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Rapid inactivation and apoptosis of osteoclasts in the maternal skeleton during the bone remodeling reversal at the end of lactation.

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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
Division of Radiobiology, Department of Radiology, School of Medicine,
University of Utah, Salt Lake City, Utah
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:
Received 26 July 2006; Accepted 11 October 2006
DOI 10.1002/ar.a.20403
Published online 28 November 2006 in Wiley InterScience
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,
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.
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-
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
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.
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.
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.
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
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
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.
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
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
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.,
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-
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
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
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.
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end, rapid, apoptosis, lactation, reversal, inactivation, skeleton, remodeling, maternal, bones, osteoclast
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