 1 wk after weaning despite the decline in CTx levels, suggesting that after the completion of lactation, osteoclast activity was suppressed before osteoclast numbers were actively reduced. Histomorphometry also demonstrated an increase in osteoblast numbers and bone formation rates in lactating compared with virgin mice. This difference was even more striking when measurements made at the end of pregnancy were compared with those obtained during lactation. At d 18 of gestation, we found that osteoblast numbers and bone formation rates were suppressed compared with both lactating and virgin mice. Thus, bone turnover becomes uncoupled at the end of pregnancy, with an increase in bone resorption and a reduction in bone formation. During lactation, bone formation rates increased dramatically, but given the progressive loss of bone mass, bone turnover must have remained relatively uncoupled, with bone resorption continuing to outstrip bone formation. Curiously, we found discordance between the circulating levels of osteocalcin and parameters of osteoblast function on histomorphometry. Our data regarding a reduction in circulating osteocalcin levels conflict with data from human studies, which reported elevations in osteocalcin levels during lactation (32). We do not understand why osteocalcin levels declined in lactating mice, but speculate that it may signify qualitative as well as quantitative differences in osteoblast function during lactation.

PTHrP levels were clearly elevated during lactation and declined back to virgin values after weaning. Urine CTx levels correlated positively with PTHrP levels, and BMD at all sites correlated negatively with plasma PTHrP concentrations. Thus, it is likely that the elevation in PTHrP serves as an important stimulus for increased bone resorption during lactation. Although earlier reports were conflicting, most recent studies using more sensitive assays have also reported elevated circulating PTHrP levels in lactating animals and women (5, 11, 37, 38, 39). PTHrP levels during lactation have been shown to correlate positively with calcium levels and negatively with PTH levels (4, 38, 39), and one prior study in humans demonstrated that the presence of measurable PTHrP in the circulation of lactating women correlated with greater degrees of bone loss (11). Because PTH and PTHrP are equipotent at stimulating the type I PTH/PTHrP (PTH1R) receptor, it is of interest to consider the combined molar concentrations of circulating PTH and PTHrP during lactation. The addition of 1 pM PTHrP (1–74) to the approximately 2 pM intact PTH in the circulation represents a 50% increase in total circulating ligands for the PTH1R. In fact, this may be an underestimation, for recent studies have shown that only 65–70% of the PTH measured in the intact 1–84 assays is bioactive (45, 46). Thus, the percentage increase in the combined bioactive ligands for the PTH1R during lactation may be much larger. Finally, in support of the importance of circulating PTHrP to lactational bone loss, we have recently used a conditional deletion strategy to remove the PTHrP gene from the mammary gland of lactating mice. These mice have lower levels of circulating PTHrP and lose significantly less bone, clearly demonstrating that mammary gland-derived PTHrP circulates in an endocrine fashion to stimulate bone resorption during lactation (47).

Our data also support an important role for estrogen deficiency in stimulating accelerated bone resorption and bone loss during lactation. We found that circulating estrogen levels were low during midlactation and correlated negatively with bone resorption markers. Furthermore, treating lactating mice with estrogen lowered urinary CTx levels and histomorphometric parameters of bone resorption, and reduced the amount of bone lost by 50–60%. It is worth mention that pamidronate, but not estrogen, lowered osteoblast numbers, osteoblast surface, and bone formation rates. This might explain the ability of estrogen, and the inability of pamidronate, to cause significant reductions in plasma and milk calcium levels. Alternatively, estrogen treatment might directly affect calcium handling by the lactating mammary gland. Nevertheless, the capacity of estrogen treatment to preserve bone mass during lactation was most likely secondary to its antiresorptive effects, because treating lactating mice with pamidronate had very similar effects. Estrogen deficiency is well known to stimulate osteoclast differentiation and activity and to inhibit osteoclast apoptosis (42). In addition, several studies have demonstrated that estrogen deficiency can magnify the bone-resorbing effects of PTH (48, 49, 50). Thus, it is likely that elevations in PTHrP during lactation act synergistically with estrogen deficiency to trigger accelerated bone resorption.

In conclusion, our data suggest that most, but not all, of the bone loss that occurs during lactation is due to accelerated bone resorption. An important trigger for bone resorption at this time appears to be the combination of increased circulating levels of PTHrP and decreased circulating levels of estrogen. These findings have direct implications for human disease. First, the bone resorption that occurs as a consequence of estrogen withdrawal after the menopause could well represent an inappropriate reactivation of physiology designed to provide calcium for milk production, because lactation is the only physiological state of estrogen deficiency during a woman’s reproductive life (2, 4, 5, 51). Second, lactation appears to be the only time in normal physiology that PTHrP circulates to act in a truly endocrine fashion. Given the importance of lactation as a reproductive strategy in mammals, this function of PTHrP may have served as a powerful selective pressure to maintain through evolution the ability of amino-terminal PTHrP to act in a PTH-like fashion.


   Acknowledgments
 
We appreciate the excellent technical help of Pamela Dann, Nancy Troiano, Sherril Nieman, Tracy Nelson, and Kimberley Wilson. We are especially grateful to Dr. Roland Baron for his insights. We also thank Drs. Arthur Broadus and Karl Insogna for their critical reading of the manuscript.


   Footnotes
 
This work was supported by NIH Grants DK059719, CA94175, and DK55501.This work was facilitated by the Yale Core Center for Musculoskeletal Diseases (NIH AR 46032).

Abbreviations: BMD, Bone mineral density; BPm, bone perimeter millimeters; BS, bone surface; BV/TV, trabecular bone volume; CTx, collagen C-telopeptide; DEXA, dual-energy x-ray absorptiometry; E2, 17-ß-estradiol; ObN, number of osteoblasts; OcN, number of osteoclasts; ObS, osteoblast surface; OcS, osteoclast surface; PTH1R, type I PTH/PTHrP receptor; TbN, trabecular number; TbTh, trabecular thickness.

Received July 17, 2003.

Accepted for publication September 8, 2003.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Sowers M 1996 Pregnancy and lactation as risk factors for subsequent bone loss and osteoporosis. J Bone Miner Res 11:1052–1060[Medline]
  2. Wysolmerski JJ 2002 The evolutionary origins of maternal calcium and bone metabolism during lactation. J Mammary Gland Biol Neoplasia 7:267–276[CrossRef][Medline]
  3. Horst RL, Goff JP, Reinhardt TA 1997 Calcium and vitamin D metabolism. J Mammary Gland Biol Neoplasia 2:253–263[CrossRef][Medline]
  4. Kovacs CS 2001 Calcium and bone metabolism in pregnancy and lactation. J Clin Endocrinol Metab 86:2344–2348[Free Full Text]
  5. Kovacs CS, Kronenberg HM 1997 Maternal-fetal calcium and bone metabolism during pregnancy, puerperium, and lactation. Endocr Rev 18:832–872[Abstract/Free Full Text]
  6. Kalkwarf HJ 1999 Hormonal and dietary regulation of changes in bone density during lactation and after weaning in women. J Mammary Gland Biol Neoplasia 4:319–329[CrossRef][Medline]
  7. Albright F, Reifenstein E 1948 The parathyroid glands and metabolic bone disease. Baltimore: Williams and Wilkins Co.; 1–26
  8. Peng TC, Garner SC, Kusy RP, Hirsch PF 1988 Effect of number of suckling pups and dietary calcium on bone mineral content and mechanical properties of femurs of lactating rats. Bone Miner 3:293–304[Medline]
  9. Laskey MA, Prentice A, Hanratty LA, Jarjou LM, Dibba B, Beavan SR, Cole TJ 1998 Bone changes after 3 mo of lactation: influence of calcium intake, breast-milk output, and vitamin D-receptor genotype. Am J Clin Nutr 67:685–692[Abstract]
  10. Krebs NF, Reidinger CJ, Robertson AD, Brenner M 1997 Bone mineral density changes during lactation: maternal, dietary, and biochemical correlates. Am J Clin Nutr 65:1738–1746[Abstract/Free Full Text]
  11. Sowers MF, Hollis BW, Shapiro B, Randolph J, Janney CA, Zhang D, Schork A, Crutchfield M, Stanczyk F, Russell-Aulet M 1996 Elevated parathyroid hormone-related peptide associated with lactation and bone density loss. JAMA 276:549–554[Abstract/Free Full Text]
  12. Matsumoto I, Kosha S, Noguchi S, Kojima N, Oki T, Douchi T, Nagata Y 1995 Changes of bone mineral density in pregnant and postpartum women. J Obstet Gynaecol 21:419–425
  13. Hayslip CC, Klein TA, Wray HL, Duncan WE 1989 The effects of lactation on bone-mineral content in healthy postpartum women. Obstet Gynecol 73:588–592[Medline]
  14. Laskey MA, Prentice A 1999 Bone mineral changes during and after lactation. Obstet Gynecol 94:608–615[CrossRef][Medline]
  15. More C, Bettembuk P, Bhattoa HP, Balogh A 2001 The effects of pregnancy and lactation on bone mineral density. Osteoporos Int 12:732–737[CrossRef][Medline]
  16. Karlsson C, Obrant KJ, Karlsson M 2001 Pregnancy and lactation confer reversible bone loss in humans. Osteoporos Int 12:828–834[CrossRef][Medline]
  17. Rasmussen P 1977 Calcium deficiency, pregnancy, and lactation in rats. Some effects on blood chemistry and the skeleton. Calcif Tissue Res 23:87–94[CrossRef][Medline]
  18. Miller SC, Halloran BP, DeLuca HF, Jee WS 1982 Role of vitamin D in maternal skeletal changes during pregnancy and lactation: a histomorphometric study. Calcif Tissue Int 34:245–252[CrossRef][Medline]
  19. Boass A, Toverud SU, Pike JW, Haussler MR 1981 Calcium metabolism during lactation: enhanced intestinal calcium absorption in vitamin D-deprived, hypocalcemic rats. Endocrinology 109:900–907[Abstract/Free Full Text]
  20. Hagaman JR, Ambrose WW, Hirsch PF 1990 A scanning electron microscopic and photon absorptiometric study of the development, prolongation, and pattern of recovery from lactation-induced osteopenia in rats. J Bone Miner Res 5:123–131[Medline]
  21. Brommage R, Deluca HF 1985 Regulation of bone-mineral loss during lactation. Am J Physiol 248:E182–E187
  22. Honda A, Kurabayashi T, Yahata T, Tomita M, Matsushita H, Takakuwa K, Tanaka K 2000 Effects of pregnancy and lactation on trabecular bone and marrow adipocytes in rats. Calcif Tissue Int 67:367–372[CrossRef][Medline]
  23. Zeni SN, Di Gregorio S, Mautalen C 1999 Bone mass changes during pregnancy and lactation in the rat. Bone 25:681–685[Medline]
  24. Bowman BM, Miller SC 1999 Skeletal mass, chemistry, and growth during and after multiple reproductive cycles in the rat. Bone 25:553–559[Medline]
  25. Vajda EG, Bowman BM, Miller SC 2001 Cancellous and cortical bone mechanical properties and tissue dynamics during pregnancy, lactation, and postlactation in the rat. Biol Reprod 65:689–695[Abstract/Free Full Text]
  26. Bowman BM, Siska CC, Miller SC 2002 Greatly increased cancellous bone formation with rapid improvements in bone structure in the rat maternal skeleton after lactation. J Bone Miner Res 17:1954–1960[CrossRef][Medline]
  27. Polatti F, Capuzzo E, Viazzo F, Colleoni R, Klersy C 1999 Bone mineral changes during and after lactation. Obstet Gynecol 94:52–56[CrossRef][Medline]
  28. Hopkinson JM, Butte NF, Ellis K, Smith EO 2000 Lactation delays postpartum bone mineral accretion and temporarily alters its regional distribution in women. J Nutr 130:777–783[Abstract/Free Full Text]
  29. Miller SC, Shupe JG, Redd EH, Miller MA, Omura TH 1986 Changes in bone-mineral and bone-formation rates during pregnancy and lactation in rats. Bone 7:283–287[Medline]
  30. Miller SC, Bowman BM 1998 Comparison of bone loss during normal lactation with estrogen deficiency osteopenia and immobilization osteopenia in the rat. Anat Rec 251:265–274[CrossRef][Medline]
  31. Sowers M, Eyre D, Hollis BW, Randolph JF, Shapiro B, Jannausch ML, Crutchfield M 1995 Biochemical markers of bone turnover in lactating and nonlactating postpartum women. J Clin Endocrinol Metab 80:2210–2216[Abstract]
  32. Cole DEC, Gundberg CM, Stirk LJ, Atkinson SA, Hanley DA, Ayer LM, Baldwin LS 1987 Changing osteocalcin concentrations during pregnancy and lactation: implications for maternal mineral metabolism. J Clin Endocrinol Metab 65:290–294[Abstract/Free Full Text]
  33. Uemura H, Yasui T, Kiyokawa M, Kuwahara A, Ikawa H, Matsuzaki T, Maegawa M, Furumoto H, Irahara M 2002 Serum osteoprotegerin/osteoclastogenesis-inhibitory factor during pregnancy and lactation and the relationship with calcium-regulating hormones and bone turnover markers. J Endocrinol 174:353–359[Abstract]
  34. Garner SC, Boass A, Toverud SU 1990 Parathyroid-hormone is not required for normal milk-composition or secretion or lactation-associated bone loss in normocalcemic rats. J Bone Miner Res 5:69–75[Medline]
  35. Halloran BP, DeLuca HF 1980 Skeletal changes during pregnancy and lactation: the role of vitamin D. Endocrinology 107:1923–1929[Abstract/Free Full Text]
  36. Anai T, Tomiyasu T, Takai N, Miyakawa I 1999 Remission of idiopathic hypoparathyroidism during lactation: a case report. J Obstet Gynaecol Res 25:271–273[Medline]
  37. Grill V, Hillary J, Ho PM, Law FM, MacIsaac RJ, MacIsaac IA, Moseley JM, Martin TJ 1992 Parathyroid hormone-related protein: a possible endocrine function in lactation. Clin Endocrinol (Oxf) 37:405–410[Medline]
  38. Dobnig H, Kainer F, Stepan V, Winter R, Lipp R, Schaffer M, Kahr A, Nocnik S, Patterer G, Leb G 1995 Elevated parathyroid hormone-related peptide levels after human gestation: relationship to changes in bone and mineral metabolism. J Clin Endocrinol Metab 80:3699–3707[Abstract]
  39. Lippuner K, Zehnder HJ, Casez JP, Takkinen R, Jaeger P 1996 PTH-related protein is released into the mother’s bloodstream during lactation: evidence for beneficial effects on maternal calcium-phosphate metabolism. J Bone Miner Res 11:1394–1399[Medline]
  40. Melton ME, D’Anza JJ, Wimbiscus SA, Grill V, Martin TJ, Kukreja SC 1990 Parathyroid hormone-related protein and calcium homeostasis in lactating mice. Am J Physiol 259:E792–E796
  41. Clement-Lacroix P, Ormandy C, Lepescheux L, Ammann P, Damotte D, Goffin V, Bouchard B, Amling M, Gaillard-Kelly M, Binart N, Baron R, Kelly PA 1999 Osteoblasts are a new target for prolactin: analysis of bone formation in prolactin receptor knockout mice. Endocrinology 140:96–105[Abstract/Free Full Text]
  42. Manolagas SC 2000 Birth and death of bone cells: basic regulatory mechanisms and implications for the pathogenesis and treatment of osteoporosis. Endocr Rev 21:115–137[Abstract/Free Full Text]
  43. Karsenty G, Wagner EF 2002 Reaching a genetic and molecular understanding of skeletal development. Dev Cell 2:389–406[CrossRef][Medline]
  44. Gundberg CM, Clough ME, Carpenter TO 1992 Development and validation of a radioimmunoassay for mouse osteocalcin: paradoxical response in the Hyp mouse. Endocrinology 130:1909–1915[Abstract/Free Full Text]
  45. John MR, Goodman WG, Gao P, Cantor TL, Salusky IB, Juppner H 1999 A novel immunoradiometric assay detects full-length human PTH but not amino-terminally truncated fragments: implications for PTH measurements in renal failure. J Clin Endocrinol Metab 84:4287–4290[Abstract/Free Full Text]
  46. Gao P, Scheibel S, D’Amour P, John MR, Rao SD, Schmidt-Gayk H, Cantor TL 2001 Development of a novel immunoradiometric assay exclusively for biologically active whole parathyroid hormone 1–84: implications for improvement of accurate assessment of parathyroid function. J Bone Miner Res 16:6005–6014
  47. VanHouten J, Dann P, Stewart A, Watson CJ, Pollak M, Karaplis AC, Wysolmerski JJ, Cre-mediated deletion of PTHrP from the mammary gland reduces bone turnover and preserves bone mass during lactation. J Clin Invest, in press
  48. Masiukiewicz U, Grey A, Mitnick MA, Insogna K 2000 Estrogen modulates parathyroid hormone-induced interleukin-6 production in vivo and in vitro. Endocrinology 141:2526–2531[Abstract/Free Full Text]
  49. Masiukiewicz US, Mitnik M, Gulanski BI, Insogna KL 2002 Evidence that the IL6/IL-6 soluble receptor cytokine system plays a role in the increased skeletal sensitivity to PTH in estrogen-deficient women. J Clin Endocrinol Metab 87:2892–2898[Abstract/Free Full Text]
  50. Cosman F, Shen V, Fang X, Sebel M, Ratcliffe A, Lindsay R 1993 Estrogen protection against bone resorbing effects of parathyroid hormone infusion: assessment by use of biochemical markers. Ann Intern Med 118:337–343[Abstract/Free Full Text]
  51. Stini WA 1998 Calcium homeostasis and human evolution. Coll Antropol 22:411–425[Medline]



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