Influence of sex steroids on development of cultured fetal rat metatarsal bones

The effects of 17 beta-estradiol (E), dihydrotestosterone (D, a non aromatisable androgen), and progesterone (P) on osteogenesis were studied on fetal rat cartilaginous anlagues cultivated in vitro. The three medial metatarsal rudiments were harvested at day 19 of gestation and grown in 1% BSA MEM medium (MO 643, Sigma) without serum nor antibiotics. After a 18h preincubation period, hormones were added for 8 days. Paired controls were incubated in the same volume of medium. The length, the metacarpal thickness and the size of the mineralized zone were measured every day, using a calibrated eyepiece (magnification X 40). DNA and protein synthesis, cartilage metabolism and mineralization were evaluated by monitoring the incorporation of 3H-Thymidine, 3H-Proline, 35S and 45Ca into anlagues for the last three hours of incubation, respectively. The dose/response effect of each steroid was studied at the concentrations of 10(-4) M, 10(-6) M, 10(-7) M and 10(-9) M. No difference was observed between male and female fetuses. A significant positive effect on total length (% of length measured at harvesting day) was observed with the 10(-7) M dose of E (163% +/- 2 vs 148% +/- 4 in controls) or D (158% +/- 3). Endochondral growth was not modified by P treatment. The effect of the three steroids (given at a dose of 10(-7) M) alone or as combinations (E, D, P, EP, ED, PD, EPD) confirmed the positive effect of E on endochondral growth and, to a lesser extend, of D and the association ED. Nevertheless, D had a better effect than E on endomembranous growth. On the contrary, P did not affect growth neither administrated alone nor in combination with E or D, while a positive effect of P on mineralization was demonstrated. The treatment associating the three steroids slowed down all the parameters concerning growth but strongly stimulated calcification.