0013-7227/79/1044-1188$02.00/0 Endocrinology Copyright © 1979 by The Endocrine Society
Vol. 104, No. 4 Printed in U.S.A.
The Effect of Long Term Estrogen Administration on Bone Metabolism in the Female Rat* R. L. CRUESS AND K. C. HONG Orthopedic Research Laboratories, Royal Victoria Hospital, McGill University, Montreal, Quebec, Canada
ABSTRACT. To determine the long term effect of estrogen administration upon some biochemical indices of bone formation and resorption as well as serum calcium and phosphorus, the following experiments were carried out. Female rats were divided into the following groups: intact, intact and estrogen-treated, oophorectomized, and oophorectomized and estrogen-treated. Treatment consisted of 400 /xg 17/8-estradiol/100 g BW twice a week for 1-12 months. Estrogen administration to the intact animals coused no consistent significant changes in any of the parameters measured. It was found that oophorectomy caused a decrease in the serum calcium concentration, a decrease in ash content, an increase in the total hydroxyproline content, an increase in incorporation of [3H]proline into collagen, a decrease in total hexosamine, an increase in the incorporation of [14C]glucose into
D
ESPITE the widespread therapeutic use of estrogen in the management of osteoporosis, its effect on bone metabolism still remains to be clarified. Albright (1) first reported the clinical entity of postmenopausal osteoporosis and many investigators have recommended estrogen in the therapy of this disorder (2-4). It is well established that estrogen can prevent or significantly decrease the loss of bone mass in the postmenopausal female (5, 6), but the beneficial effect of its use in the treatment of established postmenopausal osteoporosis is only temporary (7), with a compensatory decrease in bone formation at least partially offsetting the useful effect of the hormone on bone resorption (8). To date, no long term experiment reporting the effect of estrogen on bone metabolism in rats under experimental conditions has appeared. To obtain biochemical indices of rates of bone formation and resorption as well as compositional data on bone itself, the following long term experiments were carried out.
hexosamine, an increase in the uptake of 4r>Ca into bone, and an increase in bone collagenolytic activity. The administration of estrogen to the oophorectomized rat corrected the serum calcium changes to normal and returned the decreased ash content, collagen, and hexosamine contents to normal. Estrogen administration only partially reversed the increased uptake of [3H]proline into collagen and the increased incorporation of [I4C]glucose into hexosamine. The administration of estrogen to oophorectomized animals also returned the 4r'Ca uptake of bone to normal and caused normalization of the bone collagenolytic activity. The data indicate a sustained decrease in serum calcium and an increase in both bone formation and resorption in the oophorectomized animal. The administration of estrogen largely corrects these abnormalities and the beneficial effects are sustained over a 12-month period. (Endocrinology 104: 1188, 1979)
Materials and Methods
Received August 23, 1977. Address all correspondence and requests for reprints to: Dr. R. L. Cruess, Orthopedic Surgeon-in-Charge, Royal Victoria Hospital, 687 Pine Avenue West, Montreal, P.Q. H3A 1A1, Canada. * This work was supported by Grant MA-1571 from the MRC of Canada.
Female rats (150 g) were divided into four groups. The first group was left intact and served as control. Group 2 included intact animals treated with 400 fig 17/?-estradiol/100 g BW in sesame oil twice a week. The hormone was introduced directly into the gastric lumen. The rats in group 3 were surgically oophorectomized, and those in group 4 were oophorectomized and treated with 17/?-estradiol according to the same dosage schedule as group 2. The control groups received similar amounts of sesame oil. Sacrifice of five rats in each group for each set of chemical determinations took place at 1, 3, 6, 9, and 12 months after the institution of therapy. Sacrifice was by decapitation and serum was collected for chemical determinations. The femora and tibiae were removed immediately and dissected free of soft tissues and periosteum. The epiphyses were discarded and the bone marrow was removed by flushing with ice-cold saline. The metaphysis was separated from the diaphyseal portion of the bone and only metaphyseal bone was used for chemical analysis. Body weights were recorded monthly. Serum calcium and phosphorus determinations were carried out on an autoanalyser. The pooled metaphyseal bones of a single rat were lyophilized and used for each set of determinations. Lipid was extracted and washed by the method of Folch et al. (9). The ash content was determined after heating a sample of dried defatted bone powder in a furnace at 680 C for 20 h. The hydroxyproline content was measured after hydrolysis with 6 N HC1 at 100 C
1188
The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 18 November 2015. at 08:24 For personal use only. No other uses without permission. . All rights reserved.
ESTROGEN EFFECT ON BONE METABOLISM for 17 h, according to the method of Stegemann (10). Hexosamine was estimated after hydrolysis in 3 N HC1 at 100 C for 17 h by a modification of the method of Boas (11). Incubation studies were carried out according to the method of Deiss et al. (12). Minced metaphyseal fragments were incubated in buffered Krebs-Ringer bicarbonate medium at pH 7.4 in a Dubnoff incubator under 95% oxygen-5% CO2 at 37 C for 4 h. The incubation medium contained either 10 juCi L-[14C]proline (SA, 232 mCi/mmol) or 10 juCi D-[uC]glucose (SA, 4.06 mCi/mmol). After incubation, the bones were washed with saline and cold water several times and hydrolyzed at 100 C for 17 h with 6 N HC1 for hydroxyproline or with 3 N HC1 for hexosamine. The [l4C]hydroxyproline was separated on paper and the specific activity of the hydroxyproline fraction was determined according to methods previously described (10). To determine the specific activity of [uC]hexosamine, the hydrolysate was treated with ion exchange resin (Dowex 50 W) according to the method of Boas (11). An aliquot was dissolved in 15 ml Aquasol (New England Nuclear Corp., Boston, MA) and the radioactivity was determined in a liquid scintillation counter. The degree of quenching was estimated by internal standardization and the data were corrected. Collagenolytic activity was determined according to the method of Kaufman et al. (13). Metaphyseal bone (50 mg) was cut into four pieces and placed in a tube containing 100 jul purified neutral soluble rat skin collagen labeled with [:)H]proline and [3H]hydroxyproline (approximately 5000 cpm) with 400 /xl 0.05 M Tris-HCl buffer at pH 7.5. The tubes were incubated at 35 C for 3 days and the collagenolytic activity of the bone was determined by counting the release of radioactivity into the medium. Blank values were obtained by parallel incubation of heat-inactivated metaphyseal bone (boiled at 100 C for 3 min). To judge the uptake of mineral into bone, rats were injected sc 5 days before sacrifice with 10 juCi 45Ca. The rats were sacrificed at 1, 6, and 12 months after the start of the experiment, and the tibiae and femora were removed immediately. The bone marrow was removed as before and the metaphyseal regions were separated and ashed in a furnace at 680 C for 20 h. The ashed metaphysis was dissolved with 1 ml 2 N HC1 and 100 jul of the solution were mixed with 10 ml Aquasol and counted in a liquid scintillation counter. Quenching was corrected with a quench correction curve. Statistics were compiled by performing an analysis of variance and applying Scheffe's test (14).
Results Table 1 outlines the changes in body weight, serum calcium and phosphorus, ash content, and calcium uptake into bone. Estrogen decreased the body weight gains in intact rats, but these changes were not significant. Oophorectomy caused an increase in body weight gain throughout the entire year, while estrogen administration to the oophorectomized animals decreased body weight gain. Estrogen administration to the intact animal caused no consistent change in serum calcium concentration.
1189
Oophorectomy led to a decrease in serum calcium which was statistically significant by 6 months, with this change persisting through 12 months. However, estrogen administration to the oophorectomized animal prevented the fall in serum calcium. Neither oophorectomy nor estrogen administration caused any significant change in serum phosphorus concentration. Estrogen administration to the intact animal caused no alteration in the ash content of bones. Oophorectomy caused a statistically significant decrease in ash content which was apparent at the first month and persisted for 12 months. Estrogen administration to the oophorectomized animal reversed this; the reversal was statistically significant and consistent. Estrogen administration to the intact animal caused a slight decrease in uptake of labeled calcium to bone, and castration caused an early and persistent increase in this value. Estrogen administration returned it to normal. Table 2 presents the data on the contents and synthesis rates of hydroxyproline and hexosamine and on collagenolytic activity. The effect on collagen appeared clear-cut. While estrogen administration to the intact animal appeared initially to decrease total hydroxyproline, this value had returned to normal by 12 months. However, oophorectomy increased the hydroxyproline content. The increase persisted for 12 months and was statistically significant throughout the entire experiment. Estrogen administration to the oophorectomized animal decreased the hydroxyproline content, but it never entirely returned to normal when compared to the intact animal. The incorporation of [14C]proline into collagen was used as an index of collagen synthesis. Estrogen administration to the intact animal initially caused no change, but by 6 months there appeared to be a slight increase in collagen synthesis. The 12-month value indicated a slight but significant decrease. Oophorectomy by 3 months led to a great increase in collagen synthesis, and this persisted for the entire 12 months. With the exception of the values at 6 months, estrogen administration to the oophorectomized animal returned the values to normal and, by 12 months, collagen synthesis in the treated oophorectomized animal was identical with that of the intact animal. Estrogen had no long term effect on bone hexosamine content in the intact animal. Oophorectomy initially increased the total hexosamine, then decreased it, and estrogen administration to the oophorectomized animal returned this value to normal. Oophorectomy significantly increased the synthesis of proteoglycan and estrogen administration returned it toward normal, but it always remained elevated. Collagenolytic activity was decreased by the adminis-
The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 18 November 2015. at 08:24 For personal use only. No other uses without permission. . All rights reserved.
1190
Endo • 1979 Vol 104 • No 4
CRUESS AND HONG
TABLE 1. Effect of estrogen on body weight, serum phosphorus and calcium levels, and the ash and calcium uptake of bones of animals in the various treatment groups Month
BW(g) Intact Intact ± E2 Ovariectomy Ovariectomy ± E2 Serum phosphorus (mg/100 ml) Intact Intact ± E2 Ovariectomy Ovariectomy ± E2 Serum calcium (mg/100 ml)
1
3
6
9
188.0 ± 6.9 178.6 ± 9.3 218.4 ± 9.3" 166.0 ± 16.4"' *
256.6 ± 8.7 237.0 ± 21.0 284.4 ± 31.2 245.4 ± 16.8
305.0 ± 54.7 285.7 ± 25.0 332.6 ± 45.3 298.3 ± 28.6
324.8 ± 17.3 321.4 ± 54.0 407.4 ± 26.9" 316.2 ± 15.7*
7.1 ±0.4 7.2 ± 0.4 7.1 ±0.6 7.5 ±0.7
5.7 ±0.3 6.1 ±0.6 5.6 ± 0.6 6.4 ± 0.3
9.6 ± 0.3 10.0 ± 0.1" 9.7 ± 0.3 10.0 ± 0.2"' *
10.4 ± 0.3 10.5 ± 0.3 9.8 ± 0.3" 10.7 ± 0.36
9.7 ± 0.2 10.1 ±0.2" 9.2 ± 0.2" 9.9 ± 0.4"
10.0 ± 0.3 10.0 ±0.3 9.3 ± 0.2" 9.9 ± 0.2"
9.9 ±0.1 9.7 ± 0.4 9.3 ± 0.2" 9.2 ± 0.8
60.2 ± 0.2 61.7 ± 0.5" 57.9 ± 0.1" 59.4 ± 0.2"' *
65.3 ± 1.4 67.0 ± 0.7 63.7 ± 0.8 67.9 ± 0.6*
66.8 ± 0.9 67.0 ± 0.6 63.5 ± 1.1" 66.6 ±0.1*
66.3 ± 1.1 66.4 ±1.1 61.8 ± 1.0" 64.1 ±0.8"'
65.3 ± 0.8 66.9 ± 0.8 61.8 ± 1.2" 66.5 ± 0.4"' *
Intact Intact ± E2 Ovariectomy Ovariectomy ± E2 Ash (%) Intact Intact ± E2 Ovariectomy Ovariectomy ± E2 4n Ca uptake
8.2 ± 0.6 8.9 ± 0.5 9.5 ± 0.8 8.8 ± 0.8
5.9 5.8 5.4 5.4
± 0.3 ± 0.5 ±0.1 ±0.5
12
353.1 ± 11.4 304.1 ± 22.4 436.3 ± 66.4" 389.4 ± 46.7 5.7 ± 0.2 6.0 ± 0.1 5.7 ±0.1 5.3 ± 0.5
6028 ±516 5973 ± 553 7390 ± 663" 6385 ± 297"
1859 ± 230 1022 ± 97 914 ± 112 1470 ± 100.' 2761 ± 412" 1397 ± 128" 1154 ± 135* 2051 ± 305* Results are given as the mean ± SD of five determinations for one group (five animals were used in one group. E2, 17/?-Estradiol., " P < 0.05 between intact group and intact plus estrogen group, ovariectomy group, or ovariectomy plus estrogen group. * P < 0.05 between ovariectomy and estrogen groups.
Intact
Intact ± E2 Ovariectomy Ovariectomy ± E2
tration of estrogen to the intact animal. This decrease, while present, was only statistically significant at 1 month. Oophorectomy initially caused no statistical change in the level of collagenolytic activity but, by 3 months, there was a significant increase in this value which persisted. It was still present at 12 months but was not statistically significant. Estrogen administration to the oophorectomized animal caused a statistically significant decrease in this value which persisted throughout the experiment.
Discussion We believe that our data indicate that loss of ovarian function in the rat leads to a decrease in serum calcium. This is accompanied by an increase in both resorption and formation of bone (as indicated by increased levels of collagenolytic activity), increased rates of synthesis of both collagen and proteoglycan, and an increased uptake of radioactive calcium. Estrogen administration to the intact animal leads to a slight but insignificant decrease in levels of resorption, with no change in formation. However, estrogen administration to the oophorectomized animal returns the serum calcium to normal and
decreases the levels of resorption found in the oophorectomized animal, with the formation rates returning to normal. In the human female, postmenopausal osteoporosis is characterized by a decrease in bone mass per U volume, with changes becoming apparent at about the time of the menopause (15-18). Similar changes occur in the oophorectomized rat. Saville (19) reported a decrease in femoral calcium per U volume. Aitken et al. (20) showed that oophorectomy led to classical osteoporosis 11 months after removal of the ovaries. Their experiments showed a reduction in ash per U length of bone and a decrease in the cortical width of the midshaft of the femur. Thus, our data are consistent with other reports in the literature. It is worth noting that Aitken et al. (20) were able to demonstrate changes in both parameters, regardless of the body weight of the rats, after either oophorectomy or estrogen therapy. Therefore, it appears that the suggestion of Saville and Lieber (21), that alterations in food intake were partially responsible for the changes, are not in fact correct. A short term decrease in serum calcium concentration after oophorectomy has been noted previously (22). The long term results reported here indicate that the change
The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 18 November 2015. at 08:24 For personal use only. No other uses without permission. . All rights reserved.
ESTROGEN EFFECT ON BONE METABOLISM
1191
TABLE 2. Effect of estrogen on the hydroxyproline and hexosamine contents, the synthetic rates of collagen and hexosamine, and the collagenolytic activity of bone from animals in the various treatment groups Month
Hydroxyproline (//,g/mg bone) Intact Intact ± E2 Ovariectomy Ovariectomy ± E2 [l4C]Hydroxyproline SA (dpm/fig) Intact Intact ± E2 Ovariectomy Ovariectomy ± E2 Hexosamine (Ug/mg bone) Intact Intact ± E2 Ovariectomy Ovariectomy ± E2 [uC]Hexosamine SA
Intact Intact ± E2 Ovariectomy Ovariectomy ± E2 Collagenolytic activity, degraded [:tH]collagen (cpm/50 mg bone over blank) Intact Intact ± E2 Ovariectomy Ovariectomy ± E2
1
3
6
9
12
16.39 ± 0.53 16.13 ±0.23 17.72 ± 0.24" 17.16 ±0.23"*
19.22 ± 1.10 18.10 ±0.62 21.91 ± 0.70" 17.25 ± 0.33"' *
12.88 ± 0.46 12.11 ±0.05" 17.51 ± 0.59" 15.43 ± 0.16"'*
14.06 ± 0.20 12.92 ± 0.52" 18.65 ± 0.45" 15.43 ± 0.70"' *
14.40 ± 0.19 14.15 ± 0.33 18.78 ± 0.73" 15.19 ± 0.23"' *
246 ± 16 247 ± 20 220 ± 13 225 ± 11
179 ± 20 179 ± 11 291 ± 16" 175 ± 8"
31.5 ±6.7 46.3 ± 8.5" 78.1 ± 7.7" 84.1 ±4.0"
15.0 ± 3.5 13.1 ±2.5 39.5 ± 4.0" 31.6 ±2.9"*
24.9 ± 5.0 14.5 ± 2.4" 34.9 ± 3.6" 26.4 ± 5.2*
4.40 ±0.12 4.65 ± 0.15" 5.12 ±0.15" 5.38 ± 0.07"-*
3.78 ± 0.15 3.90 ± 0.01 3.18 ± 0.04" 4.09 ±0.10"*
4.09 ±0.10 4.03 ±0.17 2.71 ± 0.21" 4.03 ± 0.59*
2.12 ±0.05 2.28 ± 0.27 1.54 ± 0.06" 1.81 ±0.04"*
2.01 ± 0.04 2.18 ±0.15 1.25 ± 0.41" 2.14 ±0.35*
30.5 ± 2.0 20.5 ± 4.0" 41.5 ± 5.5" 20.3 ± 3.7"
14.3 ± 3.0 15.8 ± 2.2 35.5 ± 4.8" 23.5 ± 3.6"
17.6 ± 1.2 17.6 ± 1.2 23.0 ± 2.0" 12.5 ±2.3"lA
6.9 ± 1.6 7.6 ± 2.6 24.2 ± 1.1" 14.1 ±1.8"*
6.7 ± 6.4 ± 23.1 ± 12.4 ±
513 ± 74 379 ± 80" 394 ±110 293 ± 100°
312 ± 106 203 ± 50 382 ±118 205 ± 73*
256 ± 59 237 ± 40 366 ± 60" 283 ± 45*
243 ± 64 231 ± 44 356 ± 71" 259 ± 72*
162 ± 70 117 ±21 210 ± 84 121 ± 39
1.1 1.6 1.2" 1.0"' *
Results are given as the mean ± SD of five determinations for one group (five animals were used in one group). E2, 17-/?-Estradiol. " P < 0.05 between intact group and intact plus estrogen group, ovariectomy group or ovariectomy plus estrogen group. * P < 0.05 between ovariectomy and ovariectomy plus estrogen groups.
persists. It is of note that the serum calcium changes in the oophorectomized rat differ from those reported in the postmenopausal female (23-25), in whom an increase in serum calcium is found which is reversed by the administration of estrogen. It has been recognized since the initial report of Budy, Urist, and McLean (26) that the primary effect of estrogen in the experimental animal is one of inhibition of bone resorption, and this effect appears also to be present in the human (7). As resorption of bone must consist of the removal of the mineral and enzymatic degradation of the matrix, it should be possible to utilize the activity of degradative enzymes as an index of matrix resorption. Our laboratory has reported (27) increased collagenolytic activity after oophorectomy and a decrease after short term estrogen administration to both the intact and oophorectomized rat. The increase in collagenolytic activity after oophorectomy, associated with a decrease in ash content and bone mass as reported by others, makes increased resorption an almost inescapable conclusion, particularly in view of the direct correlation between bone resorption and collagenase release reported by Sak-
amoto et al. (28). The reversal of this state by estrogen administration is striking and, we believe, must also indicate a prolonged decrease in both calcium mobilization from bone and matrix resorption. The long term estrogen effect upon formation rates reported in these experiments is consistent with and parallel to other short term reports in the literature. Oophorectomy leads to an increased formation of collagen and proteoglycan and an increased uptake of radiocalcium. Estrogen administration reverses this effect. Previous short term studies by Langeland (29) and Langeland and Teig (30, 31) have indicated that removal of the ovaries leads to an increase in collagen synthesis and that this increase is suppressed by estrogen treatment. They also have noted a decrease in collagen breakdown after estrogen administration. Their data thus serve to confirm the alteration both in collagenolytic activity and in synthesis rates. Very little information is available with regard to the effect of estrogen upon proteoglycan metabolism in bone. Estrogen does appear to inhibit (35SO4) incorporation into chondroitin sulfate at the epiphyseal line (32, 33) and has a similar effect
The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 18 November 2015. at 08:24 For personal use only. No other uses without permission. . All rights reserved.
1192
CRUESS AND HONG
upon chondroitin sulfate synthesis in fibroblast cultures (34). The means by which estrogen exerts its effects upon the musculoskeletal system remain unclear. It has been demonstrated that tritiated estradiol is taken up by the rat femur (35), although it is not known whether this is nonspecific binding or if this binding is to the cell or to other areas of the bone. No receptor was demonstrated in osseous tissue, making it unlikely that bone is a primary target organ for the hormone (36). There are contradictory data on the influence of estrogen in tissue or organ culture of bone. In some experimental situations, estrogen fails to inhibit bone resorption (37), while other investigators have reported such an action (38). There is a suggestion (39) that the reason for any measurable effect is direct toxicity of estrogen on cell metabolism.
9.
10.
11. 12. 13.
14. 15.
16.
There have been several suggestions that estrogen affects
bone metabolism by decreasing the sensitivity of the osteocyte or osteoclast to parathyroid hormone (7, 22, 40). Our data are consistent with this hypothesis or could be explained by the reported (41) estrogen-mediated enhancement of calcium absorption by the gut with resultant decreased parathyroid activity. The possible effect of hormonal interrelationships, such as an estrogen-mediated increase in circulating vitamin D metabolites (42), awaits further investigation. Specifically, the question of whether estrogen deficiency leads either to a decreased sensitivity of bone to parathyroid hormone or to a decrease in parathyroid hormone secretion may be answered only by direct measurement of parathyroid function under controlled conditions. The data presented here indicate that this should be done in a long term experiment to determine whether the suggestion (8) that the estrogen effect is not prolonged and that decreased formation ultimately diminishes its therapeutic effectiveness is valid. In the rat it appears that this is not true. References 1. Albright, F. P., H. Smith, and A. M. Richardson, Postmenopausal osteoporosis; its clinical features, JAMA 116: 2465, 1941. 2. Albright, F., The effect of hormones on osteogenesis in man, Recent Prog Horm Res 1: 293, 1947. 3. Wallach, S., and P. H. Henneman, Prolonged estrogen therapy in postmenopausal women, JAMA 171: 1637, 1959. 4. Davis, M. E., N. M. Strandjord, and L. H. Lanzl, Estrogens and the aging process, JAMA 196: 219, 1966. 5. Meema, H. E., and S. Meema, Prevention of postmenopausal osteoporosis by hormone treatment of the menopause, Can Med Assoc J 99: 248, 1968. 6. Lindsay, R , J. M. Aitken, J. B. Anderson, D. M. Hart, E. B. MacDonald, and A. C. Clarke, Long-term prevention of postmenopausal osteoporosis by estrogen: evidence for an increased bone mass after delayed onset of estrogen treatment, Lancet 1: 1038, 1976. 7. Riggs, B. L., J. Jowsey, P. J. Kelly, J. D. Jones, and F. T. Maher, Effex of sex hormones on bone in primary osteoporosis, J Clin Invest 48: 1065,1969. 8. Riggs, B. L., J. Jowsey, P. J. Kelly, D. L. Hoffman, and C. D.
17. 18.
19. 20.
21.
22.
23.
24.
25.
26.
27. 28.
29.
30.
31.
32.
Endo • 1979 Vol 104 • No 4
Arnaud, Studies on pathogenesis and treatment in postmenopausal and senile osteoporosis, Clin Endocrinol Metab 2: 317, 1973. Folch, J., M. Lees, and G. H. S. Stanley, A simple method for the isolation and purification of total lipids from animal tissues, J Biol Chem 226: 497, 1957. Stegemann, H., Mikrobestimmung von Hydroxyproline mit Chloramin-T und /j-Dimethylamino-Benzaldehyd. Hoppe Seylers Z Physiol Chem 311: 41, 1958. Boas, N. F., Method for the determination of hexosamines in tissues, J Biol Chem 204: 553, 1953. Deiss, W. P., L. B. Holmes, and C. C. Johnston, Jr., Bone matrix biosynthesis in vitro, J Biol Chem 237: 3555, 1962. Kaufman, E. J., M. J. Glimcher, G. L. Mechanic, and P. Goldhaber, Collagenolytic activity during active bone resorption in tissue culture, Proc Soc Exp Biol Med 120: 632, 1965. Armitage, P., Statistical Methods in Medical Research, John Wiley and Sons, New York, 1971, pp. 202-207. Meema, H. E., Cortical bone atrophy and osteoporosis as a manifestation of aging, Am J Roentgenol Radium Ther Nucl Med 89: 1287, 1963. Nordin, B. E. C, J. MacGregor, and D. A. Smith, The incidence of osteoporosis in normal women: its relation to age and the menopause, QJ Med 137: 25, 1966. Garn, S. M., C. G. Rohmann, and B. Wagner, Bone loss as a general phenomenon in man, Fed Proc 26: 1729, 1967. Smith, D. A., J. B. Anderson, J. Shimmins, C. F. Spens, and E. Barnett, Changes in metacarpal mineral content and density in normal male and female subjects with age, Clin Radiol 20: 23, 1969. Saville, P. D., Changes in skeletal mass and fragility with castration in the rat; a model of osteoporosis, J Am Geriatr Soc 17: 155,1969. Aitken, J. M., E. Armstrong, and J. B. Anderson, Osteoporosis after oophorectomy in the mature female rat and the effect of estrogen and/or progestrogen replacement therapy in its prevention, J Endocrinol 55: 79, 1972. Saville, P. D., and C. S. Lieber, Increases in skeletal calcium and femur cortex thickness produced by undernutrition, J Nutr 99: 141, 1969. Orimo, H., T. Fujita, and M. Yoshikawa, Increased sensitivity of bone to parathyroid hormone in ovariectomized rats, Endocrinology 90: 760, 1972. Gallagher, J. C, M. Young, and B. E. C. Nordin, Effects of artificial menopause on plasma and urine calcium and phosphate, Clin Endocrinol 1: 57, 1972. Aitken, J. M., D. McKay Hart, and D. A. Smitty, The effect of longterm mestranol administration on calcium and phosphorous homeostasis in oophorectomized women, Clin Sci 41: 233, 1971. Aitken, J. M., D. M. Hart, and R. Lindsay, Estrogen replacement therapy for prevention of osteoporosis after oophorectomy, BrMed J 3 : 515, 1973. Budy, A. M., M. R. Urist, and F. C. MacLean, The effect of estrogens on the growth apparatus of the bones of immature rats, Am JPathol 28: 1143, 1952. Cruess, R. L., and K. C. Hong, Effect of estrogen on the collagenolytic activity of rat bone, Calcif Tissue Res 20: 317, 1976. Sakamoto, S., M. Sakamoto, P. Goldhaber, and M. Glimcher, Collagenase and bone resorption: isolation of collagenase from culture medium containing serum after stimulation of bone resorption by addition of parathyroid hormone extract, Biochem Biophys Res Commun 63: 172, 1975. Langeland, N., In vitro studies on collagen metabolism in metaphyseal rat bone. A. The effect of pre-treatment with estradiol-17/?, Acta Endocrinol (Kbh) 80: 775, 1975. Langeland, N., and V. Teig, In vitro studies on collagen metabolism in metaphyseal rat bone. B. The early effects of estradiol-17/?, Acta Endocrinol {Kbh) 80: 784, 1975. Langeland, N., and V. Teig, In vitro studies on collagen metabolism in metaphyseal rat bone. C. The effect of estradiol-17/8 hypophysectomized and in thyroparathyroidectomized animals, Acta Endocrinol (Kbh) 80: 795,1975. Endo, H., S. Murota, and H. Enomoto, Effect of hormones on the chondroitin sulfate metabolism of chick embryo femora growing in
The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 18 November 2015. at 08:24 For personal use only. No other uses without permission. . All rights reserved.
ESTROGEN EFFECT ON BONE METABOLISM
33. 34. 35.
36.
37.
vitro. III. Effect of sex steroids on chondroitin sulfate synthesis in the cartilagenous bones growing in the natural medium, Endocrinol Jap 16: 115, 1969. Herbai, G., Autoradiographic studies with S-sulfate on somatotrophin and estrogen sensitive growth zones in rat and mouse costal cartilage, Ada Physiol Scand 79: 351, 1970. Nordbo, H., The effect of estradiol-17/? on the metabolism of chondroitin sulphate B, Steroidologia 2: 7, 1971. Anderson, J. J. B., and J. M. Floeckher, Skeletal uptake of H3estradiol by rats primed with nonradioactive estrogen and parathyroid extract, Isr J Med Sci 7: 353, 1971. Nutik, G., and R. L. Cruess, Estrogen receptors in bone. An evaluation of the uptake of estrogen into bone cells, Proc Soc Exp Biol Med 146: 265, 1974. Caputo, C. B., D. Meadows, and L. G. Raisz, Failure of estrogens
38.
39. 40. 41.
42.
1193
and androgens to inhibit bone resorption in tissue culture, Endocrinology 98: 1065, 1976. Atkins, D., J. M. Zanelli, M. Peacock, and B. E. C. Nordin, The effect of estrogens on the response of bone to parathyroid hormone in vitro, J Endocrinol 54: 107, 1972. Liskova, M., Influence of estrogens on bone resorption in organ culture, Calcif Tissue Res 22: 207, 1976. Heaney, R. P., A unified concept of osteoporosis, Am J Med 39: 877, 1965. Caniggia, A., Intestinal absorption of calcium-47 after treatment with oral estrogen-gestogens in senile osteoporosis, Br Med J 4: 30, 1970. Castillo, L., Y. Tanaka, H. F. deLuca, and M. L. Sunde, The stimulation of 25-hydroxyvitamin D,)-l,,-hydroxylase by estrogen, Arch Biochem Biophys 179: 211, 1977.
The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 18 November 2015. at 08:24 For personal use only. No other uses without permission. . All rights reserved.
Vol. 104, No. 4 Printed in U.S.A.
The Effect of Long Term Estrogen Administration on Bone Metabolism in the Female Rat* R. L. CRUESS AND K. C. HONG Orthopedic Research Laboratories, Royal Victoria Hospital, McGill University, Montreal, Quebec, Canada
ABSTRACT. To determine the long term effect of estrogen administration upon some biochemical indices of bone formation and resorption as well as serum calcium and phosphorus, the following experiments were carried out. Female rats were divided into the following groups: intact, intact and estrogen-treated, oophorectomized, and oophorectomized and estrogen-treated. Treatment consisted of 400 /xg 17/8-estradiol/100 g BW twice a week for 1-12 months. Estrogen administration to the intact animals coused no consistent significant changes in any of the parameters measured. It was found that oophorectomy caused a decrease in the serum calcium concentration, a decrease in ash content, an increase in the total hydroxyproline content, an increase in incorporation of [3H]proline into collagen, a decrease in total hexosamine, an increase in the incorporation of [14C]glucose into
D
ESPITE the widespread therapeutic use of estrogen in the management of osteoporosis, its effect on bone metabolism still remains to be clarified. Albright (1) first reported the clinical entity of postmenopausal osteoporosis and many investigators have recommended estrogen in the therapy of this disorder (2-4). It is well established that estrogen can prevent or significantly decrease the loss of bone mass in the postmenopausal female (5, 6), but the beneficial effect of its use in the treatment of established postmenopausal osteoporosis is only temporary (7), with a compensatory decrease in bone formation at least partially offsetting the useful effect of the hormone on bone resorption (8). To date, no long term experiment reporting the effect of estrogen on bone metabolism in rats under experimental conditions has appeared. To obtain biochemical indices of rates of bone formation and resorption as well as compositional data on bone itself, the following long term experiments were carried out.
hexosamine, an increase in the uptake of 4r>Ca into bone, and an increase in bone collagenolytic activity. The administration of estrogen to the oophorectomized rat corrected the serum calcium changes to normal and returned the decreased ash content, collagen, and hexosamine contents to normal. Estrogen administration only partially reversed the increased uptake of [3H]proline into collagen and the increased incorporation of [I4C]glucose into hexosamine. The administration of estrogen to oophorectomized animals also returned the 4r'Ca uptake of bone to normal and caused normalization of the bone collagenolytic activity. The data indicate a sustained decrease in serum calcium and an increase in both bone formation and resorption in the oophorectomized animal. The administration of estrogen largely corrects these abnormalities and the beneficial effects are sustained over a 12-month period. (Endocrinology 104: 1188, 1979)
Materials and Methods
Received August 23, 1977. Address all correspondence and requests for reprints to: Dr. R. L. Cruess, Orthopedic Surgeon-in-Charge, Royal Victoria Hospital, 687 Pine Avenue West, Montreal, P.Q. H3A 1A1, Canada. * This work was supported by Grant MA-1571 from the MRC of Canada.
Female rats (150 g) were divided into four groups. The first group was left intact and served as control. Group 2 included intact animals treated with 400 fig 17/?-estradiol/100 g BW in sesame oil twice a week. The hormone was introduced directly into the gastric lumen. The rats in group 3 were surgically oophorectomized, and those in group 4 were oophorectomized and treated with 17/?-estradiol according to the same dosage schedule as group 2. The control groups received similar amounts of sesame oil. Sacrifice of five rats in each group for each set of chemical determinations took place at 1, 3, 6, 9, and 12 months after the institution of therapy. Sacrifice was by decapitation and serum was collected for chemical determinations. The femora and tibiae were removed immediately and dissected free of soft tissues and periosteum. The epiphyses were discarded and the bone marrow was removed by flushing with ice-cold saline. The metaphysis was separated from the diaphyseal portion of the bone and only metaphyseal bone was used for chemical analysis. Body weights were recorded monthly. Serum calcium and phosphorus determinations were carried out on an autoanalyser. The pooled metaphyseal bones of a single rat were lyophilized and used for each set of determinations. Lipid was extracted and washed by the method of Folch et al. (9). The ash content was determined after heating a sample of dried defatted bone powder in a furnace at 680 C for 20 h. The hydroxyproline content was measured after hydrolysis with 6 N HC1 at 100 C
1188
The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 18 November 2015. at 08:24 For personal use only. No other uses without permission. . All rights reserved.
ESTROGEN EFFECT ON BONE METABOLISM for 17 h, according to the method of Stegemann (10). Hexosamine was estimated after hydrolysis in 3 N HC1 at 100 C for 17 h by a modification of the method of Boas (11). Incubation studies were carried out according to the method of Deiss et al. (12). Minced metaphyseal fragments were incubated in buffered Krebs-Ringer bicarbonate medium at pH 7.4 in a Dubnoff incubator under 95% oxygen-5% CO2 at 37 C for 4 h. The incubation medium contained either 10 juCi L-[14C]proline (SA, 232 mCi/mmol) or 10 juCi D-[uC]glucose (SA, 4.06 mCi/mmol). After incubation, the bones were washed with saline and cold water several times and hydrolyzed at 100 C for 17 h with 6 N HC1 for hydroxyproline or with 3 N HC1 for hexosamine. The [l4C]hydroxyproline was separated on paper and the specific activity of the hydroxyproline fraction was determined according to methods previously described (10). To determine the specific activity of [uC]hexosamine, the hydrolysate was treated with ion exchange resin (Dowex 50 W) according to the method of Boas (11). An aliquot was dissolved in 15 ml Aquasol (New England Nuclear Corp., Boston, MA) and the radioactivity was determined in a liquid scintillation counter. The degree of quenching was estimated by internal standardization and the data were corrected. Collagenolytic activity was determined according to the method of Kaufman et al. (13). Metaphyseal bone (50 mg) was cut into four pieces and placed in a tube containing 100 jul purified neutral soluble rat skin collagen labeled with [:)H]proline and [3H]hydroxyproline (approximately 5000 cpm) with 400 /xl 0.05 M Tris-HCl buffer at pH 7.5. The tubes were incubated at 35 C for 3 days and the collagenolytic activity of the bone was determined by counting the release of radioactivity into the medium. Blank values were obtained by parallel incubation of heat-inactivated metaphyseal bone (boiled at 100 C for 3 min). To judge the uptake of mineral into bone, rats were injected sc 5 days before sacrifice with 10 juCi 45Ca. The rats were sacrificed at 1, 6, and 12 months after the start of the experiment, and the tibiae and femora were removed immediately. The bone marrow was removed as before and the metaphyseal regions were separated and ashed in a furnace at 680 C for 20 h. The ashed metaphysis was dissolved with 1 ml 2 N HC1 and 100 jul of the solution were mixed with 10 ml Aquasol and counted in a liquid scintillation counter. Quenching was corrected with a quench correction curve. Statistics were compiled by performing an analysis of variance and applying Scheffe's test (14).
Results Table 1 outlines the changes in body weight, serum calcium and phosphorus, ash content, and calcium uptake into bone. Estrogen decreased the body weight gains in intact rats, but these changes were not significant. Oophorectomy caused an increase in body weight gain throughout the entire year, while estrogen administration to the oophorectomized animals decreased body weight gain. Estrogen administration to the intact animal caused no consistent change in serum calcium concentration.
1189
Oophorectomy led to a decrease in serum calcium which was statistically significant by 6 months, with this change persisting through 12 months. However, estrogen administration to the oophorectomized animal prevented the fall in serum calcium. Neither oophorectomy nor estrogen administration caused any significant change in serum phosphorus concentration. Estrogen administration to the intact animal caused no alteration in the ash content of bones. Oophorectomy caused a statistically significant decrease in ash content which was apparent at the first month and persisted for 12 months. Estrogen administration to the oophorectomized animal reversed this; the reversal was statistically significant and consistent. Estrogen administration to the intact animal caused a slight decrease in uptake of labeled calcium to bone, and castration caused an early and persistent increase in this value. Estrogen administration returned it to normal. Table 2 presents the data on the contents and synthesis rates of hydroxyproline and hexosamine and on collagenolytic activity. The effect on collagen appeared clear-cut. While estrogen administration to the intact animal appeared initially to decrease total hydroxyproline, this value had returned to normal by 12 months. However, oophorectomy increased the hydroxyproline content. The increase persisted for 12 months and was statistically significant throughout the entire experiment. Estrogen administration to the oophorectomized animal decreased the hydroxyproline content, but it never entirely returned to normal when compared to the intact animal. The incorporation of [14C]proline into collagen was used as an index of collagen synthesis. Estrogen administration to the intact animal initially caused no change, but by 6 months there appeared to be a slight increase in collagen synthesis. The 12-month value indicated a slight but significant decrease. Oophorectomy by 3 months led to a great increase in collagen synthesis, and this persisted for the entire 12 months. With the exception of the values at 6 months, estrogen administration to the oophorectomized animal returned the values to normal and, by 12 months, collagen synthesis in the treated oophorectomized animal was identical with that of the intact animal. Estrogen had no long term effect on bone hexosamine content in the intact animal. Oophorectomy initially increased the total hexosamine, then decreased it, and estrogen administration to the oophorectomized animal returned this value to normal. Oophorectomy significantly increased the synthesis of proteoglycan and estrogen administration returned it toward normal, but it always remained elevated. Collagenolytic activity was decreased by the adminis-
The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 18 November 2015. at 08:24 For personal use only. No other uses without permission. . All rights reserved.
1190
Endo • 1979 Vol 104 • No 4
CRUESS AND HONG
TABLE 1. Effect of estrogen on body weight, serum phosphorus and calcium levels, and the ash and calcium uptake of bones of animals in the various treatment groups Month
BW(g) Intact Intact ± E2 Ovariectomy Ovariectomy ± E2 Serum phosphorus (mg/100 ml) Intact Intact ± E2 Ovariectomy Ovariectomy ± E2 Serum calcium (mg/100 ml)
1
3
6
9
188.0 ± 6.9 178.6 ± 9.3 218.4 ± 9.3" 166.0 ± 16.4"' *
256.6 ± 8.7 237.0 ± 21.0 284.4 ± 31.2 245.4 ± 16.8
305.0 ± 54.7 285.7 ± 25.0 332.6 ± 45.3 298.3 ± 28.6
324.8 ± 17.3 321.4 ± 54.0 407.4 ± 26.9" 316.2 ± 15.7*
7.1 ±0.4 7.2 ± 0.4 7.1 ±0.6 7.5 ±0.7
5.7 ±0.3 6.1 ±0.6 5.6 ± 0.6 6.4 ± 0.3
9.6 ± 0.3 10.0 ± 0.1" 9.7 ± 0.3 10.0 ± 0.2"' *
10.4 ± 0.3 10.5 ± 0.3 9.8 ± 0.3" 10.7 ± 0.36
9.7 ± 0.2 10.1 ±0.2" 9.2 ± 0.2" 9.9 ± 0.4"
10.0 ± 0.3 10.0 ±0.3 9.3 ± 0.2" 9.9 ± 0.2"
9.9 ±0.1 9.7 ± 0.4 9.3 ± 0.2" 9.2 ± 0.8
60.2 ± 0.2 61.7 ± 0.5" 57.9 ± 0.1" 59.4 ± 0.2"' *
65.3 ± 1.4 67.0 ± 0.7 63.7 ± 0.8 67.9 ± 0.6*
66.8 ± 0.9 67.0 ± 0.6 63.5 ± 1.1" 66.6 ±0.1*
66.3 ± 1.1 66.4 ±1.1 61.8 ± 1.0" 64.1 ±0.8"'
65.3 ± 0.8 66.9 ± 0.8 61.8 ± 1.2" 66.5 ± 0.4"' *
Intact Intact ± E2 Ovariectomy Ovariectomy ± E2 Ash (%) Intact Intact ± E2 Ovariectomy Ovariectomy ± E2 4n Ca uptake
8.2 ± 0.6 8.9 ± 0.5 9.5 ± 0.8 8.8 ± 0.8
5.9 5.8 5.4 5.4
± 0.3 ± 0.5 ±0.1 ±0.5
12
353.1 ± 11.4 304.1 ± 22.4 436.3 ± 66.4" 389.4 ± 46.7 5.7 ± 0.2 6.0 ± 0.1 5.7 ±0.1 5.3 ± 0.5
6028 ±516 5973 ± 553 7390 ± 663" 6385 ± 297"
1859 ± 230 1022 ± 97 914 ± 112 1470 ± 100.' 2761 ± 412" 1397 ± 128" 1154 ± 135* 2051 ± 305* Results are given as the mean ± SD of five determinations for one group (five animals were used in one group. E2, 17/?-Estradiol., " P < 0.05 between intact group and intact plus estrogen group, ovariectomy group, or ovariectomy plus estrogen group. * P < 0.05 between ovariectomy and estrogen groups.
Intact
Intact ± E2 Ovariectomy Ovariectomy ± E2
tration of estrogen to the intact animal. This decrease, while present, was only statistically significant at 1 month. Oophorectomy initially caused no statistical change in the level of collagenolytic activity but, by 3 months, there was a significant increase in this value which persisted. It was still present at 12 months but was not statistically significant. Estrogen administration to the oophorectomized animal caused a statistically significant decrease in this value which persisted throughout the experiment.
Discussion We believe that our data indicate that loss of ovarian function in the rat leads to a decrease in serum calcium. This is accompanied by an increase in both resorption and formation of bone (as indicated by increased levels of collagenolytic activity), increased rates of synthesis of both collagen and proteoglycan, and an increased uptake of radioactive calcium. Estrogen administration to the intact animal leads to a slight but insignificant decrease in levels of resorption, with no change in formation. However, estrogen administration to the oophorectomized animal returns the serum calcium to normal and
decreases the levels of resorption found in the oophorectomized animal, with the formation rates returning to normal. In the human female, postmenopausal osteoporosis is characterized by a decrease in bone mass per U volume, with changes becoming apparent at about the time of the menopause (15-18). Similar changes occur in the oophorectomized rat. Saville (19) reported a decrease in femoral calcium per U volume. Aitken et al. (20) showed that oophorectomy led to classical osteoporosis 11 months after removal of the ovaries. Their experiments showed a reduction in ash per U length of bone and a decrease in the cortical width of the midshaft of the femur. Thus, our data are consistent with other reports in the literature. It is worth noting that Aitken et al. (20) were able to demonstrate changes in both parameters, regardless of the body weight of the rats, after either oophorectomy or estrogen therapy. Therefore, it appears that the suggestion of Saville and Lieber (21), that alterations in food intake were partially responsible for the changes, are not in fact correct. A short term decrease in serum calcium concentration after oophorectomy has been noted previously (22). The long term results reported here indicate that the change
The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 18 November 2015. at 08:24 For personal use only. No other uses without permission. . All rights reserved.
ESTROGEN EFFECT ON BONE METABOLISM
1191
TABLE 2. Effect of estrogen on the hydroxyproline and hexosamine contents, the synthetic rates of collagen and hexosamine, and the collagenolytic activity of bone from animals in the various treatment groups Month
Hydroxyproline (//,g/mg bone) Intact Intact ± E2 Ovariectomy Ovariectomy ± E2 [l4C]Hydroxyproline SA (dpm/fig) Intact Intact ± E2 Ovariectomy Ovariectomy ± E2 Hexosamine (Ug/mg bone) Intact Intact ± E2 Ovariectomy Ovariectomy ± E2 [uC]Hexosamine SA
Intact Intact ± E2 Ovariectomy Ovariectomy ± E2 Collagenolytic activity, degraded [:tH]collagen (cpm/50 mg bone over blank) Intact Intact ± E2 Ovariectomy Ovariectomy ± E2
1
3
6
9
12
16.39 ± 0.53 16.13 ±0.23 17.72 ± 0.24" 17.16 ±0.23"*
19.22 ± 1.10 18.10 ±0.62 21.91 ± 0.70" 17.25 ± 0.33"' *
12.88 ± 0.46 12.11 ±0.05" 17.51 ± 0.59" 15.43 ± 0.16"'*
14.06 ± 0.20 12.92 ± 0.52" 18.65 ± 0.45" 15.43 ± 0.70"' *
14.40 ± 0.19 14.15 ± 0.33 18.78 ± 0.73" 15.19 ± 0.23"' *
246 ± 16 247 ± 20 220 ± 13 225 ± 11
179 ± 20 179 ± 11 291 ± 16" 175 ± 8"
31.5 ±6.7 46.3 ± 8.5" 78.1 ± 7.7" 84.1 ±4.0"
15.0 ± 3.5 13.1 ±2.5 39.5 ± 4.0" 31.6 ±2.9"*
24.9 ± 5.0 14.5 ± 2.4" 34.9 ± 3.6" 26.4 ± 5.2*
4.40 ±0.12 4.65 ± 0.15" 5.12 ±0.15" 5.38 ± 0.07"-*
3.78 ± 0.15 3.90 ± 0.01 3.18 ± 0.04" 4.09 ±0.10"*
4.09 ±0.10 4.03 ±0.17 2.71 ± 0.21" 4.03 ± 0.59*
2.12 ±0.05 2.28 ± 0.27 1.54 ± 0.06" 1.81 ±0.04"*
2.01 ± 0.04 2.18 ±0.15 1.25 ± 0.41" 2.14 ±0.35*
30.5 ± 2.0 20.5 ± 4.0" 41.5 ± 5.5" 20.3 ± 3.7"
14.3 ± 3.0 15.8 ± 2.2 35.5 ± 4.8" 23.5 ± 3.6"
17.6 ± 1.2 17.6 ± 1.2 23.0 ± 2.0" 12.5 ±2.3"lA
6.9 ± 1.6 7.6 ± 2.6 24.2 ± 1.1" 14.1 ±1.8"*
6.7 ± 6.4 ± 23.1 ± 12.4 ±
513 ± 74 379 ± 80" 394 ±110 293 ± 100°
312 ± 106 203 ± 50 382 ±118 205 ± 73*
256 ± 59 237 ± 40 366 ± 60" 283 ± 45*
243 ± 64 231 ± 44 356 ± 71" 259 ± 72*
162 ± 70 117 ±21 210 ± 84 121 ± 39
1.1 1.6 1.2" 1.0"' *
Results are given as the mean ± SD of five determinations for one group (five animals were used in one group). E2, 17-/?-Estradiol. " P < 0.05 between intact group and intact plus estrogen group, ovariectomy group or ovariectomy plus estrogen group. * P < 0.05 between ovariectomy and ovariectomy plus estrogen groups.
persists. It is of note that the serum calcium changes in the oophorectomized rat differ from those reported in the postmenopausal female (23-25), in whom an increase in serum calcium is found which is reversed by the administration of estrogen. It has been recognized since the initial report of Budy, Urist, and McLean (26) that the primary effect of estrogen in the experimental animal is one of inhibition of bone resorption, and this effect appears also to be present in the human (7). As resorption of bone must consist of the removal of the mineral and enzymatic degradation of the matrix, it should be possible to utilize the activity of degradative enzymes as an index of matrix resorption. Our laboratory has reported (27) increased collagenolytic activity after oophorectomy and a decrease after short term estrogen administration to both the intact and oophorectomized rat. The increase in collagenolytic activity after oophorectomy, associated with a decrease in ash content and bone mass as reported by others, makes increased resorption an almost inescapable conclusion, particularly in view of the direct correlation between bone resorption and collagenase release reported by Sak-
amoto et al. (28). The reversal of this state by estrogen administration is striking and, we believe, must also indicate a prolonged decrease in both calcium mobilization from bone and matrix resorption. The long term estrogen effect upon formation rates reported in these experiments is consistent with and parallel to other short term reports in the literature. Oophorectomy leads to an increased formation of collagen and proteoglycan and an increased uptake of radiocalcium. Estrogen administration reverses this effect. Previous short term studies by Langeland (29) and Langeland and Teig (30, 31) have indicated that removal of the ovaries leads to an increase in collagen synthesis and that this increase is suppressed by estrogen treatment. They also have noted a decrease in collagen breakdown after estrogen administration. Their data thus serve to confirm the alteration both in collagenolytic activity and in synthesis rates. Very little information is available with regard to the effect of estrogen upon proteoglycan metabolism in bone. Estrogen does appear to inhibit (35SO4) incorporation into chondroitin sulfate at the epiphyseal line (32, 33) and has a similar effect
The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 18 November 2015. at 08:24 For personal use only. No other uses without permission. . All rights reserved.
1192
CRUESS AND HONG
upon chondroitin sulfate synthesis in fibroblast cultures (34). The means by which estrogen exerts its effects upon the musculoskeletal system remain unclear. It has been demonstrated that tritiated estradiol is taken up by the rat femur (35), although it is not known whether this is nonspecific binding or if this binding is to the cell or to other areas of the bone. No receptor was demonstrated in osseous tissue, making it unlikely that bone is a primary target organ for the hormone (36). There are contradictory data on the influence of estrogen in tissue or organ culture of bone. In some experimental situations, estrogen fails to inhibit bone resorption (37), while other investigators have reported such an action (38). There is a suggestion (39) that the reason for any measurable effect is direct toxicity of estrogen on cell metabolism.
9.
10.
11. 12. 13.
14. 15.
16.
There have been several suggestions that estrogen affects
bone metabolism by decreasing the sensitivity of the osteocyte or osteoclast to parathyroid hormone (7, 22, 40). Our data are consistent with this hypothesis or could be explained by the reported (41) estrogen-mediated enhancement of calcium absorption by the gut with resultant decreased parathyroid activity. The possible effect of hormonal interrelationships, such as an estrogen-mediated increase in circulating vitamin D metabolites (42), awaits further investigation. Specifically, the question of whether estrogen deficiency leads either to a decreased sensitivity of bone to parathyroid hormone or to a decrease in parathyroid hormone secretion may be answered only by direct measurement of parathyroid function under controlled conditions. The data presented here indicate that this should be done in a long term experiment to determine whether the suggestion (8) that the estrogen effect is not prolonged and that decreased formation ultimately diminishes its therapeutic effectiveness is valid. In the rat it appears that this is not true. References 1. Albright, F. P., H. Smith, and A. M. Richardson, Postmenopausal osteoporosis; its clinical features, JAMA 116: 2465, 1941. 2. Albright, F., The effect of hormones on osteogenesis in man, Recent Prog Horm Res 1: 293, 1947. 3. Wallach, S., and P. H. Henneman, Prolonged estrogen therapy in postmenopausal women, JAMA 171: 1637, 1959. 4. Davis, M. E., N. M. Strandjord, and L. H. Lanzl, Estrogens and the aging process, JAMA 196: 219, 1966. 5. Meema, H. E., and S. Meema, Prevention of postmenopausal osteoporosis by hormone treatment of the menopause, Can Med Assoc J 99: 248, 1968. 6. Lindsay, R , J. M. Aitken, J. B. Anderson, D. M. Hart, E. B. MacDonald, and A. C. Clarke, Long-term prevention of postmenopausal osteoporosis by estrogen: evidence for an increased bone mass after delayed onset of estrogen treatment, Lancet 1: 1038, 1976. 7. Riggs, B. L., J. Jowsey, P. J. Kelly, J. D. Jones, and F. T. Maher, Effex of sex hormones on bone in primary osteoporosis, J Clin Invest 48: 1065,1969. 8. Riggs, B. L., J. Jowsey, P. J. Kelly, D. L. Hoffman, and C. D.
17. 18.
19. 20.
21.
22.
23.
24.
25.
26.
27. 28.
29.
30.
31.
32.
Endo • 1979 Vol 104 • No 4
Arnaud, Studies on pathogenesis and treatment in postmenopausal and senile osteoporosis, Clin Endocrinol Metab 2: 317, 1973. Folch, J., M. Lees, and G. H. S. Stanley, A simple method for the isolation and purification of total lipids from animal tissues, J Biol Chem 226: 497, 1957. Stegemann, H., Mikrobestimmung von Hydroxyproline mit Chloramin-T und /j-Dimethylamino-Benzaldehyd. Hoppe Seylers Z Physiol Chem 311: 41, 1958. Boas, N. F., Method for the determination of hexosamines in tissues, J Biol Chem 204: 553, 1953. Deiss, W. P., L. B. Holmes, and C. C. Johnston, Jr., Bone matrix biosynthesis in vitro, J Biol Chem 237: 3555, 1962. Kaufman, E. J., M. J. Glimcher, G. L. Mechanic, and P. Goldhaber, Collagenolytic activity during active bone resorption in tissue culture, Proc Soc Exp Biol Med 120: 632, 1965. Armitage, P., Statistical Methods in Medical Research, John Wiley and Sons, New York, 1971, pp. 202-207. Meema, H. E., Cortical bone atrophy and osteoporosis as a manifestation of aging, Am J Roentgenol Radium Ther Nucl Med 89: 1287, 1963. Nordin, B. E. C, J. MacGregor, and D. A. Smith, The incidence of osteoporosis in normal women: its relation to age and the menopause, QJ Med 137: 25, 1966. Garn, S. M., C. G. Rohmann, and B. Wagner, Bone loss as a general phenomenon in man, Fed Proc 26: 1729, 1967. Smith, D. A., J. B. Anderson, J. Shimmins, C. F. Spens, and E. Barnett, Changes in metacarpal mineral content and density in normal male and female subjects with age, Clin Radiol 20: 23, 1969. Saville, P. D., Changes in skeletal mass and fragility with castration in the rat; a model of osteoporosis, J Am Geriatr Soc 17: 155,1969. Aitken, J. M., E. Armstrong, and J. B. Anderson, Osteoporosis after oophorectomy in the mature female rat and the effect of estrogen and/or progestrogen replacement therapy in its prevention, J Endocrinol 55: 79, 1972. Saville, P. D., and C. S. Lieber, Increases in skeletal calcium and femur cortex thickness produced by undernutrition, J Nutr 99: 141, 1969. Orimo, H., T. Fujita, and M. Yoshikawa, Increased sensitivity of bone to parathyroid hormone in ovariectomized rats, Endocrinology 90: 760, 1972. Gallagher, J. C, M. Young, and B. E. C. Nordin, Effects of artificial menopause on plasma and urine calcium and phosphate, Clin Endocrinol 1: 57, 1972. Aitken, J. M., D. McKay Hart, and D. A. Smitty, The effect of longterm mestranol administration on calcium and phosphorous homeostasis in oophorectomized women, Clin Sci 41: 233, 1971. Aitken, J. M., D. M. Hart, and R. Lindsay, Estrogen replacement therapy for prevention of osteoporosis after oophorectomy, BrMed J 3 : 515, 1973. Budy, A. M., M. R. Urist, and F. C. MacLean, The effect of estrogens on the growth apparatus of the bones of immature rats, Am JPathol 28: 1143, 1952. Cruess, R. L., and K. C. Hong, Effect of estrogen on the collagenolytic activity of rat bone, Calcif Tissue Res 20: 317, 1976. Sakamoto, S., M. Sakamoto, P. Goldhaber, and M. Glimcher, Collagenase and bone resorption: isolation of collagenase from culture medium containing serum after stimulation of bone resorption by addition of parathyroid hormone extract, Biochem Biophys Res Commun 63: 172, 1975. Langeland, N., In vitro studies on collagen metabolism in metaphyseal rat bone. A. The effect of pre-treatment with estradiol-17/?, Acta Endocrinol (Kbh) 80: 775, 1975. Langeland, N., and V. Teig, In vitro studies on collagen metabolism in metaphyseal rat bone. B. The early effects of estradiol-17/?, Acta Endocrinol {Kbh) 80: 784, 1975. Langeland, N., and V. Teig, In vitro studies on collagen metabolism in metaphyseal rat bone. C. The effect of estradiol-17/8 hypophysectomized and in thyroparathyroidectomized animals, Acta Endocrinol (Kbh) 80: 795,1975. Endo, H., S. Murota, and H. Enomoto, Effect of hormones on the chondroitin sulfate metabolism of chick embryo femora growing in
The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 18 November 2015. at 08:24 For personal use only. No other uses without permission. . All rights reserved.
ESTROGEN EFFECT ON BONE METABOLISM
33. 34. 35.
36.
37.
vitro. III. Effect of sex steroids on chondroitin sulfate synthesis in the cartilagenous bones growing in the natural medium, Endocrinol Jap 16: 115, 1969. Herbai, G., Autoradiographic studies with S-sulfate on somatotrophin and estrogen sensitive growth zones in rat and mouse costal cartilage, Ada Physiol Scand 79: 351, 1970. Nordbo, H., The effect of estradiol-17/? on the metabolism of chondroitin sulphate B, Steroidologia 2: 7, 1971. Anderson, J. J. B., and J. M. Floeckher, Skeletal uptake of H3estradiol by rats primed with nonradioactive estrogen and parathyroid extract, Isr J Med Sci 7: 353, 1971. Nutik, G., and R. L. Cruess, Estrogen receptors in bone. An evaluation of the uptake of estrogen into bone cells, Proc Soc Exp Biol Med 146: 265, 1974. Caputo, C. B., D. Meadows, and L. G. Raisz, Failure of estrogens
38.
39. 40. 41.
42.
1193
and androgens to inhibit bone resorption in tissue culture, Endocrinology 98: 1065, 1976. Atkins, D., J. M. Zanelli, M. Peacock, and B. E. C. Nordin, The effect of estrogens on the response of bone to parathyroid hormone in vitro, J Endocrinol 54: 107, 1972. Liskova, M., Influence of estrogens on bone resorption in organ culture, Calcif Tissue Res 22: 207, 1976. Heaney, R. P., A unified concept of osteoporosis, Am J Med 39: 877, 1965. Caniggia, A., Intestinal absorption of calcium-47 after treatment with oral estrogen-gestogens in senile osteoporosis, Br Med J 4: 30, 1970. Castillo, L., Y. Tanaka, H. F. deLuca, and M. L. Sunde, The stimulation of 25-hydroxyvitamin D,)-l,,-hydroxylase by estrogen, Arch Biochem Biophys 179: 211, 1977.
The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 18 November 2015. at 08:24 For personal use only. No other uses without permission. . All rights reserved.
