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Endocrinology, doi:10.1210/en.2006-1467
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Endocrinology Vol. 148, No. 8 3875-3886
Copyright © 2007 by The Endocrine Society

Weaning Triggers a Decrease in Receptor Activator of Nuclear Factor-{kappa}B Ligand Expression, Widespread Osteoclast Apoptosis, and Rapid Recovery of Bone Mass after Lactation in Mice

Laleh Ardeshirpour, Pamela Dann, Douglas J. Adams, Tracey Nelson, Joshua VanHouten, Mark C. Horowitz and John J. Wysolmerski

Section of Pediatric Endocrinology (L.A.), Department of Pediatrics, Section of Endocrinology and Metabolism (P.D., J.V., J.J.W.), Department of Internal Medicine, and Department of Orthopaedics and Rehabilitation (T.N., M.C.H.), Yale University School of Medicine, New Haven, Connecticut 06520-8020; and Department of Orthopaedic Surgery (D.J.A.), University of Connecticut Health Center, Farmington, Connecticut 06034-4037

Address all correspondence and requests for reprints to: John J. Wysolmerski, M.D., Section of Endocrinology and Metabolism, Yale University School of Medicine, TAC S131, 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
 
A significant portion of milk calcium comes from the mother’s skeleton, and lactation is characterized by rapid bone loss. The most remarkable aspect of this bone loss is its complete reversibility, and the time after weaning is the most rapid period of skeletal anabolism in adults. Despite this, little is known of the mechanisms by which the skeleton repairs itself after lactation. We examined changes in bone and calcium metabolism defining the transition from bone loss to bone recovery at weaning in mice. Bone mass decreases during lactation and recovers rapidly after weaning. Lactation causes changes in bone microarchitecture, including thinning and perforation of trabecular plates that are quickly repaired after weaning. Weaning causes a rapid decline in urinary C-telopeptide levels and stimulates an increase in circulating levels of osteocalcin. Bone histomorphometry documented a significant reduction in the numbers of osteoclasts on d 3 after weaning caused by a coordinated wave of osteoclast apoptosis beginning 48 h after pup removal. In contrast, osteoblast numbers and bone formation rates, which are elevated during lactation, remain so 3 d after weaning. The cessation of lactation stimulates an increase in circulating calcium levels and a reciprocal decrease in PTH levels. Finally, weaning is associated with a decrease in levels of receptor activator of nuclear factor-{kappa}B ligand mRNA in bone. In conclusion, during lactation, bone turnover is elevated, and bone loss is rapid. Weaning causes selective apoptosis of osteoclasts halting bone resorption. The sudden shift in bone turnover favoring bone formation subsequently contributes to the rapid recovery of bone mass.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
LACTATION IS CENTRAL to successful mammalian reproduction because newborns depend on milk to provide all the nutrients, including calcium, needed for survival. The average nursing woman secretes 300–400 mg of calcium into milk each day (1). The mother’s skeleton serves as one of the main sources of the calcium used for milk production, and rapid maternal bone loss during lactation has been documented in several species (1, 2, 3, 4, 5, 6, 7, 8, 9). Nursing humans lose 6–10% of their bone mass over 6 months (1, 4, 7, 8). The degree of bone loss is related, in part, to suckling intensity and the amount of milk produced. Rodents, which nurse many more offspring than humans, lose between 20% and 30% of their skeletal mass over 3 wk of lactation (3, 6, 9).

Lactation is characterized by increased rates of bone turnover. Human and animal studies have demonstrated elevations in both bone resorption and formation, although as evidenced by the rapid decline in bone mass, resorption outstrips formation (1, 7). Biochemical markers of bone resorption are elevated 2- to 3-fold in mice and humans, and markers of formation have been reported to be increased in nursing humans (1, 5, 7, 9, 10, 11). Studies using histomorphometry in animals have documented increased osteoclast numbers at trabecular, endocortical, and cortical sites (1, 5, 9). Bone formation rates have been shown to be elevated by dynamic histomorphometry using fluorescent labeling (1, 5, 9).

The regulation of bone metabolism and the systemic and/or local triggers that elevate bone turnover during lactation are not completely understood. Neither vitamin D nor PTH is needed to trigger lactation-associated bone loss (1). However, estrogen deficiency contributes. Suckling directly inhibits GnRH secretion and induces a state of hypothalamic hypogonadism (12, 13). In nursing women, the degree of bone loss correlates with the duration of amenorrhea postpartum (1, 14). In addition, estrogen levels correlate negatively with rates of bone resorption in lactating mice, and pharmacological estrogen replacement in mice reduces rates of bone resorption and ameliorates, but does not completely prevent, bone loss during lactation (9). Another contributor is the secretion of PTH-related protein (PTHrP) by the lactating breast (15, 16, 17). Circulating levels of PTHrP are elevated in both lactating humans and rodents, and correlate positively with bone resorption rates in mice and negatively with bone mass in humans and mice (9, 15, 16, 17, 18, 19, 20, 21, 22, 23). Moreover, mammary specific disruption of the PTHrP gene lowers rates of bone resorption and preserves bone mass during lactation (17). Finally, several studies have suggested that calcitonin may help to constrain the amount of bone lost during lactation (24, 25, 26). Thus, multiple systemic factors likely participate in the regulation of bone metabolism during this period.

The most remarkable aspect of the bone loss associated with lactation is its rapid and complete reversibility after weaning (1). Bone mineral density (BMD) in humans is restored to baseline within 6–12 months after the cessation of nursing. This suggests that BMD accrues at a rate of 0.5–2%/month after weaning, making this the period of the most robust increase in bone mass in the adult skeleton (1, 4, 27, 28). Epidemiological data have shown that neither the duration of lactation nor the number of offspring nursed represents a risk for lower bone mass or higher rates of osteoporotic fracture later in life (1, 7, 29). The lack of cumulative damage from multiple periods of lactation speaks to the efficient nature by which bone mass is restored after each individual reproductive cycle. Surprisingly, there have been relatively few studies addressing the mechanisms that underlie this dramatic burst of anabolic bone growth. Bowman et al. (30), Miller et al. (31), Miller and Bowman (32), and Vajda et al. (33) have undertaken a series of histomorphometric studies in rats showing that bone mass recovers over a period of 4–8 wk after the end of lactation. Their studies revealed increases in trabecular number, thickness, and connectivity by 2 wk after lactation had ended. This was accompanied by a substantial increase (800%) in the rate of bone formation at this point (30). Despite the wealth of genetic tools available in mice, no studies have examined postlactation recovery in this species. In addition, nothing is known of the systemic or local regulation, or the molecular mechanisms that lead to the recovery of bone mass after lactation. To begin to define these issues, we examined the transition from bone loss to bone recovery at the end of lactation in mice in detail. Our studies demonstrate rapid recovery of bone mass after weaning that may be the result of a sudden halt in bone resorption coupled with a continuation of the increased rate of bone formation observed during lactation. These shifts in bone turnover are associated with changes in the levels of expression of receptor activator of nuclear factor-{kappa}B ligand (RANKL) and widespread osteoclast apoptosis upon weaning.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Experimental animals
Female, 12-wk-old, CD1 mice were purchased from Charles River Laboratories (Wilmington, MA). Experimental mice were allowed to become pregnant, deliver, and lactate. Litter size was adjusted to 8–12 pups to equalize suckling intensity between dams. Pups were removed on the 12th day of lactation to trigger weaning. Age-matched, virgin CD1 mice were used as controls.

Blood and urine sample collection
Mice were anesthetized with methoxyflurane (Medical Developments Australia, Springvale, Victoria, Australia), and blood was collected by cardiac puncture into syringes containing 20-µl protease inhibitor cocktail (Nichols Institute Diagnostics, San Juan Capistrano, CA). Blood was centrifuged at 750 x g for 15 min at 4 C, and plasma was removed, aliquoted, and stored at –70 C. To prepare serum, blood was collected into a plain syringe, allowed to clot at room temperature for 30 min, and then centrifuged at 750 x g for 15 min at 4 C. Serum was removed, aliquoted, and stored at –70 C. Urine was collected immediately before terminal bleeding.

Dual-energy x-ray absorptiometry (DEXA)
BMD measurements were performed by DEXA using a Lunar PIXImus (G.E. Medical Systems, Lunar Division, Madison, WI) operated by the Yale Core Center for Musculoskeletal Disorders. 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. BMD measurements included the entire thoracic and lumbar spine, the entire femur, and the total body (excluding the head).

X-ray microcomputed tomographic (CT) imaging
Trabecular morphometry within the metaphyseal region of the proximal tibia and centrum of the third lumbar vertebra (L3) was quantified using x-ray micro-CT (µCT40; Scanco Medical AG, Bassersdorf, Switzerland). Specimens were scanned in 70% ethyl alcohol at 55 kV (145 µA), using 1000 cone beam projections per revolution and an integration time of 300 msec within a 12.3-mm-diameter field of view. Three-dimensional images were reconstructed at 12-µm resolution using standard convolution backprojection algorithms with Shepp and Logan filtering, and rendered at a discrete voxel density of 578,704 voxels/mm3 (isometric 12-µm voxels). Segmentation of bone from marrow and soft tissue was performed at a global threshold of 470 mg/cm3 in conjunction with a constrained gaussian filter to reduce noise. Volumetric analysis regions of trabecular bone were selected within the endosteal borders to include the central 80% of vertebral height and secondary spongiosa of tibial metaphyses located 480 µm from the growth plate and extending 480 µm distally. Trabecular morphometric parameters were measured directly, without imposing a structural model (e.g. neither rod nor plate) (34). Trabecular morphometry was characterized by measuring the bone volume fraction, trabecular thickness, trabecular number, trabecular spacing, connectivity density, tissue mineral density, and structure model index (SMI), a numerical measure of trabecular geometry representative of increasingly rod-like (higher SMI) or plate-like architecture (lower SMI).

Cortical bone morphometry was averaged from 50 serial cross-sectional images (600 µm) centered at the longitudinal midpoint of the tibia between proximal growth plate and distal tibia-fibular junction, applying a density threshold of 710 mg/cm3. Cortical measurements included average cortical thickness, cross-sectional area of cortical bone, subperiosteal cross-sectional area, marrow area, and intracortical porosity.

Bone histology and histomorphometry
Routine bone histology was performed on 4-µm, toluidine blue-stained, methylmethacrylate-embedded, nondecalcified sections of tibia and vertebra as previously described (35). Static and dynamic histomorphometric analysis of the proximal tibia and lumbar vertebra was performed using the Osteomeasure system (OsteoMetrics, Inc., Decatur, GA). Bones were labeled by injecting mice ip with 30 mg/kg calcein (EM Science, Gibbstown, NJ) at 5 and 1 d before being killed.

Acid phosphatase activity and terminal deoxynucleotidyl transferase-mediated deoxyuridine 5-triphosphate-biotin end labeling of fragmented DNA (TUNEL) assays were performed on decalcified sections of bone prepared as follows. Freshly dissected bones were fixed in 4% paraformaldehyde in PBS at 4 C for 2 h and were then decalcified in a 6% EDTA solution in PBS at 4 C for 2 wk. Samples were washed in PBS and incubated in 100 mM magnesium chloride overnight before dehydration and embedding in paraffin. Paraffin sections (5 µm) were then dewaxed, rehydrated, and stained for acid phosphatase activity as previously described (35). Immediately after acid phosphatase staining, the slides were washed in water and subjected to TUNEL assay using the S7100 ApopTag In Situ Apoptosis Detection Kit from Chemicon (Temecula, CA). We used 3,3' diaminobenzidine with Cobalt Chloride enhancement (Sigma, St. Louis, MO) as the chromagen for the TUNEL assay. After the sequential acid phosphatase and TUNEL staining, slides were dehydrated in graded alcohol and coverslipped. The rate of osteoclast apoptosis was determined by enumerating the number of acid phosphatase-positive cells that were also TUNEL positive in sections from lumbar vertebrae and proximal tibia at d 12 of lactation and 48 h after weaning. For d 12 of lactation, we counted 639 osteoclasts from randomly selected sections taken from three separate mice. For 48 h after weaning, we counted 660 osteoclasts from three separate mice.

Biochemical measurements
PTH was measured using a two-site immunoradiometric assay for rat PTH (Immutopics International, San Clemente, CA) as per the manufacturer’s instructions. Plasma calcium was measured with an atomic absorptiometer (model 2380; PerkinElmer, Norwalk, CT) by the Clinical Chemistry laboratory at Yale-New Haven Hospital. Osteocalcin concentrations were measured using a previously described RIA (9). 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 (BioAssay System, Hayward, CA).

Bone marrow (BM) cultures
To culture osteoblast precursors, BM cells (107) derived from long bones were plated in 12-well tissue culture plates and cultured for 4 d in the presence of ascorbate-containing {alpha}-MEM culture medium. After 4 d, the nonadherent cells were removed by washing, and new medium was added. Cultures were fed again at 7 and 10 d. To determine the number of osteoblast colonies, cultures were rinsed twice with PBS to remove nonadherent cells, fixed, and stained for alkaline phosphatase using a commercially available kit (86-R alkaline phosphatase staining kit; Sigma Chemical Co., St. Louis, MO). Colonies containing more than 50 cells were counted using a dissecting microscope. Because each osteoblast colony theoretically occurred from a single osteoblast precursor, we estimated the number of osteoblast precursors by counting the number of osteoblast colonies per 107 BM cells originally plated. In each experiment, three wells were counted for each data point, and the data presented are representative of three independent experiments at each time point.

To culture osteoclast precursors, BM cells (106) were cultured in 48-well tissue culture plates in the presence of M-CSF (30 ng/ml; PeproTech, Inc., Rocky Hill, NJ) and RANKL (50 ng/ml; PeproTech, Inc.) for 12 d. The cultures were fixed with 2.5% glutaraldehyde, stained for tartrate resistant acid phosphatase (TRAP) using a commercially available kit (Leukocyte Acid Phosphatase 387-A; Sigma Diagnostics, Inc., St. Louis, MO), and large, multinucleated (>5 nuclei), TRAP-positive cells were counted using light microscopy. As before, three wells were counted for each data point, and the data presented are representative of three independent experiments at each time point.

Quantitative real-time RT-PCR
Bone samples were collected immediately after killing, snap-frozen in an ethanol/dry ice bath, and stored at –70 C until processing. Calvarial RNA was prepared from the dome of the skull. The metaphyseal samples included that portion of the long bone between the growth plate and shaft. Bone samples were pulverized in liquid nitrogen, and bone powder was added to Trizol reagent (Invitrogen, Carlsbad, CA). Samples were then homogenized using the Dispersing Tool S25N-10G (IKA, Wilmington, NC), and total RNA was isolated as per the Trizol manufacturer’s instructions. Contaminating DNA was removed using the RNeasy Minikit and DNase 1 (QIAGEN, Inc., Valencia, CA). Two-step quantitative real-time-PCRs were performed using the High Capacity cDNA archive kit (Applied Biosystems, Foster City, CA) and the Full-Velocity SYBR-Green QPCR Master Mix kit (Stratagene, La Jolla, CA) in the Opticon 2 DNA Engine (MJ Research, Waltham, MA). The relative expression levels were determined using the comparative 2–{Delta}{Delta}CT method. Glyceraldehyde-3-phosphate dehydrogenase was the endogenous reference gene, and the average 2–{Delta}{Delta}CT of the samples from virgin mice served as a calibrator sample to which all individual samples were normalized. Each sample was run in triplicate. We used the following primers: mouse glyceraldehyde-3-phosphate dehydrogenase, forward, 5'-CGTCCCGTAGACAAAAATGGT-3' and reverse, 5'-TCAATGAAGGGGTCGTTGAT-3'; mouse RANKL, forward, 5'-CATTTGCACACCTCACCATC-3' and reverse, 5'-TCCGTTGCTTAACGTCATGT-3'; mouse receptor activator of nuclear factor-{kappa}B (RANK) forward, 5'-TGATGAGAGGGGAGCCTCAG-3' and reverse, 5'-CACGATGATGTCACCCTTGA-3'; mouse osteoprotegerin (OPG), forward, 5'-CCGAGTGTGTGAGTGTGAGG-3'and reverse, 5'-TGCAAACTGTGTTTTGCTCTG-3'; and mouse calcitonin receptor, forward, 5'-CAGAGTGAAAAGGCGGAATC-3' and reverse, 5'-CTGGAGTTGGGCTCACTAGG-3'.

Statistical analysis
Values are given as averages ± SEM, and error bars represent SEM. 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. All statistical analyses were performed using Graph Pad Prism 4.00 for Windows (GraphPad Software, San Diego, CA).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Rapid recovery of bone mass after weaning
The current study was aimed at defining the nature of skeletal recovery after lactation. We first examined changes in BMD in a cohort of 12-wk-old female mice that were allowed to become pregnant, deliver, and lactate. After 12 d of suckling, the pups were abruptly separated from their mothers to trigger mammary gland involution and terminate lactation (36). Baseline bone density measurements were obtained at 12 d of lactation, just before forced weaning, and then serial measurements on these same mice were obtained 3, 7, 14, and 28 d after weaning. A rapid increase in spine, femur, and total body BMD occurred immediately upon pup withdrawal (Fig. 1Go). The increase in BMD became statistically significant by 3 d for the spine, 7 d for the femur, and 3 d for the total body. At the end of the 28-d period, BMD had increased by 37% at the spine, 27% at the femur, and 25% for the total body.


Figure 1
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  		FIG. 1. Serial changes in bone density upon weaning. Serial DEXA measurements of the spine (A), femur (B), and total body (C) were performed on a cohort of six mice at midlactation (L12), and at 3, 7, 14, and 28 d (D3, D7, D14, and D28, respectively) after forced weaning. These measurements were compared with a separate cohort of six age-matched nulliparous mice (virgin). At all three sites, bone density was significantly lower during lactation compared with virgin controls (P < 0.001 at each site). In each mouse, bone density at all sites rapidly increased back toward the virgin baseline after weaning. Spine and total body BMD became significantly greater by 3 d after weaning (P < 0.01, P < 0.05, respectively). BMD at the femur became significantly greater than midlactation by 7 d after weaning (P < 0.01).

 
We also examined changes in bone mass and structure by quantitative micro-CT imaging. We analyzed the third lumbar vertebra and proximal tibia of separate groups of 12-wk-old mice at d 12 of lactation, and 7 and 28 d after weaning. Age-matched nulliparous mice provided a baseline for comparison. As expected, there was a decrease in vertebral trabecular bone volume fraction in lactating mice compared with nulliparous mice (Table 1Go). Vertebral bone volume rapidly increased back toward baseline after weaning, and by 7 d after weaning was no longer significantly different from baseline. There were no significant differences in trabecular number, but there was a significant decline in trabecular thickness during lactation that required the full 28-d period to return to baseline. Interestingly, lactation was also associated with a significant decline in tissue mineral density that recovered by 28 d after weaning. These data suggest that reproduction may be associated with reversible changes in bone mineralization as well as changes in bone mass. Finally, the increase in connective density, the structural modular index, and the bone surface to bone volume ratio all suggest that a significant, yet reversible, change in trabecular architecture occurs during lactation. As seen in Fig. 2Go, three-dimensional reconstructions of the vertebral bodies suggest that lactation is associated with an increase in the number of smaller, rod-like trabeculae with a corresponding decease in the number of contiguous trabecular plates. Together, these data suggest that the increases in bone resorption during lactation lead to significant trabecular thinning and ultimately perforations within trabecular plates. After lactation ceases, these defects are repaired, and the trabecular network regains a more plate-like architecture. Similar changes occurred in trabecular bone in the tibia, although they were not as pronounced. Trabecular thickness and tissue density both declined in the tibia during lactation and recovered after weaning, whereas bone surface/bone volume increased during lactation and decreased afterward (data not shown).


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  		TABLE 1. Results of quantitative micro-CT analysis of trabecular bone in the third lumbar vertebrae

 

Figure 2
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  		FIG. 2. Changes in bone microarchitecture during lactation and after weaning. Three-dimensional reconstructions of representative lumbar vertebrae from nulliparous (A) and lactating (B) mice, as well as mice 7 d (C), and 28 d (D) after weaning. Lactation is associated with trabecular thinning, trabecular perforation, and the shift from a plate-like to rod-like appearance of trabeculae (compare A and B). After weaning (C and D), trabecular architecture rapidly reverts back to that seen in nulliparous animals. Magnification, x18.

 
Lactation has also been associated with increased endocortical bone resorption (32). Table 2Go shows the results for the analysis of cortical bone in the tibia. Compared with the nulliparous controls, there was a significant decrease in cortical cross-sectional area and cortical thickness during lactation. Although cortical area returned to baseline by 28 d after weaning, cortical thickness improved but remained statistically lower than in virgins. These changes were likely the result of endocortical resorption because the endosteal perimeter increased significantly during lactation, whereas the periosteal perimeter remained stable. In addition to the changes in cortical cross-sectional area, there was also a significant increase in cortical porosity during lactation that resolved by 28 d after weaning. Thus, both endocortical resorption and intracortical tunneling are increased during lactation, and these changes are subsequently repaired after weaning.


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  		TABLE 2. Results of quantitative micro-CT analysis of cortical bone in the proximal tibia

 
Weaning is associated with a selective decrease in bone resorption
The measurements of bone mass and architecture detailed previously suggest a remarkable reversal from bone loss during lactation to bone accrual immediately upon weaning. To begin to define the cellular mechanisms underlying this transition, we next examined rates of bone resorption and formation as assessed by biochemical markers of bone turnover. We measured urinary CTx levels to assess bone resorption and serum osteocalcin levels to assess bone formation. We chose d 3 after weaning as the earliest time point to examine because mammary gland involution becomes permanent 72 h after pup withdrawal. Before that point, involution is reversible, and lactation can resume (36). As noted in Fig. 3Go, CTx levels are elevated in lactating mice, decrease dramatically 3 d after weaning, increase somewhat at d 7, and then remain decreased for the duration of the 28-d period. In contrast, osteocalcin levels are increased at 3 d after weaning compared with midlactation and then gradually decline again over the course of the 28 d.


Figure 3
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  		FIG. 3. Changes in biochemical markers of bone turnover after weaning. Urinary CTx levels (A) and serum osteocalcin levels (B) were measured in virgin mice, lactating mice (L12), and 3, 7, 14, and 28 d (D3, D7, D14, and D28, respectively) after weaning. A, Statistically significant difference compared with virgin controls is denoted by an asterisk (*), whereas a statistically significant difference compared with d 12 of lactation is denoted by a dagger ({dagger}). CTx levels are elevated during lactation (P < 0.001; L12 vs. virgin) and are suppressed at 3 d after weaning (P < 0.001; d 3 vs. L12). Levels were somewhat elevated again on d 7, but otherwise remained at baseline over the rest of the 28 d examined (P < 0.05; d 7 vs. virgin). Values represent the mean ± SEM. Sample sizes included four virgin mice, 13 mice at L12, 10 mice at d 3, six mice at d 7, eight mice at d 14, and eight mice at d 28. B, Serum osteocalcin levels tended to be increased during lactation, but the change between virgins and L12 was not significant. However, levels increased significantly after weaning (P < 0.001, d 3 vs. L12; P < 0.05, d 7 vs. L12) and gradually declined to baseline over the 28-d recovery period. Sample sizes were nine virgin mice, 16 mice at L12, nine mice at d 3, seven mice at d 7, six mice at d 14, and nine mice at d 28.

 
To define the mechanisms that trigger these abrupt changes in bone resorption and formation, we concentrated subsequent experiments on the early postweaning period. Histological examination of bones harvested from mice at midlactation and from 3 d after weaning suggested that the sudden decrease in bone resorption is due to the disappearance of osteoclasts. As shown in Fig. 4Go, both osteoclasts and osteoblasts are abundant during lactation, and are easy to identify on trabecular surfaces. Three days after weaning, we observed a highly reduced number of active osteoclasts, and, sometimes, the entire circumference of an individual trabecula was surrounded by active osteoblasts. These data suggested that weaning triggers dramatic changes in the quantity and type of bone cells present on the trabecular bone surface. This impression was substantiated by the results of quantitative bone histomorphometry performed on proximal tibiae (Table 3Go) and lumbar vertebrae (Table 4Go) harvested from mice killed at midlactation and 3 d after weaning. In the tibia, bone volume and trabecular thickness tended to increase, whereas bone surface and trabecular spacing tended to decrease after weaning. Only the bone surface to bone volume ratio was significantly different at this early time point. In the vertebrae, none of these parameters were significantly different. Osteoid volumes were not different at either site, but osteoid thickness declined significantly in the vertebrae on d 3 after weaning compared with lactation. At both sites, there was a significant and dramatic decrease in both osteoclast numbers and osteoclast surfaces 3 d after weaning as compared with midlactation. At the tibia, osteoblast numbers and surfaces, as well as the bone formation rate as assessed by double-fluorescent labeling, all tended to increase by d 3, but these changes were not statistically significant. In the vertebrae, osteoblast numbers and surfaces also tended to be slightly higher after weaning, but, as in the tibia, these changes were not significant. There were no changes in vertebral bone formation rates after weaning.


Figure 4
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  		FIG. 4. Weaning triggers osteoclast apoptosis. A and B show toluidine blue-stained sections through the proximal tibia from mice at 12 d of lactation (A) and 3 d after weaning of pups (B). During lactation, both osteoclasts (red arrowheads) and osteoblasts (green arrowheads) are plentiful on trabecular surfaces. However, 3 d after weaning (B), osteoclasts are much reduced in number, and osteoblasts surround many individual trabeculae. C and D demonstrate sections of bone that have been stained for both acid phosphatase activity and subjected to TUNEL assay. Osteoclasts stain red, and apoptotic nuclei stain black. On d 12 of lactation (C), acid phosphatase-positive osteoclasts are abundant and are TUNEL negative (black arrowheads). One can appreciate TUNEL-positive cells in the BM, which serve as an internal positive control. Forty-eight hours after weaning (D), the overall number of acid phosphatase-positive cells is reduced. In addition, acid phosphatase-positive cells are frequently separated from the bone surface, appear fragmented, and are TUNEL positive (black arrowheads), consistent with the occurrence of widespread osteoclast apoptosis.

 

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  		TABLE 3. Static and dynamic histomorphometry measurements on bone sections from the proximal tibia at midlactation and 3 d after weaning

 

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  		TABLE 4. Static and dynamic histomorphometry measurements on bone sections from lumbar vertebrae at midlactation and 3 d after weaning

 
Given the rapid disappearance of osteoclasts from the bone surfaces at 3 d after weaning, we hypothesized that they underwent apoptosis. To examine this possibility, we performed TUNEL assays on decalcified sections of vertebrae and tibiae harvested from mice on d 1, 2, and 3 after forced weaning. Sections were simultaneously assessed for acid phosphatase activity to identify osteoclasts unequivocally. We did not find clear evidence of osteoclast apoptosis on the first day after weaning, but by 48 and 72 h after weaning, there were multiple acid phosphatase-positive cells with condensed, TUNEL-positive nuclei (Fig. 4Go) in both the vertebrae and tibiae. Many of these cells appeared fragmented and were found in the BM detached from the bone surface. Only 4 ± 0.1% of osteoclasts were TUNEL positive on d 12 of lactation. Forty-eight hours after weaning, 64 ± 2.1% of osteoclasts were undergoing apoptosis as defined by TUNEL staining. Therefore, weaning is associated with a wave of coordinated osteoclast apoptosis.

We also examined the numbers of preosteoclasts and preosteoblasts present in BM cultured from virgin, lactating (L12) and weaned (D3) mice. As shown in Fig. 5Go, these results were consistent with the results of our histomorphometry studies. We found a significant increase in the number of acid phosphatase-positive, osteoclast-like cells that developed from the BM of lactating compared with virgin mice. Marrow harvested from mice on d 3 after weaning consistently produced few osteoclast-like cells. BM from lactating mice also supported the growth of more alkaline-phosphatase positive, osteoblastic colonies in culture compared with marrow from virgin mice. However, in contrast to the decrease in the number of osteoclasts, marrow from weaned mice produced more osteoblast colonies in culture. Thus, similar to the histological results for mature cells, our culture results suggest that lactation is associated with an increase in the numbers of committed osteoclast and osteoblast precursors. Weaning dramatically reduces the number of preosteoclasts and, at the same time, modestly increases the number of preosteoblasts.


Figure 5
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  		FIG. 5. Weaning leads to a decline in osteoclast (OC) precursors and an increase in osteoblast (OB) precursors. A, The number of TRAP-positive, osteoclast-like cells developing in BM cultured from virgin, lactating (L12) and weaned (D3) mice. Data are expressed as the number of osteoclasts counted on d 12 of culture per 106 cells plated. The number of osteoclasts from L12 marrow is significantly higher than from marrow from virgins (P < 0.01). D3 marrow contributed to significantly fewer osteoclasts than did L12 marrow (P < 0.001). B, The number of alkaline phosphatase-positive osteoblast colonies developing in BM cultured from virgin, lactating (L12) and weaned (D3) mice. Data are expressed as the number of osteoblast colonies counted on d 12 of culture per 107 cells plated. The number of osteoblast colonies derived from L12 marrow is significantly higher than that derived from virgin marrow (P < 0.001). D3 marrow contributed to a significantly greater number of osteoblast colonies than either virgin or L12 marrow (P < 0.001). A statistically significant difference compared with virgin controls is denoted by an asterisk (*), whereas a statistically significant difference compared with d 12 of lactation is denoted by a dagger ({dagger}).

 
Weaning is associated with transient hypercalcemia
Previous studies have shown that PTHrP levels are elevated during lactation and return to baseline after weaning (9). In this study we examined changes in systemic calcium, and PTH levels. As shown in Fig. 6Go, there was a significant, but transient, increase in circulating calcium levels to 12.1 mg/dl on the third day after weaning. This was accompanied by the simultaneous suppression of circulating PTH levels on d 3. Both calcium and PTH levels began to change on d 2 after weaning, returned to the normal range by 7 d after weaning, and remained within the normal range thereafter (Fig. 6Go; data not shown).


Figure 6
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  		FIG. 6. Weaning is associated with transient hypercalcemia. Circulating calcium (A) and PTH (B) levels were measured in virgin and lactating (L12) mice, and on d 3, 7, 14, and 28 d (D3, D7, D14, D28, respectively) after forced weaning. Graphs show the mean ± SEM. Significant differences compared with lactation are denoted by a dagger ({dagger}). Weaning led to a transient increase in circulating calcium concentrations and a simultaneous decrease in circulating levels of PTH on d 3 (P < 0.001 D3 vs. L12 for calcium; P < 0.05 D3 vs. L12 for PTH). Levels of both calcium and PTH returned to baseline by d 7 and remained normal throughout the remainder of the 28-d period. Calcium levels were determined in eight virgins, 14 mice at L12, 11 mice at d 3, 10 mice at d 7, six mice at d 14, and seven mice at d 28. PTH levels were determined in seven virgins, 19 mice at L12, 12 mice at d 3, 13 mice at d 7, eight mice at d 14, and 10 mice at d 28.

 
Weaning is associated with a decrease in RANKL expression in bone
Osteoclast differentiation, activity, and survival depend on the actions of the RANKL/RANK/OPG signaling system (37, 38, 39). RANKL, which is produced by bone cells within the stromal/osteoblastic lineage, acts through its receptor RANK to stimulate the differentiation and activity of cells within the osteoclast lineage. Given the dramatic changes in bone resorption rates and osteoclast numbers associated with weaning, we hypothesized that there might be shifts in the expression of these cytokines within the bone microenvironment. We examined the expression of RANKL, OPG, and RANK by quantitative RT-PCR in RNA prepared from calvaria and long bone metaphases harvested from mice at 12 d of lactation or at 3 d after weaning. Aged-matched, nulliparous mice served as a baseline. As one can see from Fig. 7Go, A, C, and E, calvarial bone showed clear changes in RANKL and RANK expression. RANKL mRNA levels increased during lactation and promptly returned to baseline after weaning. The level of expression of OPG mRNA tended to increase during lactation, although the differences were not statistically significant, and levels did not increase further after weaning. Thus, the RANKL to OPG ratio increased during lactation, and after weaning, it decreased below the baseline level seen in the virgin controls (data not shown). RANK mRNA levels increased significantly during lactation and decreased after lactation ended. Similar patterns were noted in RNA prepared from the metaphases of the distal femur and proximal tibia (Fig. 7Go, B, D, and F). However, only the decrease in RANKL mRNA expression from lactation to d 3 after weaning reached statistical significance. These results suggest that increased RANKL signaling likely contributes to the stimulation of systemic bone resorption during lactation, and that withdrawal of RANKL signaling may contribute to osteoclast apoptosis and decreased bone resorption after weaning.


Figure 7
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  		FIG. 7. Weaning causes a decrease in RANKL mRNA expression. Real-time RT-PCR results for the relative levels of RANKL (A and B), OPG (C and D), and RANK (E and F) gene expression in calvaria (A, C, and E) or long bone metaphyses (B, D, and F) harvested from lactating mice (L12), mice 3 d after weaning (D3), and age-matched virgin controls. Graphs represent the mean ± SEM. A statistically significant difference compared with virgin controls is denoted by asterisk (*), whereas a statistically significant difference compared with d 12 of lactation is denoted by a dagger ({dagger}). Note the decrease in RANKL expression brought about by weaning at both sites. In the calvarial samples, RANKL mRNA levels at L12 were also statistically significantly different from virgins. Levels of OPG at L12 and D3 were not significantly different from virgins. RANK mRNA levels were significantly higher at L12 compared with virgins and D3 in calvarial bone. Similar trends in RANK gene expression occurred in the metaphyses but did not reach statistical significance.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
In this report we confirm that mice, like humans and rats, experience a rapid shift from bone catabolism to bone anabolism when lactation is terminated. Measurements within both cancellous and cortical compartments revealed that bone mass begins to increase almost immediately upon weaning, and statistically significant gains at the spine and total body can be seen as early as 3 d after the end of lactation. This rapid increase in bone mass is associated with a dramatic shift in rates of bone turnover. The primary response to weaning in mice appears to be the sudden halt of bone resorption, associated with a coordinated wave of osteoclast apoptosis that occurs between 48 and 72 h after pup withdrawal. BM cultures also showed that weaning led to a significant decrease in osteoclast progenitors. In contrast to this sudden decrease in bone resorption, we found that weaning is associated with the maintenance of the already increased rates of bone formation found during lactation. The sudden decline in osteoclast numbers and function in the face of continued increases in osteoblast numbers and function define the period immediately after weaning as a time of relatively unopposed bone formation, which undoubtedly contributes to the rapid recovery of bone mass.

Another factor contributing to the rapid increase in bone density after weaning may be changes in bone mineralization. We found that lactation is associated with a reversible decrease in tissue density as measured by micro-CT. In the vertebrae, we also found a decrease in osteoid thickness on d 3 after weaning compared with midlactation. However, there were no such changes at the tibia, and there were no changes in osteoid volume at either site. Therefore, it is unlikely that the findings noted on micro-CT are due to the accumulation of large amounts of unmineralized matrix during lactation. Instead, it may be that the elevated rates of bone turnover during lactation result in a higher proportion of newly synthesized bone, which has a lower mineral density (40, 41). The decrease in bone resorption at the end of lactation may allow for consolidation of this newly formed bone and an increase in its mineral content.

An interesting aspect of the reduction in bone resorption upon weaning is its selectivity; osteoblast numbers and bone formation rates were untouched, whereas osteocalcin levels and the number of osteoblast colonies derived from BM cultures actually increased upon weaning. These conflicting data regarding osteoblast function suggest that whereas overall rates of bone formation do not change much after weaning in mice, important changes in osteoblast differentiation or signaling may indeed occur at this point. Nonetheless, we did not observe the dramatic increases in bone formation rates previously reported in rats (30, 31, 32). This may be related to species differences because the magnitude of changes in bone turnover in rats and mice has differed in other settings, such as in response to ovariectomy (42, 43). Alternatively, it may be related to differences in methodology. We used a model of abrupt withdrawal of suckling offspring at a time of peak lactation intensity, which is the standard paradigm for the study of mammary gland involution (36). In contrast, Bowman (30) and Miller (31) et al. examined a process of more gradual weaning at the end of the lactation period. Interestingly, therapeutic reductions in bone resorption usually lead to a concurrent reduction in bone formation rates. For example, estrogen replacement or treatment with bisphosphonates in ovariectomized rats or mice leads to a suppression of both osteoclast and osteoblast activity (44, 45, 46, 47, 48). Likewise, estrogen or bisphosphonate use in postmenopausal women reduces both bone resorption and bone formation as assessed by biochemical markers of bone turnover (49, 50). Therefore, there may be unique features of weaning that allow the skeleton to support ongoing bone formation in response to specific antiresorptive cues. We believe that this selectivity may be important to the rapid recovery of bone mass after lactation, and learning to mimic this response would be of great therapeutic use.

Bone resorption during lactation occurs throughout the skeleton and appears to be associated with an increase in RANKL gene expression. This was most clearly seen in calvarial bone. Although there was a similar trend in metaphyseal bone, the increase in RANKL mRNA did not reach statistical significance, perhaps obscured by the higher proportion of contaminating marrow elements in these samples. However, there was a statistically significant decline in RANKL expression at both sites upon weaning. A multitude of evidence has demonstrated that RANKL signaling is important to osteoclast differentiation, function, and survival. Therefore, it is likely that this decrease in RANKL expression acts as an important trigger for the decline in CTx levels and the increase in osteoclast apoptosis we observed after weaning, as well as the decrease in the numbers of osteoclasts we cultured from the BM of weaned mice.

Elevations in osteoclast numbers and bone resorption rates during lactation are driven by systemic signals, such as a decrease in circulating levels of estrogen and elevations in circulating levels of PTHrP (1, 9, 17, 22). It may be that hormonal changes after weaning also contribute to the termination of bone resorption. After lactation ceases, estrogen levels return to normal, and PTHrP levels decrease (1, 9, 14). Both estrogen and PTHrP regulate the expression of OPG and/or RANKL by BM stromal cells and osteoblasts (37, 38, 39). Furthermore, estrogen can trigger osteoclast apoptosis directly (51, 52, 53). In addition, we observed a transient increase in circulating calcium levels and a simultaneous decrease in PTH levels upon weaning. Elevations in extracellular calcium have been shown to inhibit osteoclast activity and to promote osteoclast apoptosis in cell culture (54, 55, 56). The decline in PTH in response to the elevation of calcium may also contribute to reductions in RANKL expression, thereby helping to trigger osteoclast apoptosis (37, 38). A recent preliminary report from Bowman and Miller (57) documented a sharp increase in circulating levels of calcitonin just after weaning, which may be an additional factor that inhibits bone resorption at the end of lactation. Thus, there are changes in several systemic factors at the end of lactation that might contribute to the inhibition of bone resorption. Future studies will need to examine whether and how each of these factors contributes individually, and how they may interact together to inhibit bone resorption and induce osteoclast apoptosis.

In closing, it is worth noting that the use of the skeleton as a source of calcium for reproductive purposes is an ancient adaptation, and postmenopausal osteoporosis may be a postreproductive and unintended consequence of this physiology (58, 59). If this is correct, the efficient recovery of bone mass after lactation holds forth the possibility that a similar complete recovery of bone mass in postmenopausal women may be possible.


   Acknowledgments
 
We thank Drs. Arthur Broadus, William Horne, and Caren Gundberg for useful discussions. We also thank Xuesong Chen for helpful advice on bone histology.


   Footnotes
 
This work was supported by Grants R01CA094175, R21DK073941, and R01DK069542 from the National Institutes of Health. Support was also provided by core facilities funded by the Yale Diabetes and Endocrine Research Center (P30DK45735), the Yale Core Center for Musculoskeletal Disorders (P30AR46032), and the University of Connecticut Health Center Core Center for Musculoskeletal Disorders (P30AR46026). Dr. Ardeshirpour received support from T32DK07058 to the Yale Section of Endocrinology and Metabolism and K12HD001401 to the Yale Department of Pediatrics.

Disclosure Statement: The authors have no conflicts to report.

First Published Online May 10, 2007

Abbreviations: BM, Bone marrow; BMD, bone mineral density; CT, computed tomography; CTx, C-telopeptide; DEXA, dual-energy x-ray absorptiometry; OPG, osteoprotegerin; PTHrP, PTH-related protein; RANK, receptor activator of nuclear factor-{kappa}B; RANKL, RANK ligand; SMI, structure model index; TRAP, tartrate resistant acid phosphatase; TUNEL, terminal deoxynucleotidyl transferase-mediated deoxyuridine 5-triphosphate-biotin end labeling of fragmented DNA.

Received November 3, 2006.

Accepted for publication May 3, 2007.


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