Sandwich Configuration of Type I Collagen Suppresses Progesterone Production in Primary Cultured Porcine Granulosa Cells by Reducing Gene Expression of Cytochrome P450 Cholesterol Side-Chain Cleavage Enzyme

Abstract

When porcine granulosa cells were cultured on type I collagen (TIC)-coated dishes, progesterone was continuously secreted in the culture medium. However, when cells were overlaid with a TIC gel, progesterone production was decreased to 34% (day 3) and 16% (day 4) of the value measured for cells without the overlay. The effect of TIC gel overlay on cytochrome P450 cholesterol side-chain cleavage enzyme (P450scc), which catalyzes the conversion of cholesterol to pregnenolone and is thought to be the rate-limiting enzyme in the conversion of cholesterol to progesterone, was examined. P450scc gene expression in cells overlaid with a TIC gel was decreased to 62% (day 3) and 36% (day 4) of the value measured for cells without the overlay. Amounts of P450scc were also reduced in the cells overlaid with a TIC gel. When pregnenolone, the direct precursor of progesterone, was added to the culture medium, the increase in progesterone production by cells overlaid with a TIC gel was much greater than that for cells without a TIC gel and a statistical difference in progesterone production was no longer observed between the two groups of cells. Treatment of the cells with human FSH (hFSH) enhanced progesterone production in a dose-dependent manner, irrespective of the presence of a TIC gel overlay. Moreover, hFSH induced P450scc gene expression in cells with and without a TIC gel overlay. These results indicate that a TIC gel overlay reduces progesterone production in granulosa cells via the suppression of P450scc gene expression. This supports the possibility that the existence of a TIC gel on the apical side of granulosa cells prevents the spontaneous luteinization of granulosa cells cultured on TIC-coated dishes. The fact that hFSH overcomes the suppressive effect of the TIC gel overlay on progesterone production may explain the mechanism for the subtle rise in serum progesterone concentration in the late follicle phase of the “in vitro fertilization” program.