Direct and Sex-Specific Enhancement of Bone Formation and Calcification by Sex Steroids in Fetal Mice Long Bonein Vitro(Biochemical and Morphometric Study)^*

Abstract

The study was carried out to examine the direct effect of the sex hormones 17 beta-estradiol (E2) and testosterone on the modeling of cultured fetal mouse long bones separated according to their sex. The culture system used allowed for the simultaneous assessment of bone growth, mineralization, and resorption on each bone. Bones from 16-day-old male and female mouse fetuses were cultured in BGJ medium, supplemented with either 10% fetal calf serum or 4 mg/ml BSA (serum-free medium) for 48 h. The bones were harvested, and their length; the length of their diaphyses; their hydroxyproline, calcium, and phosphorus contents; and their 45Ca release were measured. Histomorphometric analyses on midlongitudinal sections of bones from parallel experiments were also performed. The results indicate that in medium supplemented with 10% fetal calf serum, E2 had a dose-dependent stimulatory effect on bone formation and mineralization at 10(-7) and 10(-9) M, with no effect on bone resorption. This effect was specific to bones from female mice and to E2, since 17-alpha-estradiol had no effect. Testosterone had similar effects specific to bones from male mice, resulting in the stimulation of bone formation and mineralization at 10(-7)- and 10(-9)-M concentrations. These effects were absent when serum-free medium was used. E2 and testosterone had an anabolic effect on endochondral and periosteal bone formation and mineralization, but no effect on bone resorption. This effect is dependent on the presence of a serum factor(s).

Androgen Receptor Modulation Does Not Affect Longitudinal Growth of Cultured Fetal Rat Metatarsal Bones

“…In line with our findings, AR stimulation was previously demonstrated not to affect the length of cultured fetal rat metatarsal bones [27]. Nevertheless, androgen responsiveness might differ between species, which is supported by the observations that dihydrotestosterone increases DNA synthesis in fetal human epiphyseal chondrocytes [28], and that testosterone stimulates the growth of mouse cartilage [29, 30]. Nevertheless, in cultured fetal rat metatarsal bones, dihydrotestosterone has been demonstrated to prevent the negative effects of glucocorticoids [27], raising the possibility that rat cartilage might be androgen-responsive anyway.…”

“…This is supported by our recent demonstration that aromatase is indeed expressed in fetal rat metatarsal bones, and that endogenously produced estrogens are capable of stimulating chondrocyte proliferation and fetal rat metatarsal bone growth [18]. Interestingly, it has previously been shown that testosterone is capable of stimulating growth of fetal mouse metatarsal bones only when cultured in the presence of serum [29]. This finding, together with our present observation that oxandrolone does not affect bone growth in the presence of serum, suggest that aromatization of testosterone occurs within the growth plate.…”

Localization of estrogen receptors-alpha and -beta and androgen receptor in the human growth plate at different pubertal stages

“…This is in agreement with the findings of most previous immunohistochemical studies in human (Abu et al 1997, Kusec et al 1998, Bord et al 2001, rat (van der Eerden et al 2002a and rabbit (Kusec et al 1998 growth plate cartilage. Thus the sex-specific effects of sex steroids suggested by the findings of some in vitro studies (Corvol et al 1987, Blanchard et al 1991, Schwartz et al 1991, do not appear to be caused by sex-specific receptor expression. However, immunohistochemistry is a semi-quantitative method, and real differences could have been overlooked by this technique.…”

“…Several in vitro and in situ studies have suggested that the sex steroid responsiveness of growth plate cartilage is dependent on the sex (Carrascosa et al 1990, Schwartz et al 1991, Nasatzky et al 1993, van der Eerden et al 2002b or the age, or both (Corvol et al 1987, Blanchard et al 1991, Pinus et al 1993) of the individual. However, few studies have addressed this issue in human growth plate cartilage.…”

Dihydrotestosterone Prevents Glucocorticoid-Negative Effects on Fetal Rat Metatarsal Bone in vitro

“…4): this might partly explain why DHT had no significant direct effect per se on any parameter tested in this experiment. However, Schwartz et al [53] did not demonstrate any effect of testosterone (10 -7 M) on bone resorption measured by 45 Ca release from fetal long bones after 48 h of culture. Furthermore, the therapeutic potential should be tapered by the potential disadvantage of giving testosterone or DHT which have several potential side effects.…”