The biological effects of dihydrotestosterone (DHT) and testosterone (T) on cultured human fetal epiphyseal chondrocytes were assessed by studying the ability of these androgens to promote DNA synthesis. DNA synthesis was evaluated by measuring [3H]thymidine incorporation into DNA. After 48-h incubation in Ham's F-12 serum-free medium, chrondrocytes were incubated with or without DHT (10(-11)-10(-8) M) or T (10(-11)-10(-8) M) in MCDB-104 serum-free medium for a further 48 h, with the addition of [3H]thymidine (5 microCi/mL) for the last 24 h. In chondrocytes from five male fetuses (12-40 weeks' gestation) DHT and T significantly stimulated DNA synthesis. The maximum stimulatory effect was obtained for DHT at 10(-10) M (P less than 0.01) and for T at 10(-6) M (P less than 0.02). In chondrocytes from four female fetuses the stimulatory effect was significant only for DHT and was maximum at 10(-10) M (P less than 0.02), whereas no effect was observed for T. Cultured chondrocytes from both male and female fetuses show the presence of proteins with high affinity and limited binding capacity (Bmax) for DHT (male fetuses: Bmax, 4.9 +/- 1.9 x 10(-15) M/mg protein; Kd, 0.43 +/- 0.24 x 10(-9) M; female fetuses: Bmax, 4.8 +/- 1.6 x 10(-15) M/mg protein; Kd, 0.63 +/- 0.19 x 10(-9) M) with no significant differences between sexes. In conclusion, our results show that androgens elicit a biological response in cultured human fetal epiphyseal chondrocytes and that DHT-binding sites are present in these cells. DHT, rather than T, seems to be the active androgen. A sex difference in the degree of androgen action is also documented.

The effects of T3 on cultured human fetal epiphyseal chondrocytes were assessed by studying its effects on DNA synthesis and alkaline phosphatase activity. DNA synthesis was evaluated as follows: after 48-h incubation in Ham's F-12 serum-free medium, cultured chondrocytes were incubated with or without T3 (0.1-100 nM) in MCDB-104 serum-free medium for different periods of time (2-10 days), with the addition of [3H]thymidine (5 microCi/mL) for the last 24 h. Confluent cultured chondrocytes in 25-cm2 tissue culture flasks were incubated in Ham's F-12 serum-free medium for up to 9 days with or without T3 (0.1-100 nM); the cellular cytoplasmic fraction was obtained, and alkaline phosphatase activity was evaluated using paranitrophenylphosphate as a substrate. No significant effects of T3 (0.1-100 nM) on DNA-[3H]thymidine incorporation were observed in any experiment (n = 17) for any gestational age (12-39 weeks) or for any incubation period studied (2-10 days). However, a significant (P less than 0.025 or more) stimulatory effect of T3 (0.1-100 nM) on alkaline phosphatase activity was observed after 9 days of incubation. This effect was highest for 5 nM T3 and was present in cultured chondrocytes from human fetuses of all ages studied (13-40 weeks). Cultured human fetal epiphyseal chondrocytes from human fetuses 12-40 weeks old (n = 8) showed specific nuclear binding sites for T3. The binding capacity was 27.14 +/- 2.84 fmol/100 micrograms DNA, and the Kd was 0.66 +/- 0.14 x 0.1 nM (mean +/- SEM), with no significant differences among fetal ages. In conclusion, our results show that T3 elicits a biological response in cultured human fetal epiphyseal chondrocytes and has specific nuclear binding sites. Since alkaline phosphatase is closely related to the mineralization of epiphyseal cartilage, these results suggest that thyroid hormones could regulate this process.

ABSTRACT. Somatostatin effects on cultured human fetal epiphyseal chondrocytes were evaluated by studying the effects of somatostatin on DNA synthesis. Cultured epiphyseal chondrocytes from human fetuses (12-40 wk old) were incubated for 48 h in Ham's F-12 serum-free medium. After this, the medium was replaced by MCDB-104 serumfree medium and the cells were incubated for an additional 48 h in the presence or absence of somatostatin 1 pM to 10 pM, with the addition of 3H-thymidine (5 pCi/mL) for the last 24 h of incubation. A significant (p < 0.02) inhibitory effect of somatostatin (1 nM to 10 pM) on 3H-thymidine DNA incorporation was observed in cultured chondrocytes from fetuses of all gestational ages studied (12-40 wk), with no significant differences among fetal ages. In conclusion, our results show that somatostatin exerts a biologic effect on cultured human fetal epiphyseal chondrocytes, as it does in its target cells. These results suggest that somatostatin could regulate human skeletal growth not only by growth hormone secretion regulation, but also by acting directly on chondrocyte metabolism. However, the physiologic significance of the latter remains to be elucidated. (Pediatv Res 32: 571-573, 1992)

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