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Endocrinology, doi:10.1210/en.2008-0654
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Endocrinology Vol. 150, No. 2 561-569
Copyright © 2009 by The Endocrine Society

Parathyroid Hormone Contributes to Regulating Milk Calcium Content and Modulates Neonatal Bone Formation Cooperatively with Calcium

Guofan Cao, Zhen Gu, Yongxin Ren, Lei Shu, Chunxiang Tao, Andrew Karaplis, David Goltzman and Dengshun Miao

Laboratory of Reproductive Medicine and The Research Center for Bone and Stem Cells (G.C., Z.G., Y.R., L.S., C.T., D.M.), Department of Anatomy, Histology and Embryology, Nanjing Medical University, Nanjing, Jiangsu 210029, The People’s Republic of China; and Department of Medicine (A.K., D.G.), McGill University, Montreal, Quebec, Canada H3A 1A1

Address all correspondence and requests for reprints to: Dr. Dengshun Miao, Laboratory of Reproductive Medicine, The Research Center for Bone and Stem Cells, Department of Anatomy, Histology and Embryology, Nanjing Medical University, Nanjing, Jiangsu 210029, The People’s Republic of China. E-mail: dsmiao{at}njmu.edu.cn.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
To determine whether PTH and calcium (Ca) interact in neonatal bone formation, female lactating mice either heterozygous (PTH+/–) or homozygous (PTH–/–) for targeted deletion of the pth gene were fed either a normal (1% Ca, 0.6% phosphate) or high-Ca diet (2% Ca and 0.4% phosphate). Dietary effects on milk Ca content and Ca-regulating hormones were determined in dams, and the effects of milk content were assessed on bone turnover in 3-wk-old pups. On the normal diet, milk Ca and 1,25-dihydroxyvitamin D3 levels were lower, but milk PTH-related protein levels were higher in the PTH–/– dams compared with the PTH+/– dams. On the high-Ca diet, milk Ca levels were higher, but milk 1,25-dihydroxyvitamin D3 and PTH-related protein levels were lower in both PTH+/– and PTH–/– dams. In pups fed by PTH–/– dams compared with pups fed by PTH+/– dams on normal diets, bone mineral density, trabecular bone volume relative to tissue volume, and the number of osteoblasts were reduced in both PTH+/– (32.5 ± 1.2 vs. 39.6 ± 1.5 mg/cm2, P < 0.05; 23.3 ± 1.6 vs. 29.2 ± 2.8%, P < 0.01; and 94.2 ± 8.2 vs. 123.5 ± 3.5/mm2, P < 0.01, respectively) and PTH–/– (20.4 ± 0.9 vs. 27.0 ± 1.2 mg/mm2, P < 0.05; 16.8 ± 1.9 vs. 19.3 ± 2.1%, P < 0.05; and 48.6 ± 7.9 vs. 90.5 ± 8.6/mm2, P < 0.01, respectively) pups but were lower in the PTH–/– pups compared with the PTH+/– pups. In contrast, in pups fed by either PTH+/– or PTH–/– dams on the high-Ca diet, bone mineral density, bone volume/tissue volume, and osteoblast numbers were significantly higher, in both PTH+/– (50.5 ± 1.7 vs. 58.7 ± 2.0 mg/mm2, P < 0.05; 37.9 ± 5.2 vs. 46.1 ± 5.1, P < 0.05; and 120.5 ± 9.2 vs. 159.3 ± 14.7/mm2, P < 0.01, respectively) and PTH–/– (33.0 ± 1.2 vs. 47.5 ± 2.2 mg/mm2, P < 0.001; 23.8 ± 3.1 vs. 35.9 ± 2.0, P < 0.05; and 78.7 ± 10.1 vs. 99.8 ± 13.6/mm2, P < 0.05, respectively), and were highest in the PTH+/– pups fed by the PTH+/– dams on the high-Ca diet. These results indicate that PTH can modulate Ca content of milk, and that PTH and Ca can each exert cooperative roles on osteoblastic bone formation in the neonate.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
PTH is presently the only anabolic agent available in the clinical setting. PTH normally regulates serum calcium (Ca) levels by binding, and activating the type 1 PTH receptor via its amino-terminal domain (1), and, thus, enhancing renal Ca reabsorption and increasing bone resorption. However, by careful selection of the dose and pattern of administration, PTH can stimulate bone formation in adult and aged animals of either sex, and in animals with osteopenia induced by disuse, denervation, and immobilization (2). The anabolic action of PTH on bone has been validated in humans (3, 4, 5) and its antifracture efficacy established in postmenopausal osteoporotic women (6). However, at present the physiological basis of PTH anabolism remains unclear (7, 8).

To investigate the role of PTH in vivo, we generated PTH-deficient mice. Although these mice were viable, newborn and 2-wk-old mice had decreased osteoblast numbers and diminished trabecular bone volume (BV) (9, 10). The apoptosis levels in osteoblasts and osteocytes in these mice were increased, supporting the notion that PTH protects osteoblasts from apoptosis (9). These data demonstrate that endogenous PTH is essential for bone formation. However, adult PTH gene knockout mice, when maintained on a normal Ca intake, displayed increased rather than decreased trabecular and cortical BV. They also develop hypocalcemia, hyperphosphatemia, and low-circulating 1,25-dihydroxyvitamin D3 [1,25(OH)2D3] levels consistent with primary hypoparathyroidism (11). When mutant mice were placed on a low Ca diet, renal 1{alpha}-hydroxylase expression increased despite the absence of PTH, leading to an increase in circulating 1,25(OH)2D3 levels, marked osteoclastogenesis, and profound bone resorption. These studies demonstrate the dependence of the skeletal phenotype in animals with genetically depleted PTH on the external environment, as well as on internal hormonal and ionic circulating factors (12).

Inadequate dietary Ca intake can impair bone development in childhood and adolescence, and accelerates bone loss and may contribute to osteoporosis in older adults (13). Ca consumption is essential for bone development and maintenance throughout life (14, 15). Ca intake is especially crucial during pregnancy and lactation because of the potential adverse effect on maternal bone health if maternal Ca stores are depleted (16). There is often a transient lowered bone mineral density (BMD) and increased rate of bone resorption, with the greatest consequence during the third trimester and throughout lactation. Some studies indicate that Ca consumption should be encouraged, especially during pregnancy and lactation, to replace maternal skeletal Ca stores that are depleted during these periods, although it has also been reported that Ca supplementation does not prevent bone loss in the mother during lactation and only slightly enhances the gain in bone density after weaning (17). Because the fetus in utero and the neonate through breast-feeding are dependent on maternal sources for the total Ca load, adequate maternal Ca intake also can affect fetal bone health positively. Proper Ca consumption can ensure maternal and fetal bone health, without the danger of adverse effects on the neonate. A very large body of evidence demonstrates that sufficient Ca intake augments bone gain during growth, retards age-related bone loss, and reduces osteoporotic fracture risk (18, 19, 20, 21), but it is unknown what the mechanisms of the Ca intake are that result in a positive bone mass balance. It has not been demonstrated whether the combination of PTH and Ca supplementation is beneficial for the treatment of osteoporosis.

In the present study, we examined whether the skeletal anabolic action of PTH in neonates results only from its direct action via the PTH receptor to increase the osteoblast pool, but also by an indirect action mediated through increasing extracellular Ca concentrations. To test this hypothesis, male PTH–/– and female PTH+/– or male PTH+/– and female PTH–/– mice were mated to generate PTH+/– and PTH–/– pups. PTH+/– and PTH–/– dams were fed either a diet containing normal Ca and phosphorus or a "rescue" diet containing high Ca and moderately low phosphorus. The effects of the genetic PTH deficiency and of the dietary manipulations were then determined on the content of minerals and Ca-regulating hormones in the milk of lactating dams, and on bone turnover in 3-wk-old pups.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Animals and in vivo experiments
The derivation of the parental strain of PTH–/– mice by homologous recombination in embryonic stem cells and genotyping of mice were previously described by Miao et al. (9, 12). To determine whether PTH and Ca interact in neonatal bone formation, male PTH–/– and female PTH+/– or male PTH+/– and female PTH–/– mice were mated to generate PTH+/– and PTH–/– pups. PTH+/– and PTH–/– dams were fed purified rodent diets from Harlan Teklad (Madison, WI) that were identical in content except that they contained either a normal Ca and phosphorus level (1% Ca, 0.6% phosphate) or a high Ca and moderate phosphate level (2% Ca and 0.4% phosphate). Dietary phosphorus content in the high-Ca diet was moderately reduced to help reduce phosphate absorption, decrease hyperphosphatemia, and facilitate increasing the blood Ca in the PTH–/– mice. The high-Ca, moderate phosphate diet was found to normalize hypocalcemia and hyperphosphatemia in PTH–/– mice and is referred to as the high-Ca diet. Each diet contained 17.5% total protein content. The energy density of each diet was 3.6 kcal/g. Dams consumed similar amounts of each diet, and the body weights of dams were not significantly different in both genotypes on both diets before pregnancy (Fig. 1AGo). Litter size was equalized to five to six pups per dam to equalize suckling intensity. On d 14 postpartum, dams were injected ip with 0.1 mIU oxytocin (Sigma-Aldrich Corp., St. Louis, MO) and manually milked as previously described (22). Blood was collected from 3-wk-old PTH+/– and PTH–/– pups, and serum was isolated for biochemical analysis. Tibiae were then removed after analysis. All animal experiments were performed in compliance with and approval by the Institutional Animal Care and Use Committee.


Figure 1
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  		FIG. 1. Effects of PTH deficiency and high -dietary Ca on body weight, milk Ca contents, 1,25(OH)2D3, and PTHrP levels in dams and on serum Ca, phosphorus, PTH, and 1,25(OH)2D3 in pups. A, Body weights of PTH+/– (m-PTH+/–) and PTH–/– (m-PTH–/–) dams before pregnancy on the normal or high-Ca diet. Milk Ca (B), 1,25(OH)2D3 (C), PTHrP (D), protein levels (E), and serum PTH levels (F) were determined in the samples from PTH+/– (m-PTH+/–) or PTH–/– (m-PTH–/–) dams on d 14 lactation on the normal or high-Ca diet as described in Materials and Methods. Serum PTH (G), Ca (H), phosphorus (I), and 1,25(OH)2D3 (J) levels were determined in samples from 3-wk-old PTH+/– (p-PTH+/–) and PTH–/– (p-PTH+/–) pups fed by PTH+/– (m-PTH+/–) or PTH–/– (m-PTH–/–) dams on the normal or high-Ca diet. Each value is the mean ± SEM of determinations in five mice of each group. *, P < 0.05; P < 0.01, and ***, P < 0.001 compared with PTH+/– littermates; {Delta}, P < 0.05, {Delta}{Delta}, P < 0.01, and {Delta}{Delta}{Delta}, P < 0.001 compared with pups fed by dams on the normal diet.

 
Biochemistry and hormone analyses
Milk Ca (milk was diluted 1:100 in distilled water) was measured with an atomic absorptiometer (23). Serum Ca and phosphorus were determined by an autoanalyzer (Beckman Synchron 67; Beckman Coulter, Inc., Fullerton, CA). Milk and serum 1,25(OH)2D3 was measured by RIA (ImmunoDiagnostic Systems, Bolden, UK), and PTH-related protein (PTHrP) was measured by a two-site immunoradiometric assay (Immutopics, San Clemente, CA). Serum intact PTH was measured using an ELISA (Immutopics).

Radiography and measurement of BMD
For radiography, tibiae were removed and dissected free of soft tissue, and x-ray images were taken with a Faxitron (model 805; Faxitron X-Ray Corp., Wheeling, IL), under constant conditions (22 kV, 4 min exposure), using Kodak X-Omat TL film (Eastman Kodak Co., Rochester, NY). For measurement of tibial BMD, a PIXImus densitometer (Lunar PIXImus Corp., Madison, WI) was used (5-min image acquisition with the precision of 1% coefficient of variation for skeletal BMD). The PIXImus software automatically calculated the BMD and recorded the data in Microsoft Excel files (Microsoft Corp., Redmond, WA).

Microcomputed tomography (micro-CT)
Tibiae obtained from 3-wk-old mice were dissected free of soft tissue, fixed overnight in 70% ethanol, and analyzed by micro-CT with a SkyScan 1072 scanner and associated analysis software (SkyScan, Antwerp, Belgium) as described (24). Briefly, image acquisition was performed at 100 kV and 98 µA with a 0.9° rotation between frames. During scanning, the samples were enclosed in tightly fitting plastic wrap to prevent movement and dehydration. Thresholding was applied to the images to segment the bone from the background. Two-dimensional images were used to generate three-dimensional (3D) renderings using the 3D Creator software supplied with the instrument (SkyScan). The resolution of the micro-CT images is 18.2 µm.

Histology
Tibiae were removed and fixed in 2% paraformaldehyde containing 0.075 M lysine and 0.01 M sodium periodate overnight at 4 C, and processed histologically as described (25). Proximal ends of tibiae were decalcified in EDTA glycerol solution for 5–7 d at 4 C. Decalcified right tibiae were dehydrated and embedded in paraffin after which 5-µm sections were cut on a rotary microtome. The sections were stained with hematoxylin and eosin (HE) routinely or histochemically for tartrate-resistant acid phosphatase (TRAP) activity, or immunohistochemically as described below. Alternatively, undecalcified left tibiae were embedded in LR White acrylic resin (London Resin Co. Ltd., London, UK), and 1-µm sections were cut on an ultramicrotome. These sections were stained for mineral with the von Kossa staining procedure and counterstained with toluidine blue.

Histochemical staining for TRAP
Enzyme histochemistry for TRAP was performed as previously described (26). Dewaxed sections were preincubated for 20 min in buffer containing 50 mM sodium acetate and 40 mM sodium tartrate (pH 5.0). Sections were then incubated for 15 min at room temperature in the same buffer containing 2.5 mg/ml naphthol AS-MX phosphate (Sigma-Aldrich) in dimethylformamide as substrate, and 0.5 mg/ml fast garnet GBC (Sigma-Aldrich) as a color indicator for the reaction product. After washing with distilled water, the sections were counterstained with methyl green and mounted in Kaiser’s glycerol jelly.

Immunohistochemical staining
The type I collagens were determined by immunohistochemistry as described previously (25). Affinity purified goat antihuman type I collagen antibody (Southern Biotechnology Associates, Birmingham, AL) was applied to dewaxed paraffin sections overnight at room temperature. After washing with high-salt buffer [50 mM Tris-HCl, 2.5% NaCl, and 0.05% Tween 20 (pH 7.6)] for 10 min at room temperature followed by two 10-min washes with Tris-buffered saline, the sections were incubated with secondary antibody (biotinylated rabbit antigoat IgG; Sigma-Aldrich), washed as before, and incubated with the Vectastain ABC-AP kit (Vector Laboratories, Inc., Ontario, Canada) for 45 min. After washing as before, red pigmentation to demarcate regions of immunostaining was produced by a 10- to 15-min treatment with Fast Red TR/Naphthol AS-MX phosphate (Sigma-Aldrich; containing 1 mM levamisole as endogenous alkaline phosphatase inhibitor). After washing with distilled water, the sections were counterstained with methyl green and mounted with Kaiser’s glycerol jelly.

Computer-assisted image analysis
After HE staining or histochemical or immunohistochemical staining of sections from six mice of each genotype, images of fields were photographed with a Sony digital camera (Sony Corp., Kanagawaken, Japan). Images of micrographs from single sections were digitally recorded using a rectangular template, and recordings were processed and analyzed using Northern Eclipse image analysis software (Empix Imaging Inc., Mississauga, Ontario, Canada) as previously described (25).

Statistical analysis
Data from image analysis are presented as means ± SEM. Statistical comparisons were made using a one-way ANOVA, with P < 0.05 being considered significant.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Effects of PTH deficiency and dietary Ca on milk Ca content and Ca-regulating hormones in milk
To determine whether PTH deficiency and dietary Ca alter milk Ca content and Ca-regulating hormones, milk Ca, 1,25(OH)2D3, and PTHrP were examined in the lactating PTH+/– or PTH–/– dams fed either the normal or high-Ca diet. Milk Ca and 1,25(OH)2D3 levels were reduced, but milk PTHrP levels were increased in the PTH–/– dams compared with the PTH+/– dams when they were fed the normal diet (Fig. 1Go, B–D). Milk Ca levels were increased, but milk 1,25(OH)2D3 and PTHrP levels were decreased in both PTH+/– and PTH–/– dams on the high-Ca diet compared with the genotype-matched dams on the normal diet. Milk protein concentrations were not significantly different in both genotypes on both diets (Fig. 1EGo). Serum PTH levels were decreased in PTH+/– dams on the high-Ca diet compared with PTH+/– dams on the normal diet, and were undetectable in PTH–/– dams on any diet (Fig. 1FGo).

Effects of PTH deficiency and maternal milk contents on serum Ca, phosphorus, and 1,25(OH)2D3 in suckling pups
To determine whether maternal PTH deficiency and maternal dietary Ca affected mineral homeostasis in the suckling pups, serum Ca, phosphorus, PTH, and 1,25(OH)2D3 were measured in 3-wk-old pups fed by PTH+/– or PTH–/– dams on either the normal or high-Ca diet. Serum PTH levels were normal in both PTH+/– dams and PTH+/– pups on a normal diet and undetectable in PTH–/– on any diet (Fig. 1GGo). Serum Ca levels were decreased and serum phosphorus levels were increased in the PTH–/– pups, and the Ca was lower and phosphorus was higher than in the PTH+/– pups fed by either the PTH+/– or PTH–/– dams on the normal diet (Fig. 1Go, H and I). Serum Ca and phosphorus levels were normalized in the PTH–/– pups fed by both PTH+/– and PTH–/– dams on the high-Ca diet. Serum 1,25(OH)2D3 levels were not significantly different in the PTH–/– pups compared with the PTH+/– pups whether fed by PTH+/– or by PTH–/– dams on the normal diet, however, serum PTH levels were decreased significantly in PTH+/– dams (Fig. 1FGo) and pups (Fig. 1GGo), and serum 1,25(OH)2D3 levels were decreased significantly in both PTH+/– or PTH–/– pups fed by dams on the high-Ca diet compared with genotype-matched pups fed by dams on the normal diet (Fig. 1JGo).

Effects of PTH deficiency and maternal milk contents on growth and BMD in the suckling pups
We then assessed whether PTH deficiency and the alterations of maternal milk mineral content affected skeletal growth and BMD in the suckling pups. The weights, length of tibiae, and BMD were measured in 3-wk-old pups fed by PTH+/– or PTH–/– dams on either the normal or high-Ca diet. The weights of the PTH–/– pups were significantly lower than the PTH+/– pups fed by both PTH+/– and PTH–/– dams on any diet (Fig. 2AGo). The weights were also significantly lower in both PTH+/– and PTH–/– pups fed by PTH–/– dams compared with genotype-matched pups fed by PTH+/– dams on any diet. In contrast, the weights were increased significantly in PTH+/– and PTH–/– pups fed by both PTH+/– and PTH–/– dams on the high-Ca diet compared with genotype-matched pups fed by genotype-matched dams on the normal diet (Fig. 2AGo). The length of tibiae was significantly shorter in the PTH–/– pups compared with the PTH+/– pups fed by both PTH+/– and PTH–/– dams on the normal diet. The tibial length was also shorter in both PTH+/– and PTH–/– pups fed by PTH–/– dams compared with genotype-matched pups fed by PTH+/– dams on the normal diet (Fig. 2Go, B and C). In contrast, the length of tibiae was increased significantly in PTH–/– pups fed by both PTH+/– and PTH–/– dams on the high-Ca diet compared with PTH–/– pups fed by both genotype-matched dams on the normal diet. BMD was reduced significantly in the PTH–/– pups compared with the PTH+/– pups (Fig. 2Go, B and D). BMD was also reduced in both PTH+/– and PTH–/– pups fed by PTH–/– dams compared with genotype-matched pups fed by PTH+/– dams on normal diets. In contrast, BMD was increased significantly in both PTH+/– and PTH–/– pups fed by dams on the high-Ca diet compared with genotype-matched pups by dams on the normal diet (Fig. 2Go, B and D).


Figure 2
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  		FIG. 2. Effects of PTH deficiency and maternal milk contents on skeletal growth and BMD in 3-wk-old pups. Body weights (A), x-rays of tibiae (B), the length of tibiae (C), and BMD (D) were determined in 3-wk-old PTH+/– (p-PTH+/–) and PTH–/– (p-PTH+/–) pups fed by PTH+/– (m-PTH+/–) or PTH–/– (m-PTH–/–) dams on either a normal or high-Ca diet. Each value is the mean ± SEM of determinations in five mice of each group. *, P < 0.05 and ***, P < 0.001 compared with PTH+/– littermates; #, P < 0.05 and ###, P < 0.001 compared with pups fed by PTH+/– dams; {Delta}, P < 0.05 and {Delta}{Delta}{Delta}, P < 0.001 compared with pups fed by dams on the normal diet.

 
Effects of PTH deficiency and maternal milk content on BV in the suckling pups
To determine whether PTH deficiency and altering maternal milk mineral content affected BV in the suckling pups, the trabecular BV [tissue volume (TV)] was assessed by micro-CT (Fig. 3AGo) and histomorphometric analysis (Fig. 3Go, B and C) in 3-wk-old pups fed by PTH+/– or PTH–/– dams on either the normal or high-Ca diet. BV/TV was reduced significantly in the PTH–/– pups compared with the PTH+/– pups (Fig. 3Go). BV/TV was also reduced in both PTH+/– and PTH–/– pups fed by PTH–/– dams compared with genotype-matched pups fed by PTH+/– dams on normal diets. In contrast, BV/TV was increased significantly in both PTH+/– and PTH–/– pups fed by dams on the high-Ca diet compared with genotype-matched pups by dams on the normal diet (Fig. 3Go).


Figure 3
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  		FIG. 3. Effects of PTH deficiency and maternal milk contents on BV in 3-wk-old pups. Representative longitudinal sections of the proximal end of tibiae by micro-CT scan and 3D reconstruction (A) and stained with von Kossa procedure (B) in 3-wk-old PTH+/– (p-PTH+/–) and PTH–/– (p-PTH+/–) pups fed by PTH+/– (m-PTH+/–) or PTH–/– (m-PTH–/–) dams on either a normal or high-Ca diet. C, Trabecular BV (TV) was measured as described in Materials and Methods. Each value is the mean ± SEM of determinations in five mice of each group. ***, P < 0.001 compared with PTH+/– littermates; #, P < 0.05 and ##, P < 0.01 compared with pups fed by PTH+/– dams; {Delta}{Delta}, P < 0.01 and {Delta}{Delta}{Delta}, P < 0.001 compared with pups fed by dams on the normal diet.

 
Effects of PTH deficiency and maternal milk content on osteoblastic bone formation in the suckling pups
To determine whether the alterations of BV were associated with alterations of osteoblastic bone formation in the suckling pups, the paraffin-embedded longitudinal tibial sections were stained with HE (Fig. 4AGo) and immunohistochemically for type I collagen (Fig. 4BGo). The osteoblast number was determined by histomorphometric analysis in the HE-stained sections (Fig. 4CGo), and the type I collagen positive area was measured by image analysis (Fig. 4DGo). Osteoblast number and type I collagen positive area were reduced significantly in the PTH–/– pups compared with the PTH+/– pups, and in both PTH+/– and PTH–/– pups fed by PTH–/– dams compared with pups fed by PTH+/– dams on normal diets (Fig. 4Go). In contrast, these parameters were increased significantly in both PTH+/– and PTH–/– pups fed by dams on the high-Ca diet compared with genotype-matched pups by dams on the normal diet.


Figure 4
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  		FIG. 4. Effects of PTH deficiency and maternal milk contents on bone formation in 3-wk-old pups. Representative micrographs of decalcified paraffin embedded sections stained with HE (A) and immunostained for type I collagen (Col I) (B) in 3-wk-old PTH+/– (p-PTH+/–) and PTH–/– (p-PTH+/–) pups fed by PTH+/– (m-PTH+/–) or PTH–/– (m-PTH–/–) dams on either a normal or high-Ca diet. C, Numbers of osteoblasts per mm2 were counted in the primary spongiosa of HE-stained tibiae of the mice. D, The type I collagen positive area as a percentage of the tissue area was determined in the metaphyseal regions for each group. Each value is the mean ± SEM of determinations in five mice of each group. ***, P < 0.001 compared with PTH+/– littermates; #, P < 0.05 and ##, P < 0.01 compared with pups fed by PTH+/– dams; {Delta}, P < 0.05, {Delta}{Delta}, P < 0.01, and {Delta}{Delta}{Delta}, P < 0.001 compared with pups fed by dams on the normal diet.

 
Effects of PTH deficiency and maternal milk content on bone resorption in suckling pups
To determine whether the alterations of BV were associated with alterations of osteoclastic bone resorption in the suckling pups, the paraffin-embedded longitudinal tibial sections were stained histochemically for TRAP (Fig. 5AGo), and the TRAP-positive osteoclast number and surface were determined by histomorphometric analysis (Fig. 5Go, B and C). Osteoclast number and surface were reduced significantly in the PTH–/– pups compared with the PTH+/– pups, in both PTH+/– and PTH–/– pups fed by PTH–/– dams compared with pups fed by PTH+/– dams, and in both PTH+/– and PTH–/– pups fed by dams on the high-Ca diet compared with genotype-matched pups by both PTH+/– and PTH–/– dams on the normal diet (Fig. 5Go).


Figure 5
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  		FIG. 5. Effects of PTH deficiency and maternal milk content on bone resorption in 3-wk-old pups. A, Representative micrographs of sections of the tibial metaphysis stained histochemically for TRAP activity in 3-wk-old PTH+/– (p-PTH+/–) and PTH–/– (p-PTH+/–) pups fed by PTH+/– (m-PTH+/–) or PTH–/– (m-PTH–/–) dams on either a normal or high-Ca diet. Number of TRAP-positive osteoclasts related to tissue area [N.Oc/T.Ar (#/mm2)] (B) and osteoclastic surface relative to bone surface [Oc.S/BS (%)] (C) were counted in the metaphyseal regions for each group. Each value is the mean ± SEM of determinations in five mice of each group. ***, P < 0.001 compared with PTH+/– littermates; #, P < 0.05 and ##, P < 0.01 compared with pups fed by PTH+/– dams; {Delta}{Delta}{Delta}, P < 0.001 compared with pups fed by dams on the normal diet.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
In the present study, we assessed the interaction of PTH and Ca on neonatal skeletal growth and turnover by a combination of genetic and environmental manipulations. We used a genetic approach to modulate circulating PTH levels in the neonates, and compared mice that were homozygotes for targeted deletion of the Pth gene and that have undetectable circulating PTH levels with mice that were heterozygotes and have normal circulating PTH levels. We used environmental manipulation to modulate serum Ca in the neonates. In view of the fact that suckling neonates receive their Ca intake via maternal milk, we altered the dietary content of the dams to attempt to modulate neonatal Ca intake. In view of the fact that heterozygous and homozygous neonates were derived from heterozygous and homozygous dams, we were able to observe the effect of the presence or absence of circulating PTH in the dams on the levels of Ca in maternal milk when the dietary Ca intake was altered.

Our results show that Ca in milk is altered by altering the Ca in the diet of the lactating mothers and that for each level of dietary Ca presented to the dams, milk Ca was higher in those with circulating PTH than in those without circulating PTH. In view of the fact that the dietary levels of protein were not different and the protein levels in milk were not substantially different, dietary Ca appeared to influence milk Ca independent of changes in protein. Several studies have now implicated the Ca-sensing receptor (CaR) in the transport of Ca from the systemic circulation of lactating animals to milk (22, 27, 28). The contribution of PTH to the increase in milk Ca may well have been via the stimulation of 1,25(OH)2D3 production and the resultant increase in gut absorption of dietary Ca by the dams. Indeed, we have previously reported that 1,25(OH)2D3 levels on a normal Ca intake are reduced in PTH–/– mice, and this reduction may have impeded the capacity of Ca to be absorbed by the PTH–/– mice whether on a low or high-Ca intake. Thus, the undetectable levels of 1,25(OH)2D3 in milk of the PTH–/– dams may indeed reflect the diminished renal synthesis of 1,25(OH)2D3 or may reflect reduced mammary gland synthesis of 1,25(OH)2D3. However, although mammary gland synthesis of 1,25(OH)2D3 has been reported (29), the factors modulating mammary cell 25-hydroxyvitamin D3 1{alpha}-hydroxylase have not yet been described.

Studies in humans have suggested that milk Ca levels are independent of the vitamin D status (30) and the Ca status (31) of the lactating mother, and that the major source of milk Ca is derived from bone resorption, likely mediated by increased circulating PTHrP (32, 33, 34) rather than PTH, and by reduced estrogen levels (35, 36). Systemic hypocalcemia is thought to increase mammary cell production of PTHrP in lactating females via the CaR (27), and increased circulating PTHrP is then believed to contribute to bone resorption and, thereby, help restore serum Ca and, consequently, milk Ca levels to normal.

However, species differences do appear to exist in the relative contributions of gut absorption, bone resorption, and renal reabsorption to the Ca economy of lactating mothers and to the hormones involved in this process (37). Thus, in dairy cows, lactation from the previous pregnancy persists until very late into gestation in the subsequent pregnancy, so that Ca demands are high and are required both for formation of the fetal skeleton and milk production. In early lactation, in this setting, PTH increases and stimulates renal reabsorption of Ca and 1,25(OH)2D3 production such that active intestinal Ca transport is increased. Consequently, these hormonal changes are more consistent with the role they appear to play in our model. Bone resorption is also augmented in the bovine model. In later lactation, with the continued influence of elevated 1,25(OH)2D3, intestinal hypertrophy and increased feed intake, Ca absorption increases, and skeletal Ca lost by resorption during early lactation is replenished. In the rat, as lactational demands increase with the growth of pups, circulating PTH and 1,25(OH)2D3 increase, intestinal hypertrophy occurs, and the Ca absorption rate is doubled, but still provides only 80% of the Ca requirements for milk production, the remainder being provided by bone resorption. Therefore, our results may be most closely in line with events described in the rat in which increased PTH and increased 1,25(OH)2D3 play an important role in Ca requirements for lactation. However, parathyroidectomized lactating rats can resorb bone independent of PTH (38). In our studies in mice, PTHrP concentrations were higher in the milk of the homozygotes, and correlated inversely with Ca levels, consistent with a role for PTHrP in bone resorption in the lactating dams independent of PTH.

Serum Ca in the neonates was lowest in the PTH–/– mice that received milk Ca from PTH–/– dams that had the lowest milk Ca. Serum Ca was higher in the PTH+/– neonates, even when suckling from the same PTH–/– dams with the same low milk Ca. Consequently, PTH per se appeared to contribute to elevating the serum Ca in the neonate. However, serum 1,25(OH)2D3 concentrations in the neonates were unaffected by the presence or absence of PTH and, therefore, did not appear to contribute to the increased serum Ca in the presence of PTH.

When PTH–/– neonates were exposed to the higher Ca in the milk of the PTH+/– dams on the normal Ca intake, serum Ca increased above that seen in animals exposed to the lower milk Ca. Therefore, the levels of 1,25(OH)2D3 appeared sufficient to enhance absorption of the higher load and increase the serum Ca. Alternatively, absorption of milk Ca in the neonate may have been independent of 1,25(OH)2D3 analogous to the vitamin D-independent absorption of Ca observed with diets high in Ca and lactose. However, the PTH contribution to maintaining the serum Ca was again seen in the pups of the PTH+/– dams inasmuch as the serum Ca in the PTH+/– pups was higher than in the PTH–/– pups.

Elevating the maternal Ca intake in the lactating females via the high-Ca "rescue" diet was associated with elevated milk Ca, and increased the serum Ca in the pups despite reductions in levels of circulating 1,25(OH)2D3. However, again, serum Ca was highest in the pups that had circulating PTH.

Therefore, circulating 1,25(OH)2D3 levels appeared less dependent on PTH in the neonates than has been reported in older animals but could be suppressed partly by inducing hypercalcemia in the pups. Furthermore, the much higher levels of 1,25(OH)2D3 measured in the circulation of the pups compared with the levels measured in maternal milk suggest that it is endogenously produced by the neonates and, in keeping with previous reports (39, 40), that milk is not a major source of active vitamin D for the neonate. In addition, the undetectable levels of circulating PTHrP in the neonates suggest that maternal milk is not a major contributor to PTHrP in the neonate.

We then demonstrated that BMD and trabecular BV were more dramatically reduced in the combined presence of neonatal Ca deficiency and PTH deficiency than with PTH deficiency alone. This reduction was not mediated by excess PTHrP because PTHrP levels were undetectable in the neonatal circulation, and increased bone resorption was not observed. Instead, decreased osteoblastic bone formation was evident and was more evident in the hypocalcemic than in the normocalcemic neonates lacking PTH. Consequently, hypocalcemia appeared to contribute to the reduced bone formation. However, even in hypocalcemic neonates, the presence of circulating PTH (in the PTH+/– mice) appeared to reduce the diminished bone formation. The converse was also seen, i.e. the high maternal milk Ca observed in dams on the high-Ca "rescue" diet and that resulted in higher serum Ca in the neonates, facilitated a higher bone mass. However, here again this bone mass remained lower in PTH–/– than in PTH+/– neonates, emphasizing the importance of both factors in modulating bone. If the high-Ca diet were also richer in protein or energy, some indirect influence through the IGF-I system could perhaps have been be inferred; however, the protein and energy content of both diets were the same.

Interestingly, the elevations in blood Ca were associated with increases in osteoblast activity and numbers. Consequently, both PTH deficiency and hypocalcemia appeared to interact to cause reduced bone mass. The mechanism of the Ca effect remains unclear. The presence of CaR has been reported in osteoclastic cells, but stimulation of the CaR in vitro has been reported to increase osteoclast differentiation but also increase osteoclast apoptosis (41). Consequently, whether the net effect via the CaR of reducing Ca would be to reduce osteoclast differentiation or to reduce osteoclast apoptosis, therefore, appears uncertain. Recent studies have reported that increased signaling by the CaR in mature osteoblasts in vivo can enhance bone resorption from 6 wk of age onward, although it is unclear from that study whether effects were also observed in neonates (42). The CaR has also been localized on osteoblastic cells, and in preliminary studies, osteoblast-specific deletion of the CaR has caused reductions in BV and BMD, but also enhanced new bone formation (43). However, more recent studies by the same group have reported that deletion of both alleles of the Casr gene in osteoblasts profoundly blocked postnatal growth and skeletal development, a finding that was evident by 3 d of age (44). In tissue culture models, elevations in extracellular free ionized Ca concentrations [Cae2+] increase osteoblast chemotaxis and proliferation (45, 46), and alter the levels of expression of some differentiation markers (47, 48, 49). Furthermore, calcitonin release in rodents has been implicated in bone sparing in lactating females (50) and could help explain the reduced osteoclasts in the pups with elevations in Ca.

Consequently, whether the effect of Ca we observed is direct or indirect, whether it occurs via the CaR or other mechanisms, and whether it occurs only in the neonatal skeleton or persists into young adult and adult life remains to be clarified. Nevertheless, our studies do show an important contributory role of Ca in conjunction with PTH to bone formation in the neonate.


   Footnotes
 
This work was supported by Grant 30671009 from the National Natural Science Foundation of China (to D.M.) and a grant from the Canadian Institutes for Health Research (to D.G.).

Disclosure Statement: G.C., Z.G., Y.R., L.S., C.T., A.K., and D.G. have nothing to declare.

First Published Online October 1, 2008

Abbreviations: BMD, Bone mineral density; BV, bone volume; Ca, calcium; CaR, calcium-sensing receptor; 1,25(OH)2D3, 1,25-dihydroxyvitamin D3; HE, hematoxylin and eosin; micro-CT, microcomputed tomography; PTHrP, PTH-related protein; TRAP, tartrate-resistant acid phosphatase; 3D, three-dimensional; TV, tissue volume.

Received May 5, 2008.

Accepted for publication September 19, 2008.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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