Calcif Tissue Int (1992) 51:223-228
Calcified Tissue International 9 1992 Springer-Verlag New York Inc.
The Anabolic Effect of Estrogen on Endosteal Bone Formation in the Mouse is Attenuated by Ovariohysterectomy: A Role for the Uterus in the Skeletal Response to Estrogen? Steven D. Bain, Mason C. Bailey, and Martin W. Edwards Osteoporosis and Hard Tissue Repair Laboratory, ZymoGenetics, Inc., 4225 Roosevelt Way NE, Seattle, Washington 98105, USA Received December 13, 1991, and in revised form January 31, 1992
Summary. In the mouse, the anabolic effect of estrogen on
Key words: Bone formation - Ovariectomy - Ovariohyster-
the uterus and its stimulatory effect on endosteal bone formation are well documented. When these observations are coupled with the recent description of uterine-derived bone cell mitogens, it raises the possibility that uterine hypertrophy in response to estrogen might lead to the production and release of factors that participate in the skeleton's anabolic response to estrogen. To determine if the stimulatory effects of estrogen on endosteal bone formation and uterine tissue in the mouse are related, we have studied this specific skeletal response to ovariectomy (OVX) and ovariohysterectomy (OHTX), and to two levels of 17[3-estradiol (1713-E2). To assess treatment effects, 48 Swiss-webster mice were assigned to six groups: OHTX/oil vehicle, OVX/oil vehicle, OHTX/150 p~g 1713-E2, OHTX/300 izg 1713-E2, OVX/150 I~g 1713-E2, and OVX/300 ~Lg 1713-E2. Animals were treated once per week with vehicle or the respective 1713-E2 dose. To quantitate bone formation, fluorochrome labels were administered at the beginning and end of the experimental period. At the conclusion of the 5-week study, tibiae were processed undecalcified for embedding in methyl methacrylate plastic. Cross-sectional areal properties and bone formation rates were quantitated from 30 ~m mid-diaphyseal sections using a Bioquant Bone Morphometry system. Compared with the vehicle-treated OVX and OHTX mice, 150 ~g of 1713-E2 administered once per week significantly increased cortical bone areas (P < 0.05) but cortical bone widths and the ratio of cortical bone area to total bone area was increased only in estrogen-treated OVX mice (P < 0.01). The attenuation of bone formation in the OHTX mice was even more apparent in animals treated with 300 ~g 1713E2. Endosteal mineral apposition and bone formation, cortical bone widths, and cortical bone ratios were all significantly reduced in OHTX mice compared with OVX animals treated with the same 1713-E2 dose. Indeed, the 17[3-E2induced cortical bone increases in the OVX animals were reduced 50% by OHTX. These results suggest that the anabolic effects of high-dose 1713-E2 on endosteal bone formation in the mouse are modulated by estrogen's uterotrophic activity, and are therefore consistent with the hypothesis that the uterus may produce and release factors with the capacity to stimulate bone formation.
ectomy - Estrogen - Uterus.
In recent years, an increasing number of investigations have focused on elucidating the role of estrogen in the regulation of skeletal remodeling. The demonstration of estrogen receptors in bone tissue homogenates [1] and in bone cells [2, 3], as well as estrogen's ability to modulate bone cell function in vitro [4--6] and to stimulate bone formation in vivo [7], are part of an accumulating body of evidence that underscores estrogen's key role in skeletal metabolism. However, though these observations substantiate estrogen's capacity to influence bone directly, little is known about skeletal effects that might be mediated by estrogen indirectly via its action on other target organs. One estrogen-responsive tissue that may have the the capacity to modulate e s t r o g e n ' s effects on bone is the uterus. Hreshchyshyn et al. [8] have shown that bone densities in hysterectomized women are intermediate between normal subjects and oophorectomized patients. In addition, Koutsilieris [9] has demonstrated that acid extracts of human uterine tissue stimulate the proliferation of rat calvarial osteoblasts in a dose-dependent manner. Moreover, estrogen also stimulates IGF-1 production [10] and upregulates IGF-1 m R N A in the rat uterus [11], a hormone well known for its anabolic effects on bone [12-15]. Considered together, these observations implicate the uterus as a potentially active participant in the regulation of skeletal activity. To determine if estrogen effects on the uterus are associated with the skeleton's response to estrogen, we have studied the actions of this hormone in the mouse. In this species, estradiol is a potent inducer of uterine hypertrophy and stimulates endosteal bone formation [16--18]. Therefore, to evaluate the ability of estrogen-induced uterine hypertrophy to modulate the skeleton's anabolic response to estrogen, we have compared the endosteal bone response with estradiol in ovariectomized (OVX) and ovariohysterectomized (OHTX) mice.
Materials and Methods Animals and Experimental Design
Offprint requests to: S. D. Bain
Forty-eight, 8-week-old female Swiss-Webster mice (Simonsen Labs, Gilroy, CA) were acclimatized to wire-topped plastic cages
S. D. Bain et al.: Hysterectomy Decreases Bone Formation
224 for 2 weeks prior to the beginning of the study. During acclimatization and study periods, animals received a standard diet (Rodent BLOX #8604, Wayne Lab Animal Diets, Madison, WI) and water ad libitum, and were kept on an alternating 12-hour light/dark cycle. Following acclimatization, mice were randomly divided into six groups of eight mice each; three groups for ovariectomy plus hysterectomy (i.e., ovariohysterectomy, OHTX) and three groups for ovariectomy only (OVX). Prior to surgery, each animal was anesthetized with an i.p. injection of ketamine hydrochloride and xylazine. The abdomen was opened through a midline incision and the ovaries were located and removed. In the three groups undergoing OHTX, the base of the two uterine horns was ligated at the junction with the cervix before excision. Two days after the surgeries, animals began a treatment regimen consisting of a single weekly injection of either oil vehicle or 17[~-E2 for 5 consecutive weeks according to the following outline: OHTX/oil vehicle; OVX/oil vehicle; OHTX 150 ~g 1713-E2, OVX 150 ~g 17[~-E2, OHTX 300 p.g 1713-E2, and OVX 300 ~g 1713-E2. To document changes in bone formation during the experimental period, fluorochrome labels were administered i.p. at the initiation of the study (tetracycline 15 mg/kg), and at 7 and 2 days prior to sacrifice (calcein 15 mg/kg).
Tissue Collection and Preparation Two days after the final injection, mice were anesthetized with ether, blood was drawn via the orbital sinus, and sacrificed by cervical dislocation. The abdomen of each animal was opened to confirm the success of the initial surgery and, in the three groups with an intact uterus, the uterus was excised and weighed. Tibia were then removed, dissected free of soft tissue, and fixed in 70% ethanol (EtOH). After 24-hour fixation, specimens were dehydrated in an ascending series of EtOH, infiltrated in a methyl methacrylate solution, and embedded undecalcified in methyl methacrylate plastic [19]. Transverse, mid-diaphyseal sections 150 ~m thick were cut using a low-speed diamond-wheel saw (Struers Accutom-2, Torrance, CA) and hand ground to approximately 30 ~m. Ground sections were mounted on glass slides with Eukitt's medium (Calibrated Instruments, Inc., Ardsley, NY).
Bone Morphometry Histomorphometric parameters were determined using the Bioquant Bone Morphometry program (Biometrics, Inc., Nashville, TN) interfaced via a camera lucida with an Olympus BH-2 light/ epiflourescent microscope (Scientific Instruments, Inc., Redmond, WA). Total bone area (i.e., cross-sectional area enclosed by periosteum), medullary area, and cortical bone widths were measured by 40x magnification. Medullary bone area was subtracted from total bone area to calculate cortical bone area. In addition, cortical bone area was divided by total bone area to calculate the ratio of cortical bone to total cross-sectional bone area. Fluorochrome interlabel widths at the endosteal and periosteal surfaces were quantitated from the initial tetracycline label and the most distant portion of the calcein labels. This was necessary as resolution of both calcein labels was not possible in all specimens. The mean interlabel widths were divided by the appropriate interlabel time period to calculate periosteal and endosteal mineral apposition rates (MARp and MARe, respectively). Periosteal and endosteal bone formation rates (BFRp and BFRe, respectively) were determined by tracing the area of newly formed bone and dividing this area by the interlabel time period [20].
Statistical Analysis Analyses of body weights, uterine weights, and cortical bone histomorphometry were based on analysis of variance (ANOVA) using Statview statistical programs. When significance was indicated by the ANOVA, control and treatment means were compared using
Table 1. The effects of 1713-E2 on final body weights and uterine weights in OVX and OHTX mice
OVX/vehicle OHTX/vehicle OVX/150 p~g 1713-E2 OHTX/150 p~g 1713-E2 OVX/300 ~g 1713-E2 OHTX/300 ~g 17[~-E2
Final body weight (g)
Uterine weight (g)
36.3 35.6 35.1 34.5 34.0 34.2
0.077 --- 0.020 * 0.125 -+ 0.016 ~ * 0.198 --- 0.043 b *
- 2.8 +- 2.7 -+ 1.6 -+ 4.7 --- 1.4 +-- 2.9
Values are means +- SD, n = 8 mice per group * Not applicable. P < 0.01 significantly greater than OVX vehicle-treated mice b p < 0.01 significantly greater than OVX mice-treated with vehicle or 150 p.g 17[~-E2 Dunnett's multiple comparison procedure [21]. P values of less than 0.05 were considered significant.
Results Final body weights and uterine weights are s u m m a r i z e d in Table 1. There were no statistically significant differences b e t w e e n any treatment groups with respect to final b o d y weights. As e x p e c t e d , in the estrogen-treated animals with an i n t a c t u t e r u s , u t e r i n e w e i g h t i n c r e a s e d in a d o s e dependent manner. In the O H T X mice, the h y s t e r e c t o m y procedure was confirmed in all cases although in several animals hypertrophied cervical tissue at the uterine excision site was visible. Cortical bone h i s t o m o r p h o m e t r y data is s u m m a r i z e d in Table 2. C o m p a r e d with their respective controls, 150 and 300 ~g of 17[~-E2 increased cortical bone areas of the tibial mid-diaphysis in O V X and O H T X mice in a d o s e - d e p e n d e n t manner (Fig. 1). H o w e v e r , in mice treated with 150 p~g 1713E2, cortical bone widths and the ratio of cortical b o n e area to total bone area w e r e significantly increased only in the O V X group (Fig. 2). M o r e o v e r , the increases in b o n e widths and bone area ratios in the O H T X mice treated with 300 p~g 1713E2 were reduced 50% c o m p a r e d with O V X animals treated with the equivalent estradiol dose. Consistent with the static m o r p h o m e t r y of the diaphysis, the dynamic indices of b o n e formation (i.e., M A R e and B F R e ) w e r e also increased in a d o s e - d e p e n d e n t m a n n e r by 1713-E2 t r e a t m e n t (Fig. 3). F u r t h e r m o r e , w h e n c o m p a r e d with O V X mice treated with 300 ~g of 1713-E2, o v a r i e c t o m y combined with h y s t e r e c t o m y significantly d e c r e a s e d the endosteal bone response in animals treated with the same level of estradiol. The photomicrographs in Figure 4 d e m o n s t r a t e the morphological characteristics and f l u o r o c h r o m e labeling patterns of the cortical bone response of the different treatment groups. Interestingly, changes at the periosteal surface a p p e a r e d to be independent of estradiol's effects at the endosteal surface. T r e a t m e n t with 1713-E2 had no stimulatory effect on periosteal bone formation in either O V X or O H T X groups. H o w e v e r , w h e n c o m p a r e d with the effect of O V X alone, O H T X significantly reduced B F R p (Fig. 5). M A R p was not significantly affected by either 17f3-E2 t r e a t m e n t or the O V X and O H T X procedures.
Discussion
TM
Previously, we h a v e shown that 1713-E2 can stimulate a
S. D. Bain et al.: Hysterectomy Decreases Bone Formation
225
Table 2. Effects of 1713-E2 on cortical bone histomorphometry in the tibiae of OVX and OHTX mice
TBA (mm 2) Medullary area (mm 2) CBA (mm 1) CBW (mm) CBA/TBA BFRe (mm2/mm/day x 10 -3) MARe (ixm/day) BFRp (mmZ/mm/day x 1 0 - 3 ) c MARp (p.m/day)
OVX/Placebo
OHTX/Placebo
OVX/150 txg E2
OHTX/150 txg E2
OVX/300 Ixg E2
OHTX/300 Ixg E2
1.26 0.43 0.83 0.25 0.67 1.78 0.89 1.47 0.68
1.14 0.36 0.77 0.25 0.68 2.26 1.00 0.72 0.63
1.28 0.33 0.95 0.29 0.75 3.07 1.37 1.50 0.67
1.36 0.42 0.94 0.27 0.69 3.33 1.34 0.43 0.41
1.21 0.17 1.04 0.35 0.86 5.06 2.40 1.26 0.62
1.29 0.31 0.97 0.30 0.76 3.85 1.63 1.06 0.53
--- 0.07 -+ 0.04 -+ 0.03 -+ 0.01 -+ 0.02 -+ 0.23 -+ 0.10 -+ 0.27 - 0.06
-+ 0.03 + 0.02 -+ 0.03 -+ 0.01 -+ 0.01 -+ 0.34 -+ 0.08 -+- 0.17 b -+ 0.12
Values are means -+ SE, n = 8 mice per group Abbreviations: CBA = cortical bone area; TBA = total bone area; CBW = cortical bone width; BFR = bone formation rate; MAR = mineral apposition rate; e = endosteal; p = periosteal a Significantly different from respective placebo-treated control groups, P < 0.01
1.1 =
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1.0
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0.8'
0
0.7
I
I
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oil
150 p.g 17B-estradiol
300
Fig. 1. Cortical bone areas in OVX and OHTX mice are increased in a dose-dependent manner by 1713-estradiol; *P < 0.01. Vales represent means -+ SE, n = 8 mice per group.
d o s e - d e p e n d e n t increase in uterine weights and cortical bone areas of the tibiae in o v a r i e c t o m i z e d mice [18]. Though the increase in cortical b o n e area m a y represent the direct action of 1713-E2 on cortical b o n e formation, e v i d e n c e in the literature describing uterine-derived polypeptide growth factors [for r e v i e w see 22] and bone cell mitogens [9] raises the possibility that uterine h y p e r t r o p h y in r e s p o n s e to estrogen treatment might participate in the skeletal response to 1713E2. Therefore, as we could not rule out the possibility of an indirect effect o f estradiol on the stimulation of endosteal bone formation in this animal model, the skeletal r e s p o n s e to estrogen in O V X mice with that o f O H T X animals was compared. Results f r o m this c o m p a r i s o n indicate that the stimulation of endosteal bone formation in the m o u s e by 1713-E2 can be modulated by estradiol's uterotrophic action. The attenuation of 1713-E2's effect on cortical b o n e widths and bone area ratios, as well as d e c r e a s e s in endosteal mineral apposition and bone formation, w e r e consistently o b s e r v e d in
-+ 0.05 -+ 0.04 _+ 0.03 a --_ 0.01 a -+ 0.02 a -+ 0.35 a --- 0.13 - 0.16 + 0.07
-+ 0.06 -+ 0.02 - 0.04 a 4- 0.01 --- 0.01 --- 0.31 -+ 0.08 --- 0.08 b --- 0.02
-+ 0.05 -+ 0.03 a -+ 0.04 a --- 0.02 a - 0.02" -+ 0.50 a -+- 0.32 a -+ 0.26 --+-0.11
+ 0.06 --- 0.05 b --- 0.04 a -+ 0.02 ~'b -2-_0.03 ",b -+ 0.53 ~'b +- 0.29 "'b --- 0.23 --- 0.05
b Significantly different from OVX animals treated with the an equivalent estradiol dose or with placebo, P < 0.01 The main effect of OHTX was to significantly reduce BFRp compared with OVX mice (see Fig. 5)
O H T X mice treated with 1713-E2. M o r e o v e r , the changes in bone formation at the periosteal surface, which w e r e independent of estradiol treatment, indicate that O H T X alone may have significant effects on bone formation. Thus, these observations would appear to be consistent with the hypothesis that the uterus m a y produce and release factors in response to 1713-E2 that can participate in the regulation of cortical bone formation in the murine system. Though the aim of this investigation was not to elucidate which factor(s) might mediate a putative uterine-bone interaction, it is interesting to postulate a role for insulin-like growth factor-1 (IGF-1) in this process. Estradiol significantly increases IGF-1 m R N A in bone [23] and uterine tissue [10, 11] in vivo. In addition, the intraarterial infusion of IGF-1 into the rat hindlimb stimulates cortical bone formation [15]. Thus, given the capacity of IGF-1 to act as both an endocrine or paracrine factor in b o n e cell function (14), it is conceivable that increased systemic levels of IGF-1 as a consequence of 17[3-E2-induced uterine h y p e r t r o p h y could interact with estradiol's local effect to increase endosteal b o n e formation. The effects of O H T X at the periosteal surface of the tibiae, which appear to be independent of the action of 1713-E2 at the endosteal surface, are consistent with the hypothesis that the periosteal and endosteal e n v e l o p e s in bone are regulated independently [24, 25]. This regional specificity is further exemplified in the m o u s e by studies demonstrating that the actions of 1713-E2 on bone formation and resorption are dose d e p e n d e n t and site specific [26]. Regardless of the mechanisms that control this p h e n o m e n o n , the a b s e n c e of the uterus undoubtedly alters periosteal b o n e modeling and suggests a role for the uterus in the endocrine regulation of bone growth at this surface. H y s t e r e c t o m y increases total R N A t u r n o v e r in the anterior pituitary of adult rats [27], and uterine extracts can also modulate the production of pituitary gonadotropin [28, 29]. H o w e v e r , nothing is k n o w n of how uterine-pituitary interactions regulate skeletal activity. Obviously, an issue of importance to h u m a n medicine is w h e t h e r the protective effects of estrogen on the female skeleton are c o m p r o m i s e d by h y s t e r e c t o m y . T h o u g h estrogen r e p l a c e m e n t therapy in h y s t e r e c t o m i z e d w o m e n appears to remain effective in reducing fracture incidence [30], w o m e n w h o h a v e u n d e r g o n e h y s t e r e c t o m y do h a v e a d e c r e a s e d bone mass c o m p a r e d with normal subjects [8]. T h e s e data notwithstanding, the relationship b e t w e e n bone mass, uter-
226
S.D. Bain et al.: Hysterectomy Decreases Bone Formation
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B 0.38 -
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4
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/
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0.32
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0.26
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0.24
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Fig. 2. Treatment of OVX and OHTX mice with 1713-E2 increased cortical bone widths (A), and the ratio of cortical bone areas to total bone areas (B) in a dose-dependent manner. However, when compared with their respective vehicle-treated control groups, increases in bone widths and area ratios in OHTX mice were significantly only in animals treated with the highest 1713-E2 dose. Furthermore, the increases in bone widths and area ratios in OHTX stimulated by 300
I
150 l.tg 178-estradiol
I
300
Ixg of 1713-E2 were significantly reduced compared with OVX animals treated with the same estradiol dose. Values represent means -+ SE, n = 8 mice per group, a = P < 0.01 significantly increased compared with vehicle-treated controls and b = P < 0.01 significantly reduced compared with OVX mice treated with the equivalent dose of estradiol.
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Calcified Tissue International 9 1992 Springer-Verlag New York Inc.
The Anabolic Effect of Estrogen on Endosteal Bone Formation in the Mouse is Attenuated by Ovariohysterectomy: A Role for the Uterus in the Skeletal Response to Estrogen? Steven D. Bain, Mason C. Bailey, and Martin W. Edwards Osteoporosis and Hard Tissue Repair Laboratory, ZymoGenetics, Inc., 4225 Roosevelt Way NE, Seattle, Washington 98105, USA Received December 13, 1991, and in revised form January 31, 1992
Summary. In the mouse, the anabolic effect of estrogen on
Key words: Bone formation - Ovariectomy - Ovariohyster-
the uterus and its stimulatory effect on endosteal bone formation are well documented. When these observations are coupled with the recent description of uterine-derived bone cell mitogens, it raises the possibility that uterine hypertrophy in response to estrogen might lead to the production and release of factors that participate in the skeleton's anabolic response to estrogen. To determine if the stimulatory effects of estrogen on endosteal bone formation and uterine tissue in the mouse are related, we have studied this specific skeletal response to ovariectomy (OVX) and ovariohysterectomy (OHTX), and to two levels of 17[3-estradiol (1713-E2). To assess treatment effects, 48 Swiss-webster mice were assigned to six groups: OHTX/oil vehicle, OVX/oil vehicle, OHTX/150 p~g 1713-E2, OHTX/300 izg 1713-E2, OVX/150 I~g 1713-E2, and OVX/300 ~Lg 1713-E2. Animals were treated once per week with vehicle or the respective 1713-E2 dose. To quantitate bone formation, fluorochrome labels were administered at the beginning and end of the experimental period. At the conclusion of the 5-week study, tibiae were processed undecalcified for embedding in methyl methacrylate plastic. Cross-sectional areal properties and bone formation rates were quantitated from 30 ~m mid-diaphyseal sections using a Bioquant Bone Morphometry system. Compared with the vehicle-treated OVX and OHTX mice, 150 ~g of 1713-E2 administered once per week significantly increased cortical bone areas (P < 0.05) but cortical bone widths and the ratio of cortical bone area to total bone area was increased only in estrogen-treated OVX mice (P < 0.01). The attenuation of bone formation in the OHTX mice was even more apparent in animals treated with 300 ~g 1713E2. Endosteal mineral apposition and bone formation, cortical bone widths, and cortical bone ratios were all significantly reduced in OHTX mice compared with OVX animals treated with the same 1713-E2 dose. Indeed, the 17[3-E2induced cortical bone increases in the OVX animals were reduced 50% by OHTX. These results suggest that the anabolic effects of high-dose 1713-E2 on endosteal bone formation in the mouse are modulated by estrogen's uterotrophic activity, and are therefore consistent with the hypothesis that the uterus may produce and release factors with the capacity to stimulate bone formation.
ectomy - Estrogen - Uterus.
In recent years, an increasing number of investigations have focused on elucidating the role of estrogen in the regulation of skeletal remodeling. The demonstration of estrogen receptors in bone tissue homogenates [1] and in bone cells [2, 3], as well as estrogen's ability to modulate bone cell function in vitro [4--6] and to stimulate bone formation in vivo [7], are part of an accumulating body of evidence that underscores estrogen's key role in skeletal metabolism. However, though these observations substantiate estrogen's capacity to influence bone directly, little is known about skeletal effects that might be mediated by estrogen indirectly via its action on other target organs. One estrogen-responsive tissue that may have the the capacity to modulate e s t r o g e n ' s effects on bone is the uterus. Hreshchyshyn et al. [8] have shown that bone densities in hysterectomized women are intermediate between normal subjects and oophorectomized patients. In addition, Koutsilieris [9] has demonstrated that acid extracts of human uterine tissue stimulate the proliferation of rat calvarial osteoblasts in a dose-dependent manner. Moreover, estrogen also stimulates IGF-1 production [10] and upregulates IGF-1 m R N A in the rat uterus [11], a hormone well known for its anabolic effects on bone [12-15]. Considered together, these observations implicate the uterus as a potentially active participant in the regulation of skeletal activity. To determine if estrogen effects on the uterus are associated with the skeleton's response to estrogen, we have studied the actions of this hormone in the mouse. In this species, estradiol is a potent inducer of uterine hypertrophy and stimulates endosteal bone formation [16--18]. Therefore, to evaluate the ability of estrogen-induced uterine hypertrophy to modulate the skeleton's anabolic response to estrogen, we have compared the endosteal bone response with estradiol in ovariectomized (OVX) and ovariohysterectomized (OHTX) mice.
Materials and Methods Animals and Experimental Design
Offprint requests to: S. D. Bain
Forty-eight, 8-week-old female Swiss-Webster mice (Simonsen Labs, Gilroy, CA) were acclimatized to wire-topped plastic cages
S. D. Bain et al.: Hysterectomy Decreases Bone Formation
224 for 2 weeks prior to the beginning of the study. During acclimatization and study periods, animals received a standard diet (Rodent BLOX #8604, Wayne Lab Animal Diets, Madison, WI) and water ad libitum, and were kept on an alternating 12-hour light/dark cycle. Following acclimatization, mice were randomly divided into six groups of eight mice each; three groups for ovariectomy plus hysterectomy (i.e., ovariohysterectomy, OHTX) and three groups for ovariectomy only (OVX). Prior to surgery, each animal was anesthetized with an i.p. injection of ketamine hydrochloride and xylazine. The abdomen was opened through a midline incision and the ovaries were located and removed. In the three groups undergoing OHTX, the base of the two uterine horns was ligated at the junction with the cervix before excision. Two days after the surgeries, animals began a treatment regimen consisting of a single weekly injection of either oil vehicle or 17[~-E2 for 5 consecutive weeks according to the following outline: OHTX/oil vehicle; OVX/oil vehicle; OHTX 150 ~g 1713-E2, OVX 150 ~g 17[~-E2, OHTX 300 p.g 1713-E2, and OVX 300 ~g 1713-E2. To document changes in bone formation during the experimental period, fluorochrome labels were administered i.p. at the initiation of the study (tetracycline 15 mg/kg), and at 7 and 2 days prior to sacrifice (calcein 15 mg/kg).
Tissue Collection and Preparation Two days after the final injection, mice were anesthetized with ether, blood was drawn via the orbital sinus, and sacrificed by cervical dislocation. The abdomen of each animal was opened to confirm the success of the initial surgery and, in the three groups with an intact uterus, the uterus was excised and weighed. Tibia were then removed, dissected free of soft tissue, and fixed in 70% ethanol (EtOH). After 24-hour fixation, specimens were dehydrated in an ascending series of EtOH, infiltrated in a methyl methacrylate solution, and embedded undecalcified in methyl methacrylate plastic [19]. Transverse, mid-diaphyseal sections 150 ~m thick were cut using a low-speed diamond-wheel saw (Struers Accutom-2, Torrance, CA) and hand ground to approximately 30 ~m. Ground sections were mounted on glass slides with Eukitt's medium (Calibrated Instruments, Inc., Ardsley, NY).
Bone Morphometry Histomorphometric parameters were determined using the Bioquant Bone Morphometry program (Biometrics, Inc., Nashville, TN) interfaced via a camera lucida with an Olympus BH-2 light/ epiflourescent microscope (Scientific Instruments, Inc., Redmond, WA). Total bone area (i.e., cross-sectional area enclosed by periosteum), medullary area, and cortical bone widths were measured by 40x magnification. Medullary bone area was subtracted from total bone area to calculate cortical bone area. In addition, cortical bone area was divided by total bone area to calculate the ratio of cortical bone to total cross-sectional bone area. Fluorochrome interlabel widths at the endosteal and periosteal surfaces were quantitated from the initial tetracycline label and the most distant portion of the calcein labels. This was necessary as resolution of both calcein labels was not possible in all specimens. The mean interlabel widths were divided by the appropriate interlabel time period to calculate periosteal and endosteal mineral apposition rates (MARp and MARe, respectively). Periosteal and endosteal bone formation rates (BFRp and BFRe, respectively) were determined by tracing the area of newly formed bone and dividing this area by the interlabel time period [20].
Statistical Analysis Analyses of body weights, uterine weights, and cortical bone histomorphometry were based on analysis of variance (ANOVA) using Statview statistical programs. When significance was indicated by the ANOVA, control and treatment means were compared using
Table 1. The effects of 1713-E2 on final body weights and uterine weights in OVX and OHTX mice
OVX/vehicle OHTX/vehicle OVX/150 p~g 1713-E2 OHTX/150 p~g 1713-E2 OVX/300 ~g 1713-E2 OHTX/300 ~g 17[~-E2
Final body weight (g)
Uterine weight (g)
36.3 35.6 35.1 34.5 34.0 34.2
0.077 --- 0.020 * 0.125 -+ 0.016 ~ * 0.198 --- 0.043 b *
- 2.8 +- 2.7 -+ 1.6 -+ 4.7 --- 1.4 +-- 2.9
Values are means +- SD, n = 8 mice per group * Not applicable. P < 0.01 significantly greater than OVX vehicle-treated mice b p < 0.01 significantly greater than OVX mice-treated with vehicle or 150 p.g 17[~-E2 Dunnett's multiple comparison procedure [21]. P values of less than 0.05 were considered significant.
Results Final body weights and uterine weights are s u m m a r i z e d in Table 1. There were no statistically significant differences b e t w e e n any treatment groups with respect to final b o d y weights. As e x p e c t e d , in the estrogen-treated animals with an i n t a c t u t e r u s , u t e r i n e w e i g h t i n c r e a s e d in a d o s e dependent manner. In the O H T X mice, the h y s t e r e c t o m y procedure was confirmed in all cases although in several animals hypertrophied cervical tissue at the uterine excision site was visible. Cortical bone h i s t o m o r p h o m e t r y data is s u m m a r i z e d in Table 2. C o m p a r e d with their respective controls, 150 and 300 ~g of 17[~-E2 increased cortical bone areas of the tibial mid-diaphysis in O V X and O H T X mice in a d o s e - d e p e n d e n t manner (Fig. 1). H o w e v e r , in mice treated with 150 p~g 1713E2, cortical bone widths and the ratio of cortical b o n e area to total bone area w e r e significantly increased only in the O V X group (Fig. 2). M o r e o v e r , the increases in b o n e widths and bone area ratios in the O H T X mice treated with 300 p~g 1713E2 were reduced 50% c o m p a r e d with O V X animals treated with the equivalent estradiol dose. Consistent with the static m o r p h o m e t r y of the diaphysis, the dynamic indices of b o n e formation (i.e., M A R e and B F R e ) w e r e also increased in a d o s e - d e p e n d e n t m a n n e r by 1713-E2 t r e a t m e n t (Fig. 3). F u r t h e r m o r e , w h e n c o m p a r e d with O V X mice treated with 300 ~g of 1713-E2, o v a r i e c t o m y combined with h y s t e r e c t o m y significantly d e c r e a s e d the endosteal bone response in animals treated with the same level of estradiol. The photomicrographs in Figure 4 d e m o n s t r a t e the morphological characteristics and f l u o r o c h r o m e labeling patterns of the cortical bone response of the different treatment groups. Interestingly, changes at the periosteal surface a p p e a r e d to be independent of estradiol's effects at the endosteal surface. T r e a t m e n t with 1713-E2 had no stimulatory effect on periosteal bone formation in either O V X or O H T X groups. H o w e v e r , w h e n c o m p a r e d with the effect of O V X alone, O H T X significantly reduced B F R p (Fig. 5). M A R p was not significantly affected by either 17f3-E2 t r e a t m e n t or the O V X and O H T X procedures.
Discussion
TM
Previously, we h a v e shown that 1713-E2 can stimulate a
S. D. Bain et al.: Hysterectomy Decreases Bone Formation
225
Table 2. Effects of 1713-E2 on cortical bone histomorphometry in the tibiae of OVX and OHTX mice
TBA (mm 2) Medullary area (mm 2) CBA (mm 1) CBW (mm) CBA/TBA BFRe (mm2/mm/day x 10 -3) MARe (ixm/day) BFRp (mmZ/mm/day x 1 0 - 3 ) c MARp (p.m/day)
OVX/Placebo
OHTX/Placebo
OVX/150 txg E2
OHTX/150 txg E2
OVX/300 Ixg E2
OHTX/300 Ixg E2
1.26 0.43 0.83 0.25 0.67 1.78 0.89 1.47 0.68
1.14 0.36 0.77 0.25 0.68 2.26 1.00 0.72 0.63
1.28 0.33 0.95 0.29 0.75 3.07 1.37 1.50 0.67
1.36 0.42 0.94 0.27 0.69 3.33 1.34 0.43 0.41
1.21 0.17 1.04 0.35 0.86 5.06 2.40 1.26 0.62
1.29 0.31 0.97 0.30 0.76 3.85 1.63 1.06 0.53
--- 0.07 -+ 0.04 -+ 0.03 -+ 0.01 -+ 0.02 -+ 0.23 -+ 0.10 -+ 0.27 - 0.06
-+ 0.03 + 0.02 -+ 0.03 -+ 0.01 -+ 0.01 -+ 0.34 -+ 0.08 -+- 0.17 b -+ 0.12
Values are means -+ SE, n = 8 mice per group Abbreviations: CBA = cortical bone area; TBA = total bone area; CBW = cortical bone width; BFR = bone formation rate; MAR = mineral apposition rate; e = endosteal; p = periosteal a Significantly different from respective placebo-treated control groups, P < 0.01
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Fig. 1. Cortical bone areas in OVX and OHTX mice are increased in a dose-dependent manner by 1713-estradiol; *P < 0.01. Vales represent means -+ SE, n = 8 mice per group.
d o s e - d e p e n d e n t increase in uterine weights and cortical bone areas of the tibiae in o v a r i e c t o m i z e d mice [18]. Though the increase in cortical b o n e area m a y represent the direct action of 1713-E2 on cortical b o n e formation, e v i d e n c e in the literature describing uterine-derived polypeptide growth factors [for r e v i e w see 22] and bone cell mitogens [9] raises the possibility that uterine h y p e r t r o p h y in r e s p o n s e to estrogen treatment might participate in the skeletal response to 1713E2. Therefore, as we could not rule out the possibility of an indirect effect o f estradiol on the stimulation of endosteal bone formation in this animal model, the skeletal r e s p o n s e to estrogen in O V X mice with that o f O H T X animals was compared. Results f r o m this c o m p a r i s o n indicate that the stimulation of endosteal bone formation in the m o u s e by 1713-E2 can be modulated by estradiol's uterotrophic action. The attenuation of 1713-E2's effect on cortical b o n e widths and bone area ratios, as well as d e c r e a s e s in endosteal mineral apposition and bone formation, w e r e consistently o b s e r v e d in
-+ 0.05 -+ 0.04 _+ 0.03 a --_ 0.01 a -+ 0.02 a -+ 0.35 a --- 0.13 - 0.16 + 0.07
-+ 0.06 -+ 0.02 - 0.04 a 4- 0.01 --- 0.01 --- 0.31 -+ 0.08 --- 0.08 b --- 0.02
-+ 0.05 -+ 0.03 a -+ 0.04 a --- 0.02 a - 0.02" -+ 0.50 a -+- 0.32 a -+ 0.26 --+-0.11
+ 0.06 --- 0.05 b --- 0.04 a -+ 0.02 ~'b -2-_0.03 ",b -+ 0.53 ~'b +- 0.29 "'b --- 0.23 --- 0.05
b Significantly different from OVX animals treated with the an equivalent estradiol dose or with placebo, P < 0.01 The main effect of OHTX was to significantly reduce BFRp compared with OVX mice (see Fig. 5)
O H T X mice treated with 1713-E2. M o r e o v e r , the changes in bone formation at the periosteal surface, which w e r e independent of estradiol treatment, indicate that O H T X alone may have significant effects on bone formation. Thus, these observations would appear to be consistent with the hypothesis that the uterus m a y produce and release factors in response to 1713-E2 that can participate in the regulation of cortical bone formation in the murine system. Though the aim of this investigation was not to elucidate which factor(s) might mediate a putative uterine-bone interaction, it is interesting to postulate a role for insulin-like growth factor-1 (IGF-1) in this process. Estradiol significantly increases IGF-1 m R N A in bone [23] and uterine tissue [10, 11] in vivo. In addition, the intraarterial infusion of IGF-1 into the rat hindlimb stimulates cortical bone formation [15]. Thus, given the capacity of IGF-1 to act as both an endocrine or paracrine factor in b o n e cell function (14), it is conceivable that increased systemic levels of IGF-1 as a consequence of 17[3-E2-induced uterine h y p e r t r o p h y could interact with estradiol's local effect to increase endosteal b o n e formation. The effects of O H T X at the periosteal surface of the tibiae, which appear to be independent of the action of 1713-E2 at the endosteal surface, are consistent with the hypothesis that the periosteal and endosteal e n v e l o p e s in bone are regulated independently [24, 25]. This regional specificity is further exemplified in the m o u s e by studies demonstrating that the actions of 1713-E2 on bone formation and resorption are dose d e p e n d e n t and site specific [26]. Regardless of the mechanisms that control this p h e n o m e n o n , the a b s e n c e of the uterus undoubtedly alters periosteal b o n e modeling and suggests a role for the uterus in the endocrine regulation of bone growth at this surface. H y s t e r e c t o m y increases total R N A t u r n o v e r in the anterior pituitary of adult rats [27], and uterine extracts can also modulate the production of pituitary gonadotropin [28, 29]. H o w e v e r , nothing is k n o w n of how uterine-pituitary interactions regulate skeletal activity. Obviously, an issue of importance to h u m a n medicine is w h e t h e r the protective effects of estrogen on the female skeleton are c o m p r o m i s e d by h y s t e r e c t o m y . T h o u g h estrogen r e p l a c e m e n t therapy in h y s t e r e c t o m i z e d w o m e n appears to remain effective in reducing fracture incidence [30], w o m e n w h o h a v e u n d e r g o n e h y s t e r e c t o m y do h a v e a d e c r e a s e d bone mass c o m p a r e d with normal subjects [8]. T h e s e data notwithstanding, the relationship b e t w e e n bone mass, uter-
226
S.D. Bain et al.: Hysterectomy Decreases Bone Formation
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Fig. 2. Treatment of OVX and OHTX mice with 1713-E2 increased cortical bone widths (A), and the ratio of cortical bone areas to total bone areas (B) in a dose-dependent manner. However, when compared with their respective vehicle-treated control groups, increases in bone widths and area ratios in OHTX mice were significantly only in animals treated with the highest 1713-E2 dose. Furthermore, the increases in bone widths and area ratios in OHTX stimulated by 300
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Ixg of 1713-E2 were significantly reduced compared with OVX animals treated with the same estradiol dose. Values represent means -+ SE, n = 8 mice per group, a = P < 0.01 significantly increased compared with vehicle-treated controls and b = P < 0.01 significantly reduced compared with OVX mice treated with the equivalent dose of estradiol.
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