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BC van der Eerden, CW Lowik, JM Wit and M Karperien

Estrogens are essential for bone mass accrual but their role before sexual maturation has remained elusive. Using in situ hybridization and immunohistochemistry, we investigated the expression of both estrogen receptor (ER) alpha and beta mRNA and protein as well as several mRNAs coding for enzymes involved in sex steroid metabolism (aromatase, type I and II 17 beta-hydroxysteroid dehydrogenase (17 beta-HSD), steroid sulfatase (STS) and type I 5 alpha-reductase) on sections of tibial metaphyses before (1- and 4-week-old), during (7-week-old) and after (16-week-old) sexual maturation in female and male rats. ER alpha and ER beta mRNA and protein were detected in metaphyseal bone in lining cells, osteoblasts, osteoclasts and some osteocytes with no apparent differences in expression during development or between the sexes. In contrast, aromatase, type I and II 17 beta-HSD and type I 5 alpha-reductase mRNAs were first detected in osteoblasts, osteoclasts and occasionally in osteocytes from sexual maturation (7-week-old rat) and onwards. Only STS was present before sexual maturation. To study the significance of ER alpha and beta expression in bone before sexual maturation when circulating sex steroid levels are low, 26-day-old female and male rats underwent gonadectomy or 17 beta-estradiol (E(2)) supplementation (0.5 mg/21 days) during 3 weeks. Following gonadectomy, trabecular bone volume (TBV) was lower in males (P=0.03) and there was a trend towards reduction in females (P=0.057). E(2) supplementation increased tibial TBV compared with controls in both genders as assessed by Masson-Goldner staining. These data suggest that the presence of ERs in bone cells before sex maturation might be of significance for bone mass accrual. Furthermore, based on the mRNA expression of the crucial enzymes aromatase and type I 17 beta-HSD, we suggest that bone cells in the tibial metaphysis acquire the intrinsic capacity to metabolize sex steroids from sexual maturation onwards. This process may contribute to the beneficial effects of estrogen on bone mass accrual, possibly by intracrinology.

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RL van Bezooijen, I Que, AG Ederveen, HJ Kloosterboer, SE Papapoulos and CW Lowik

Nitric oxide (NO) is a mediator of bone metabolism and its production is under the control of gender hormones in several cell types or tissues. Changes in endogenous NO production, measured as plasma nitrate+nitrite levels, may therefore contribute to ovariectomy (OVX)-induced bone loss. We studied plasma nitrate+nitrite levels and trabecular bone mineral density (TBMD) 4 weeks after sham-operation or OVX in rats receiving various hormonal treatments. OVX decreased plasma nitrate+nitrite levels significantly and this was accompanied by a significant decrease in TBMD. Treatment with oral ethinyl oestradiol (EE) and subcutaneous 17beta-oestradiol dose-dependently prevented the decrease in plasma nitrate+nitrite levels after OVX, but treatment with oral 17beta-oestradiol did not. Oestrogen treatment, 17beta-oestradiol (s. c. or orally) or EE (orally), prevented the OVX-induced decrease in TBMD. Treatment of sham-operated rats with the anti-oestrogen ICI164, 384 induced a significant decrease in TBMD that corresponded to 54% of the decrease observed after OVX, but did not affect plasma nitrate+nitrite levels. Treatment of ovariectomized rats with Org 2058, a pure progestagen, did not prevent bone loss, but prevented the decrease in plasma nitrate+nitrite levels dose-dependently. Treatment with tibolone, a synthetic steroid with combined weak oestrogenic, progestagenic, and androgenic properties, or with progestagen in combination with EE completely prevented bone loss after OVX. These treatments, however, only partly prevented the OVX-induced decrease in plasma nitrate+nitrite levels. In conclusion, OVX decreased both TBMD and plasma nitrate+nitrite levels. Although plasma nitrate+nitrite levels were under the control of both oestrogen and progesterone, TBMD was affected by oestrogen only. Decreased systemic production of NO is, therefore, not involved in OVX-induced bone loss in rats.

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BC van der Eerden, J Emons, S Ahmed, HW van Essen, CW Lowik, JM Wit and M Karperien

Recently, both estrogen receptor (ER) alpha and beta were detected in growth plate chondrocytes of rats before sexual maturation, implying a role for estrogen at this stage. In this study, therefore, we investigated the effects of ovariectomy (OVX) or estrogen supplementation on parameters of longitudinal growth in 26-day-old rats, which were sexually immature at the start of the experiment. OVX caused an increase in body weight gain, tibial length and growth plate width due to an increased proliferating zone. This increase correlated with an increase in cell number, with a decrease in cell diameter and with increased proliferating cell nuclear antigen (PCNA) immunostaining compared with sham. Interestingly, the increase in proliferation was not caused by an increase in insulin-like growth factor-I (IGF-I) mRNA expression in the growth plate as assessed by real-time PCR. In contrast to OVX, 17beta-estradiol (E(2)) supplementation (0.5 mg/21 days) of 26-day-old female rats caused a strong decrease in body weight gain, tibial length and growth plate width. The latter was explained by a reduction of the proliferating zone width, which correlated with a reduced number of PCNA-positive cells (not significant) and by a reduction of the hypertrophic zone width. In male rats supplemented with E(2), similar effects were observed compared with the females. ERalpha and beta immunostaining was found predominantly in late proliferating and early hypertrophic chondrocytes. OVX did not affect ER expression but E(2) supplementation strongly decreased immunostaining for both ERalpha and beta in both sexes. Besides E(2), desoxyestrone (DE), an activator of nongenomic estrogen-like signaling (ANGEL) and 2-methoxyestradiol (2-MeO-E(2)), a tissue-selective naturally occurring metabolite of E(2), were administered to female and male rats of the same age. Compared with E(2), these compounds had less pronounced, though significant, effects on some parameters of longitudinal growth in both sexes, especially on growth plate characteristics. In conclusion, E(2) may exert effects on longitudinal growth before and at the onset of sexual maturation, despite very low endogenous serum levels at these stages. There may be a role for nongenomic signaling in body weight gain, tibial length and growth plate width but genomic signaling prevails.