Progesterone secretion and proliferation in cultured rabbit granulosa cells under conditions of β-D-xyloside-induced inhibition of proteoglycan synthesis

Abstract

Proteoglycans present in follicular fluid are synthesized by granulosa cells under gonadotropin control. An inhibitor of proteoglycan synthesis, p- nitrophenyl-β-D-xyloside (β-D-xyloside) was used as a probe to study rabbit granulosa cell steroidogenesis and proliferation under abrogated proteoglycan synthesis. Granulosa cells isolated from rabbit preovulatory follicles were cultured 24 h in Minimum Essential Medium plus 2.5% fetal calf serum in the presence or absence of β-D-xyloside and were then treated with FSH or dibutyryl cAMP (db-cAMP) alone or in combination with β-D-xyloside for a further 24 h. The exposure for 48 h of granulosa cells to 1 mM β-D-xyloside in the absence or presence of FSH inhibited proteoglycan synthesis and increased the amount of glycosaminoglycans (GAG). FSH-stimulated progesterone production was significantly correlated only with proteoglycan synthesis and not with GAG production. The addition of various concentrations of β-D- xyloside (0.1-4 mM) for 48 h to granulosa cells induced a dose-dependent inhibition of FSH-stimulated progesterone secretion and [3H]thymidine incorporation into DNA. β-D-Xyloside concentrations lower than 1 mM induced an inhibition of FSH-stimulated progesterone secretion but had no significant effect on FSH-induced proliferation. One millimolar β-D-xyloside did not modify basal progesterone production, but in the presence of various doses (0.1-2.5 ng/ml) of FSH or hCG (0.1-1 IU/ml) it exerted a significant inhibitory effect on steroid secretion. Fifty percent inhibition was obtained for doses of FSH above 0.5 ng/ml. When cells were stimulated by db cAMP instead of FSH, the inhibition of progesterone synthesis induced by 1 mM β- D-xyloside was slightly significant. This dose of β-D-xyloside markedly inhibited FSH-induced cAMP production. Thus, it seems that the major consequence of proteoglycan synthesis abrogation was at a step beyond cAMP synthesis. These results suggest a role of proteoglycan metabolism in maintaining important functions of granulosa cell steroidogenesis and proliferation.