Rapid inactivation and apoptosis of osteoclasts in the maternal skeleton during the bone remodeling reversal at the end of lactation.код для вставкиСкачать
THE ANATOMICAL RECORD 290:65–73 (2007) Rapid Inactivation and Apoptosis of Osteoclasts in the Maternal Skeleton During the Bone Remodeling Reversal at the End of Lactation SCOTT C. MILLER* AND BETH M. BOWMAN Division of Radiobiology, Department of Radiology, School of Medicine, University of Utah, Salt Lake City, Utah ABSTRACT There is a rapid reversal in maternal skeletal metabolism and bone remodeling from accelerated bone resorption during lactation to skeletal rebuilding after lactation. The purpose was to determine the changes that occur in maternal osteoclasts during the transition from lactation to postlactation. Skeletal samples were taken from female rats on days 10 and 19 of lactation and 1 and 7 days after lactation. The pups were weaned on day 20. There was a rapid change in the osteoclast population after weaning, resulting in less resorption surface. Osteoclasts detached from bone surfaces, lost their rufﬂed borders, and became fragmented with immunocytochemical evidence of apoptosis within 24 hr after lactation. Concomitant with the rapid regression in the osteoclast population was an over ﬁvefold increase in maternal calcitonin (CT) levels at 24 hr after weaning. Serum calcium and estrogen (E2) increased, but prolactin (PRL) and PTH decreased after weaning. The hormone changes, particularly that of CT, are consistent with the rapid regression of the osteoclast population at the end of lactation. These changes are similar to a reversal phase of a bone remodeling cycle where bone formation commences when resorption ceases on bone surfaces and suggests that the fate of osteoclasts during bone remodeling is programmed cell death. These results also suggest that bone remodeling is well synchronized prior to, during, and after lactation to accommodate the mineral requirements of the offspring as well as the mother. Anat Rec, 290:65–73, 2007. Ó 2006 Wiley-Liss, Inc. Key words: lactation; calcium; osteoclasts; bone resorption; bone remodeling; apoptosis; calcitonin; estrogen Fetal skeletal mineralization during pregnancy and milk production during lactation place considerable demands on mineral homeostasis in the mother (Prentice, 2000). Lactation is a period of substantial metabolic adaptations with considerable changes in energy balance and mineral metabolism. In the skeleton, lactation is a period of high bone turnover, with excess bone resorption that results in a loss of skeletal mass (Sowers et al., 1993). This loss of bone during lactation has been observed in all mammals studied and includes humans, monkeys, dogs, sheep, rats, and mice (Bowman and Miller, 2001). Consistent with observations made in humans (Kent et al., 1990), the majority of bone that is lost occurs predominantly at cancellous bone sites in exÓ 2006 WILEY-LISS, INC. perimental animals (Bowman and Miller, 2001), but changes in cortical bone are also observed (Miller et al., 1986; Vajda et al., 1999). Grant sponsor: National Institute of Arthritis and Musculoskeletal and Skin Disease; Grant number: R01 AR044806. *Correspondence to: Scott C. Miller, University of Utah, 729 Arapeen Drive, Suite 2334, Salt Lake City, UT 84108. Fax: 801581-7008. E-mail: firstname.lastname@example.org Received 26 July 2006; Accepted 11 October 2006 DOI 10.1002/ar.a.20403 Published online 28 November 2006 in Wiley InterScience (www.interscience.wiley.com). 66 MILLER AND BOWMAN Many of the changes in maternal skeletal metabolism during the reproductive cycle appear to be facilitated by classical bone remodeling (Vajda et al., 1999; Miller and Bowman, 2004), although growth and modeling processes are also important. Bone remodeling is initiated when osteoclasts are activated, appear on a bone surface, and commence resorption. The resorption phase of bone remodeling is followed by a reversal period in which the osteoclasts leave the resorption domain and osteoblasts appear and ﬁll in the space created by the osteoclasts. The fate of the osteoclasts when the resorption phase is completed is not clear. The best anatomical example of bone remodeling is the formation of Haversian systems, or osteons, although a similar process can occur in the remodeling of cancellous bone. At the end of lactation, there is a rapid reversal of bone metabolism from predominantly bone resorption to a profound anabolic period characterized by greatly increased bone formation (Bowman et al., 2002; Miller and Bowman, 2004). Morphologically and functionally, this period appears to represent a classical bone remodeling reversal phase because osteogenesis rapidly commences on bone surfaces that were previously resorbing (Miller and Bowman, 2004). The initiation of this postlactational bone formation period in the maternal skeletal is characterized by a greatly increased proliferation of osteoblast progenitors resulting in a rapid expansion of osteoblasts on bone surfaces in the maternal skeleton (Miller et al., 2005). This results in greatly increased rates of bone formation with a rebuilding of the maternal skeleton and augmentation of bone strength (Vajda et al., 2001). Increases in serum markers of bone formation with increases in bone mineral density are also observed in humans after lactation (Lopez et al., 1996; Akesson et al., 2004). This postlactation phase of skeletal reconstitution is considered to be the most anabolic phase in the life history of the adult female skeleton (Bowman and Miller, 1999; Vajda et al., 2001). It has been hypothesized that the primary purpose of this profound postlactation anabolic period is to prepare the maternal skeleton for the next reproductive cycle (Bowman and Miller, 1999, 2001). There are a number of endocrine mediators that may be involved in mineral and skeletal metabolism during the reproductive cycle and include estradiol (E2), prolactin (PRL), vitamin D, parathyroid hormone (PTH), parathyroid hormone-related peptide (PTHrP), calcitonin (CT), gonadotrophins, and lactogens. Some of the changes that occur at weaning in the mother include a decrease in PRL and possibly PTHrP. There are some data suggesting roles for PRL (Lotinun et al., 1998) and PTHrP (DeMauro and Wysolmerski, 2005) in the regulation of skeletal metabolism during lactation. Levels of E2 are typically low during lactation, but will increase as the estrus (or menstrual) cycle is reinitiated after weaning with corresponding changes in progesterone. Furthermore, the reinitiation of menstrual cycles is associated with the restoration of bone mineral density in humans (Ritchie et al., 1998; Prentice, 2000). CT has been considered to be a vestigial calciotrophic hormone and a nonfunctional remnant in mammals from early evolution (Hirsch and Baruch, 2003). Some data suggest that CT may have a physiological role during periods of calcium stress (Hirsch and Baruch, 2003), with lactation being such a situation (Garel and Barlet, 1978). CT is produced in large quantities in the mammary gland during pregnancy with large amounts found in milk, suggesting a possible paracrine role in the mammary gland (Tverberg et al., 2000). CT also inhibits the secretion of PRL in lactating rats (Tohei et al., 2000) and has an antiproliferative action on pituitary lactotrophs that produce PRL (Wang et al., 2003). That CT may blunt the action of PRL led to the suggestion that CT helps maintain bone mass during lactation (Olgiati et al., 1982; Woodrow et al., 2006). While the role of endogenous CT in mammals is debated, the ability of exogenous CT to inactivate osteoclasts rapidly and slow bone resorption is well established. The ﬁrst purpose of this study was to determine the fate of the osteoclasts during the rapid reversal from bone resorption to bone formation that occurs during weaning. The changes in the osteoclast population were quantiﬁed by histomorphometry while the morphology and ultrastructure were assessed by histochemisty as well as light and electron microscopy. Apoptosis was established by immunocytochemistry. The second purpose of this study was to correlate these rapid skeletal changes with the reinitiation of estrus cycles and changes in some known endocrine mediators of bone and skeletal metabolism, including CT, PTH, PRL, and E2. MATERIALS AND METHODS Animals Female Sprague-Dawley rats (Charles River, Portage, MI) were obtained at 90–100 days of age and housed in a light- and temperature-controlled room. They were allowed to acclimate to their environment for several weeks, maintained on a 12-hr light/12-hr dark cycle and given free access to standard laboratory chow and water. Rats with at least ﬁve regular estrus cycles, established by vaginal smears, were mated with male breeders and the ﬁrst day of pregnancy was established by the presence of sperm in the vaginal smears. Following parturition, the number of pups was normalized to between 12 and 14 per dam and the rats were randomly sorted into the following groups: day 10 of lactation, which is about mid-lactation (day 10); day 19 of lactation (day 19), which was at the end of lactation, 1 day prior to weaning on day 20; 1 day following weaning (1 day postwean); 7 days following weaning (7 days postwean); and nonmated age-matched normal estrus-cycling females (designated as diestrus). Vaginal smears were continued during lactation and after weaning to determine the estrus status of the animals and to help determine if and when the estrus cycle was initiated after weaning and to correlate the vaginal cytology with endocrine proﬁles. Additionally, serum and tissues were collected from the nonmated normal estrus-cycling group during the second day of diestrus. The protocol was approved by the Institutional Animal Care and Use Committee at the University of Utah and the study conducted according to the National Institutes of Health Guidelines for the Care and Use of Laboratory Animals. Tissue Preparation for Light and Electron Microscopy Proximal tibias, distal femurs, and lumbar vertebral bodies were collected at necropsy, trimmed open to ex- MATERNAL OSTEOCLAST APOPTOSIS AT WEANING 67 pose the marrow cavities, and ﬁxed in 10% phosphatebuffered formalin for 24 hr at 48C. Bones were decalciﬁed in 14% EDTA, pH 7.4, for 4 weeks. The left femur and lumbar vertebrae 1 and 2 were dissected into smaller pieces and rinsed in 0.1 M sodium cacodylate buffer, postﬁxed in 1% osmium tetroxide, dehydrated, and embedded in epoxy resin for light and electron microscopy. Thick sections (about 1 mm) were cut, mounted on slides, and stained with toluidine blue for light microscopy. Thin sections were cut on an ultramicrotome and stained with uranyl acetate and lead citrate prior to viewing in a transmission electron microscope. The left tibia and the third and fourth lumbar vertebrae were rinsed with buffer and processed for parafﬁn embedding. Low-temperature parafﬁn was used and temperatures were kept below 608C. Tartrate-Resistant Acid Phosphatase (TRAP) Staining for Osteoclasts TRAP is a histochemical marker for osteoclasts in light microscopy. The parafﬁn sections were incubated in a media consisting of naphthol AS-BI phosphate, N,N-dimethyl formamide, and 50 mM L(þ)-tartaric acid in acetate buffer (pH 5.0). Fast Red Violet (LB) was included as the coupling agent giving the resulting red stain associated with the presence of acid phosphatase. Sections were counterstained with 1% aqueous fast green. All reagents were obtained from Sigma (St. Louis, MO). Cancellous Bone Osteoclast Perimeter The percentage of cancellous bone surfaces in the proximal tibia metaphysis covered by TRAP-stained osteoclasts was quantiﬁed. For this, the total perimeter and osteoclast surface perimeter of the cancellous bone surfaces in a 4.5 mm2 area adjacent to the growth plate and extending into the secondary spongiosa in the TRAP-stained parafﬁn sections were measured in at least one section from each animal. The sections were read blinded to the observer using a Bioquant image analysis system (R&M Biometrics, Nashville, TN). The surface perimeter covered by TRAP-stained osteoclasts is expressed as a % of the total cancellous bone perimeter. Apoptosis The presence of apoptotic cells was detected by the In Situ Oligo Ligation (ISOL) reaction using a commercial kit (ApopTag; Chemicon, Temucoula, CA). The ISOL reaction detects the 30 single-base overhang fragments of DNA. Diaminobenzidine was used as the chromogen substrate and results in a brown staining over the apoptotic nuclei, as observed by bright-ﬁeld light microscopy. Regressing mammary gland tissue was used as a positive control. Hormone and Calcium Assays Blood was collected by cardiac puncture, the serum separated by centrifugation and frozen at 208C until time of assay. Rat PTH (rPTH) was measured using a Rat Bioactive PTH ELISA (Immutopics, San Clemente, CA). CT was measured using a two-site immunoradiometric assay (Immutopics). Serum PRL was measured Fig. 1. The percentage of cancellous bone surface in the maternal proximal tibial metaphysis covered by TRAP-stained osteoclasts (% osteoclast perimeter) was measured at days 10 and 19 of lactation, days 1 and 7 after weaning, and compared with an age-matched normal estrus-cycling group taken at diestrus. Weaning occurred on day 20 of lactation. The percentage of the bone surface covered by osteoclasts was very high during lactation, as expected, but rapidly decreased by the ﬁrst day after weaning. Data are expressed as mean 6 SE. Asterisk, P < 0.05 compared with the lactation groups at day 10 and day 19. using a rat-speciﬁc enzyme immunoassay (Amersham Biosciences, Piscataway, NH). E2 was measured using a commercial RIA (Diagnostic Systems Laboratories, Webster, TX). Total serum calcium was measured by ﬂame atomic absorption spectrophotometry. All measurements were done in duplicate, and the mean value was used. Statistics Of particular interest were the speciﬁc changes that occurred during weaning. Thus, emphasis was placed on the comparisons of the data from late lactation (day 19) with the ﬁrst day after weaning (1 day postweaning). The data for all of the groups were ﬁrst tested for significance using an ANOVA with the individual groups of interest compared using the Tukey’s multiple-comparisons test. A level of P < 0.05 was considered as signiﬁcant and the data were expressed as mean 6 SE. RESULTS Osteoclast Populations During and After Lactation The relative perimeter of cancellous bone surfaces covered by osteoclasts was substantially greater during lactation when compared with nonmated normal estrus-cycling females (Fig. 1). At 24 hr after the pups were weaned, the percentage of maternal cancellous bone surface covered by osteoclasts decreased by about 80%. At 7 days after the end of lactation, the osteoclast surface remained low relative to both lactation values and those observed in nonmated normal estrus-cycling females. 68 MILLER AND BOWMAN Fig. 2. TRAP-positive osteoclasts were greatly increased on maternal cancellous bone surfaces during lactation, but rounded up and moved off of bone surfaces by the ﬁrst day after weaning. Many of these cells after weaning were undergoing apoptosis as indicated by ISOL labeling. A: TRAP staining of metaphyseal cancellous bone taken from a nonmated normal estrus-cycling rat. The osteoclasts are stained red and can be seen adjacent to the bone surfaces. B and C: TRAP-stained osteoclasts taken on day 19 of lactation, 1 day prior to the pups being weaned on day 20 of lactation. Many of the cancellous bone surfaces were covered by the TRAP-stained osteoclasts. D and E: TRAP-stained sections from metaphyseal cancellous bone taken 24 hr after weaning. The TRAP-stained cells were usually rounded up and fragments of TRAP-stained cells were often seen removed from the bone surfaces in the adjacent bone marrow. F: ISOL immunocytochemical reaction demonstrating apoptosis in the rounded multinucleated cells that were usually detached from the bone surface at 24 hr after weaning. Histological, Ultrastructural, and Apoptotic Changes in Maternal Osteoclasts With Weaning The presence of large numbers of osteoclasts on cancellous bone surfaces during lactation was also clearly evident when TRAP-stained sections from nonmated normal estrus-cycling animals (Fig. 2A) were compared with those at day 19 of lactation (Fig. 2B and C). During lactation, most cancellous bone surfaces were covered by Fig. 3. Light and electron microscope images of cancellous bone osteoclasts on day 19 of lactation and 24 hr after weaning. A: Toluidine blue-stained, 1 mm thick epoxy resin section illustrating a layer of osteoclasts (arrows) extending along a bone (B) surface at day 19 of lactation. The bone surface has a scalloped morphology, typical of eroding surfaces. B: A similarly prepared section taken at 24 hr after weaning. Several osteoclasts (double arrows) are partially detached from the bone surface and lack the foamy vacuolated appearance typically observed in active osteoclasts. C: Transmission electron micrograph (TEM) of the rufﬂed border region of an osteoclast adjacent to bone taken at day 19 of lactation. The rufﬂed border is typical of osteoclasts actively engaged in bone resorption as also illustrated in this image by the fraying of the adjacent collagenous matrix ﬁlaments. D: A TEM of a multinucleated osteoclast-like cell taken 24 hr after weaning. The cell has detached from the bone surface and lacks a rufﬂed border. In this case, the osteoclast is separated from the bone surface by several layers of thin bone-lining cell cytoplasmic extensions. TRAP-stained osteoclasts. At 1 day after weaning, most of the TRAP-stained osteoclasts were rounded, many were fragmented and partially or entirely detached from the bone surface (Fig. 2D and E). Many of these osteoclasts and associated TRAP-stained fragments in the bone marrow were undergoing apoptosis as indicated by the ISOL immunocytochemical reaction (Fig. 2F). When viewed histologically, osteoclasts were well extended along and lined most cancellous bone surfaces at day 19 of lactation (Fig. 3A). Many of the bone surfaces were scalloped with resorption pits (Howship’s lacunae), typical of active resorption surfaces. At 24 hr after MATERNAL OSTEOCLAST APOPTOSIS AT WEANING 69 the end of lactation, the osteoclasts were rounding up and retracting from bone surfaces (Fig. 3B). When observed by electron microscopy, the osteoclasts had well-developed rufﬂed borders adjacent to bone surfaces at day 19 of lactation (Fig. 3C). However, at 24 hr after the end of lactation, most maternal cancellous bone osteoclasts had lost their rufﬂed borders and were partially or completely retracted from the bone surface (Fig. 3D). Additionally, thinner, bone-lining cell cytoplasmic extensions were frequently observed separating the osteoclasts from the bone surface. These observations are consistent with the histomorphometric data on relative osteoclast surface (Fig. 1) as well as identiﬁcation by TRAP staining (Fig. 2) and apoptotic activity (Fig. 2). By 1 week after the end of lactation, most of the bone surfaces were lined with active osteoblasts that were actively forming new bone (not shown). Changes in Maternal Total Serum Calcium, Calcitonin, and Parathyroid Hormone Before and After Weaning Maternal total serum calcium levels were signiﬁcantly greater 1 day after weaning (11.3 6 0.5 mg/dl) than at the day prior to weaning, at day 19 of lactation (9.1 6 1.2). At day 10 and day 19 of lactation, maternal CT levels were about 20 and 18 pg/ml (Fig. 4A), which was less than that in nonmated normal estrus-cycling rats sampled at diestrus at about 60 pg/ml. The range of CT levels on day 19 of lactation was 8–39 pg/ml. However, at 24 hr after the end of lactation (1 day after weaning), the mean CT levels had increased by about a factor of 5 (Fig. 4A), but the range was quite large, from 57 to 106 pg/ml. By 1 week after weaning, the levels of CT had returned to those observed during lactation, but were less than observed in nonmated normal estrus-cycling rats taken at the second day of diestrus. The maternal levels of PTH were inverse to the changes observed with both total serum calcium and CT, with about a 55% decrease from day 19 of lactation to 1 day postweaning (Fig. 4B). Changes in Maternal Prolactin, Estrogen, and Indicators of Estrus Status Before and After Weaning Estradiol (E2) increased from mid-lactation (day 10) to late lactation (day 19) as expected (Fig. 5A). Vaginal cytology, however, conﬁrmed that all of the rats remained in diestrus through lactation up to and including the day of weaning on day 20 of lactation. At the day after weaning, E2 levels continued to increase and 2 of the 10 dams had already entered a proestrus phase, as assessed by vaginal cytology. At 1 week after weaning, all of the rats had reestablished estrus cycles and E2 levels varied but were consistent with the particular phase of the cycle and also similar to nonmating, normal, estrus-cycling, age-matched rats. Prolactin (PRL) was high during lactation, as expected, and decreased as lactation progressed (Fig. 5B). From day 19 of lactation to 1 day postweaning, PRL had decreased by over 50% and was further decreased at 1 week after the end of lactation. Fig. 4. Rat CT and rat PTH measured at days 10 and 19 of lactation, days 1 and 7 after weaning, and in a group of age-matched normal estrus-cycling animals taken at diestrus. A: Rat CT measured using an immunoradiometric assay was substantially greater at 1 day after weaning than during lactation, but was relatively low again by 7 days after weaning. Asterisk, signiﬁcantly greater than days 10 and 19 of lactation and 7 days postweaning (P < 0.05). B: Rat PTH was signiﬁcantly less (P < 0.05) at 1 day postweaning compared with days 10 and 19 of lactation. Data are expressed as the mean 6 SE. DISCUSSION The present study demonstrated that within 24 hr after a timed weaning, there was a dramatic change in the maternal osteoclast population that included morphological and ultrastructural evidence of osteoclast inactivation, including the disappearance of the rufﬂed borders, retraction from the bone surfaces, and apoptosis. We have recently demonstrated that this immediate postlactation period is also characterized by an increase in the formation of new osteoblasts from proliferating 70 MILLER AND BOWMAN Fig. 5. Maternal serum levels of estradiol (E2) and prolactin (PRL) measured at mid-lactation (day 10), late lactation (day 19), and 1 day after weaning. A: E2 was increased from mid-lactation through weaning and later became cyclic (not shown) as the estrus cycles were reinitiated. B: PRL decreased during lactation and after weaning, as expected. Asterisk, signiﬁcantly different from mid-lactation (day 10; P < 0.05). Data are expressed as the mean 6 SE. progenitors (Miller et al., 2005) and that this transition is reminiscent of a bone remodeling reversal where bone formation follows a period of bone resorption (Miller and Bowman, 2004). It is important to view these rapid changes in the maternal osteoclast population at the end of lactation in the context of the other changes that occur in the female skeleton prior to and during multiple reproductive cycles. There is accumulating evidence that the female skeleton preferentially accumulates bone mineral and skeletal mass on endocortical and endosteal surfaces during adolescence and puberty (Schoenau et al., 2001). In humans, this gain in skeletal mass is in excess of that needed for their particular level of physical activity (Schiessl et al., 1998; Lyritis et al., 2000). It is suggested that this excess skeletal mass is an evolutionary mechanism to ensure adequate mineral availability for the ﬁrst reproductive cycle (Bowman and Miller, 1999, 2001). This may occur because the ﬁrst reproductive cycle is considered to be metabolically less efﬁcient than later reproductions (Kunkele and Kenagy, 1997). After initiation of pregnancy, or even pseudopregnancy (Bowman and Miller, 1997), there is an accumulation of skeletal mass in a variety of mammals (Bowman and Miller, 2001). The utilization of this excess skeletal mass may begin as early as mid- to late pregnancy when the skeleton begins to form in the developing fetus. During lactation, the demand on the maternal mineral homeostatic system greatly increases and this is characterized by a period of high bone turnover (Miller et al., 1989) and typically a loss in skeletal mass regardless of maternal nutrition in experimental animals (Ellinger et al., 1952) and humans (Kalkwarf, 1999). It is during the postlactation period that the maternal skeleton rapidly rebuilds skeletal mineral stores and prepares the skeleton for the next reproductive period. While the rapidity of changes in the osteoclast population, structure and demise following weaning might seem unusual, rapid changes in osteoclast structure and function have been reported during different phases of reproductive cycles in other vertebrates. For example, it is known that there is a synchronization of bone resorption and formation during the avian egg-laying cycle that includes the rapid activation and inactivation of medullary bone osteoclasts associated with the state of egg shell calciﬁcation (Miller, 1977, 1981; van de Velde et al., 1984; Sugiyama and Kusuhara, 1993). In this situation, medullary bone osteoclasts are very active during egg shell calciﬁcation, but rapidly lose their rufﬂed borders and retract from the bone surface at the end of shell calciﬁcation. These inactive osteoclasts then remain dormant until the next phase of egg shell calciﬁcation, which in some birds may be within 8 to 12 hr. In the present study, however, many of the osteoclasts became apoptotic with the cessation of lactation after weaning, as indicated from both morphological and immunocytochemical criteria. There was a rapid decrease in the bone surface covered by osteoclasts after weaning observed in the present study. Histologically, bone-lining cells could be seen separating the osteoclasts from the surface and many of these surfaces later became bone formation domains covered by osteoblasts. We have previously demonstrated an increase in the formation of osteoblasts from proliferating progenitors immediately after weaning (Miller et al., 2005) with a subsequent bone formation phase that lasts about 2 months in the rat (Bowman et al., 2002). This postlactation bone formation phase may serve several purposes, including reconstitution of the maternal skeleton after lactation and to prepare the maternal skeleton for the next reproductive period. This MATERNAL OSTEOCLAST APOPTOSIS AT WEANING reversal from a bone resorption surface to a formation surface is reminiscent of a true bone remodeling cycle where bone resorption precedes bone formation on a speciﬁc bone surface in a bone basic multicellular unit (BMU) (Frost, 1990). If so, then there appears to be considerable synchronization of bone remodeling during the mammalian reproductive cycle to accommodate the maternal, fetal, and neonatal mineral needs during this period. The synchronization of bone remodeling activities during different phases of the reproductive cycle has been suggested from other studies conducted in experimental animals (Vajda et al., 1999). All of the endocrine changes that were observed in this study were consistent with the known or suspected individual actions of these hormones on bone resorption. However, the most dramatic and unexpected change was observed with circulating levels of CT. Maternal CT was increased by about ﬁvefold at 24 hr after the pups were weaned, but this was also accompanied by a signiﬁcant increase in total serum calcium levels, increased E2, and decreased PTH and PRL. Early studies demonstrated that exogenous CT could rapidly inhibit bone resorption by causing the osteoclast rufﬂed border rapidly to regress and disappear, with a subsequent decrease in the number of osteoclasts on bone surfaces (Kallio et al., 1972; Lucht, 1973). In vitro, the exposure of isolated, actively resorbing osteoclasts to physiological concentrations of CT resulted in cessation of lamellipoidial activity within minutes, followed by the retraction of the cell processes (Chambers and Magnus, 1982) and a decrease in the cell surface in contact with bone (Zaidi et al., 1992). CT administration also causes the rapid arrest in the synthesis and secretion of lysosomal enzymes (Lucht and Norgaard, 1977; Cao and Gay, 1985; Baron et al., 1990). The ability of CT to modulate osteoclast function rapidly is consistent with the high density of CT receptors found on the osteoclast surface (Nicholson et al., 1986). The stimulus for the large increase in CT that was observed at 1 day after weaning was not determined in this study, but it could have been in response to the increased in maternal serum calcium levels that was also observed at this same time. The increase in maternal serum calcium after weaning was likely due to the continued efﬂux of calcium from the bone into blood and the cessation of calcium uptake by the mammary gland. It would be presumed that this source of the increased circulating levels of CT at 24 hr after weaning would be from the C-cells of the thyroid gland. However, there is the intriguing possibility that the source of perhaps some of the CT may have been from the mammary gland itself. Large amounts, compared with serum concentrations, of CT are found in milk (Werner et al., 1982), and recent studies have demonstrated the production and secretion of CT from luminal epithelial cells in the gland (Ismail et al., 2004). The greatest concentrations of CT, however, were found in colostrum and then decrease as lactation continued. This is consistent with a greater expression of CT mRNA observed during mid- to late pregnancy; however, expression of the CT receptor continued through lactation (Tverberg et al., 2000). The possibility that the mammary gland may contribute to the serum pool of CT comes from earlier studies in thyroidectomized lactating women. In these studies, measurable amounts of serum CT could be detected in thyroi- 71 dectomized women during pregnancy and lactation, but not in thyroidectomized women in whom lactation had ceased (Bucht et al., 1986). While the changes that occur in the maternal osteoclast population, described here, are consistent with the known effects of CT, there were other endocrine changes that are also consistent with these observations. PTH was signiﬁcantly decreased, perhaps in response to the increased serum calcium levels. PTH is a well-established activator of bone resorption and causes the release of calcium from the bone that results in an increase in serum calcium levels. Thus, the decrease in serum PTH after weaning is consistent with the regression of the osteoclast population. While PTH is not absolutely required for a successful lactation (Halloran and DeLuca, 1979; Hodnett et al., 1992), there is growing evidence that this role might be ﬁlled by PTHrP produced in the mammary gland (Kovacs, 2005). We attempted to measure PTHrP in this study and while some of the measured values suggested a decrease after weaning, too many of the individual values were near or below the reliable minimum detectable levels of the assay and thus were not included in this study. PRL is classically associated with initiation and maintenance of lactation; however, over 300 individual functions of the hormone have now been identiﬁed (Gofﬁn et al., 1999), including important roles in skeletal metabolism. Short-term increases in PRL are believed to promote bone turnover (resorption and formation) while prolonged increases, e.g., hyperprolactinemia, are associated with uncoupling of remodeling with an excess bone resorption (Abraham et al., 2003). PRL also promotes the passive intestinal absorption of calcium (Krishnamra and Seemoung, 1996), perhaps contributing to the calcium economy during lactation (Quan-Sheng and Miller, 1989). Thus, the rapid inactivation of osteoclasts observed after weaning is also consistent with the expected and observed decreases in PRL. Estrogen is also known to decrease bone resorption and turnover, yet maintain bone mass, thus forming the pharmacological basis for traditional estrogen replacement therapies for osteoporosis (Krassas and Papadopoulou, 2001). E2 levels are typically low during lactation as menstrual and estrus cycles are suppressed. As lactation progresses, however, E2 typically increases, as also observed in the present study. Low levels of E2 during lactation are generally considered to be permissive of the greatly increased rates of bone turnover and resorption that occur to accommodate the mineral requirements during this period (Bowman and Miller, 2001). Likewise, the resumption of normal menstrual cycles after lactation in humans is associated with a restoration of bone mineral content and density (Ritchie et al., 1998). The highest circulating levels of E2 observed in this study were 24 hr after weaning and were greater than those observed after the animals reinitiated their estrus cycles. Estrogen receptors have been identiﬁed on both osteoclasts and osteoblasts, but at lower densities than found in most reproductive tissues (Oursler, 2003). Some direct and indirect effects of E2 on osteoclasts have been described and include the suppression of osteoclastic resorption, suppression of osteoclast formation, retraction of osteoclasts from bone surfaces, and an increase in cell fragmentation and apoptosis (Liu and Howard, 1991; Liu et al., 2002). All of the changes 72 MILLER AND BOWMAN in the osteoclast population described in the present study soon after weaning are consistent with the known effects of E2 and also those of CT, PRL, and PTH on this cell. In conclusion, this study demonstrates rapid changes in the maternal skeletal osteoclasts immediately following lactation. These changes include the disappearance of most osteoclast-rufﬂed borders, the retraction of the cells from the bone surface, cellular fragmentation, and apoptosis. These morphological, ultrastructural, and immunocytochemical changes observed in the osteoclasts are consistent with rapid endocrine changes that include a large but transient increase in CT and also E2 with decreases in PTH and PRL. These changes occur during the rapid transition from bone resorption during lactation to bone formation after lactation and are reminiscent of a true bone remodeling cycle. These observations suggest a synchronization of BMU-based bone remodeling activities to accommodate maternal mineral metabolism during the mammalian reproductive cycle. LITERATURE CITED Abraham G, Paing WW, Kaminski J, Joseph A, Kohegyi E, Josiassen RC. 2003. Effects of elevated serum prolactin on bone mineral density and bone metabolism in female patients with schizophrenia: a prospective study. Am J Psychiatry 160:1618–1620. Akesson A, Vahter M, Berglund M, Eklof T, Bremme K, Bjellerup P. 2004. Bone turnover from early pregnancy to postweaning. Acta Obstet Gynecol Scand 83:1049–1055. Baron R, Neff L, Brown W, Louvard D, Courtoy PJ. 1990. Selective internalization of the apical plasma membrane and rapid redistribution of lysosomal enzymes and mannose 6-phosphate receptors during osteoclast inactivation by calcitonin. J Cell Sci 97:439–447. Bowman BM, Miller SC. 1997. Endochondral bone growth during early pregnancy compared with pseudopregnancy in rats. Endocrine 6:173–177. Bowman BM, Miller SC. 1999. Skeletal mass, chemistry, and growth during and after multiple reproductive cycles in the rat. Bone 25:553–559. Bowman BM, Miller SC. 2001. Skeletal adaptations during mammalian reproduction. J Musculoskel Neuron Interact 1:347–355. 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. Bucht E, Telenius-Berg M, Lundell G, Sjoberg HE. 1986. Immunoextracted calcitonin in milk and plasma from totally thyroidectomized women: evidence of monomeric calcitonin in plasma during pregnancy and lactation. Acta Endocrinol (Copenh) 113:529–535. Cao H, Gay CV. 1985. Effects of parathyroid hormone and calcitonin on carbonic anhydrase location in osteoclasts of cultured embryonic chick bone. Experientia 41:1472–1474. Chambers TJ, Magnus CJ. 1982. Calcitonin alters behaviour of isolated osteoclasts. J Pathol 136:27–39. DeMauro S, Wysolmerski J. 2005. Hypercalcemia in breast cancer: an echo of bone mobilization during lactation? J Mammary Gland Biol Neoplasia 10:157–167. Ellinger GM, Duckworth J, Dalgarno AC, Quenouille MH. 1952. Skeletal changes during pregnancy and lactation in the rat: effect of different levels of dietary calcium. Br J Nutr 6:235–253. Frost HM. 1990. Skeletal structural adaptations to mechanical usage (SATMU): 2, redeﬁning Wolff ’s law—the remodeling problem. Anat Rec 226:414–422. Garel JM, Barlet JP. 1978. Calcitonin in the mother, fetus and newborn. Ann Biol Anim Bioch Biophys 18:53–68. Gofﬁn V, Touraine P, Pichard C, Bernichtein S, Kelly PA. 1999. Should prolactin be reconsidered as a therapeutic target in human breast cancer? Mol Cell Endocrinol 151:79–87. Halloran BP, DeLuca HF. 1979. Vitamin D deﬁciency and reproduction in rats. Science 204:73–74. Hirsch PF, Baruch H. 2003. Is calcitonin an important physiological substance? Endocrine 21:201–208. Hodnett DW, DeLuca HF, Jorgensen NA. 1992. Bone mineral loss during lactation occurs in absence of parathyroid tissue. Am J Physiol 262:E230–E233. Ismail PM, DeMayo FJ, Amato P, Lydon JP. 2004. Progesterone induction of calcitonin expression in the murine mammary gland. J Endocrinol 180:287–295. 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. Kallio DM, Garant PR, Minkin C. 1972. Ultrastructural effects of calcitonin on osteoclasts in tissue culture. J Ultrastruct Res 39:205–216. Kent GN, Price RI, Gutteridge DH, Smith M, Allen JR, Bhagat CI, Barnes MP, Hickling CJ, Retallack RW, Wilson SG, Devlin RD, Davies C, St John A. 1990. Human lactation: forearm trabecular bone loss, increased bone turnover, and renal conservation of calcium and inorganic phosphate with recovery of bone mass following weaning. J Bone Miner Res 5:361–369. Kovacs CS. 2005. Calcium and bone metabolism during pregnancy and lactation. J Mammary Gland Biol Neoplasia 10:105–118. Krassas GE, Papadopoulou P. 2001. Oestrogen action on bone cells. J Musculoskelet Neuronal Interact 2:143–151. Krishnamra N, Seemoung J. 1996. Effects of acute and long-term administration of prolactin on bone 45Ca uptake, calcium deposit, and calcium resorption in weaned, young, and mature rats. Can J Physiol Pharmacol 74:1157–1165. Kunkele J, Kenagy GJ. 1997. Inefﬁciency of lactation in primiparous rats: The costs of the ﬁrst reproduction. Physiol Zool 70:571– 577. Liu BY, Wu PW, Bringhurst FR, Wang JT. 2002. Estrogen inhibition of PTH-stimulated osteoclast formation and attachment in vitro: involvement of both PKA and PKC. Endocrinology 143:627–635. Liu CC, Howard GA. 1991. Bone-cell changes in estrogen-induced bone-mass increase in mice: dissociation of osteoclasts from bone surfaces. Anat Rec 229:240–250. Lopez JM, Gonzalez G, Reyes V, Campino C, Diaz S. 1996. Bone turnover and density in healthy women during breastfeeding and after weaning. Osteoporos Int 6:153–159. Lotinun S, Limlomwongse L, Krishnamra N. 1998. The study of a physiological signiﬁcance of prolactin in the regulation of calcium metabolism during pregnancy and lactation in rats. Can J Physiol Pharmacol 76:218–228. Lucht U. 1973. Effects of calcitonin on osteoclasts in vivo. Z Zellforsch Mikrosk Anat 145:75–87. Lucht U, Norgaard JO. 1977. Uptake of peroxidase by calcitonin inhibited osteoclasts. Histochemistry 54:143–148. Lyritis GP, Schoenau E, Skarantavos G. 2000. Osteopenic syndromes in the adolescent female. Ann NY Acad Sci 900:403–408. Miller MA, Omura TH, Miller SC. 1989. Increased cancellous bone remodeling during lactation in beagles. Bone 10:279–228. Miller SC. 1977. Osteoclast cell-surface changes during the egg-laying cycle in Japanese quail. J Cell Biol 75:104–118. Miller SC. 1981. Osteoclast cell-surface specializations and nuclear kinetics during egg-laying in Japanese quail. Am J Anat 162:35– 43. 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. Miller SC, Bowman BM. 2004. Rapid improvements in cortical bone dynamics and structure after lactation in established breeder rats. Anat Rec A Discov Mol Cell Evol Biol 276:143–149. Miller SC, Anderson BL, Bowman BM. 2005. Weaning initiates a rapid and powerful anabolic phase in the rat maternal skeleton. Biol Reprod 73:156–162. Nicholson GC, Moseley JM, Sexton PM, Mendelsohn FAO, Martin TJ. 1986. Abundant calcitonin receptors in isolated rat osteoclasts: biochemical and autoradiographic characterization. J Clin Invest 78:355–360. MATERNAL OSTEOCLAST APOPTOSIS AT WEANING Olgiati VR, Netti C, Guidobono F, Pecile A. 1982. High sensitivity to calcitonin of prolactin-secreting control in lactating rats. Endocrinology 111:641–644. Oursler MJ. 2003. Direct and indirect effects of estrogen on osteoclasts. J Musculoskelet Neuronal Interact 3:363–366. Prentice A. 2000. Calcium in pregnancy and lactation. Annu Rev Nutr 20:249–272. Quan-Sheng D, Miller SC. 1989. Calciotrophic hormone levels and calcium absorption during pregnancy in rats. Am J Physiol 257:E118–E123. Ritchie LD, Fung EB, Halloran BP, Turnlund JR, Van Loan MD, Cann CE, King JC. 1998. A longitudinal study of calcium homeostasis during human pregnancy and lactation and after resumption of menses. Am J Clin Nutr 67:693–701. Schiessl H, Frost HH, Jee WSS. 1998. Estrogen and bone-muscle strength and mass relationships. Bone 22:1–6. Schoenau E, Neu CM, Rauch F, Manz F. 2001. The development of bone strength at the proximal radius during childhood and adolescence. J Clin Endocrinol Metab 86:613–618. Sowers M, Corton G, Shapiro B, Jannausch ML, Crutchﬁeld M, Smith ML, Randolph JF, Hollis B. 1993. Changes in bone density with lactation. JAMA 269:3130–3135. Sugiyama T, Kusuhara S. 1993. Ultrastructural changes of osteoclasts on hen medullary bone during the egg-laying cycle. Br Poult Sci 34:471–477. Tohei A, VandeGarde B, Arbogast LA, Voogt JL. 2000. Calcitonin inhibition of prolactin secretion in lactating rats: mechanism of action. Neuroendocrinology 71:327–332. 73 Tverberg LA, Gustafson MF, Scott TL, Arzumanova IV, Provost ER, Yan AW, Rawie SA. 2000. Induction of calcitonin and calcitonin receptor expression in rat mammary tissue during pregnancy. Endocrinology 141:3696–3702. Vajda EG, Kneissel M, Muggenburg B, Miller SC. 1999. Increased intracortical bone remodeling during lactation in beagle dogs. Biol Reprod 61:1439–1444. 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. van de Velde JP, Vermeiden JP, Touw JJ, Veldhuijzen JP. 1984. Changes in activity of chicken medullary bone cell populations in relation to the egg-laying cycle. Metab Bone Dis Relat Res 5:191–193. Wang YQ, Yuan R, Sun YP, Lee TJ, Shah GV. 2003. Antiproliferative action of calcitonin on lactotrophs of the rat anterior pituitary gland: evidence for the involvement of transforming growth factor beta 1 in calcitonin action. Endocrinology 144:2164–2171. Werner S, Widstrom AM, Wahlberg V, Eneroth P, Winberg J. 1982. Immunoreactive calcitonin in maternal milk and serum in relation to prolactin and neurotensin. Early Hum Dev 6:77–82. Woodrow JP, Sharpe CJ, Fudge NJ, Hoff AO, Gagel RF, Kovacs CS. 2006. Calcitonin plays a critical role in regulating skeletal mineral metabolism during lactation. Endocrinology 147:4010–4021. Zaidi M, Alam AS, Shankar VS, Bax BE, Moonga BS, Bevis PJ, Pazianas M, Huang CL. 1992. A quantitative description of components of in vitro morphometric change in the rat osteoclast model: relationships with cellular function. Eur Biophys J 21:349–355.