Microtubules and the Calcium-Dependent Regulation of Rat Granulosa Cell Steroidogenesis1

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Abstract
The possible relationship between calcium and microtubules in the regulation of granulosa cell steroidogenesis was assessed by using agents known to alter microubule-tubulin equilibrium together with the ionophore A23187, an antibiotic that facilitates the movement of calcium across plasma membranes. Using immunofluorescence and morphometric analysis, we determined alterations in microtubule organization and overall cell shape, respectively, in response to ionophore-stimulated production of progesterone and 20.alpha.-hydroxypregn-4-en-3-one (20.alpha.-OH-progesterone) during 24 h of culture. In addition, the influences of colchicine and nocodazole, two agents known to induce microtubule depolymerization, and of taxol, an agent that stabilizes tubulin polymers, on calcium-dependent regulation of granulosa cell progestin production in vitro were examined. Cells cultured as controls were flattened, highly irregular in outline, and associated with a complexly organized, well-spread cytoplasmic network of microtubules. In contrast, those maintained in the presence of increasing concentrations of ionophore were progressively more circular and smooth in outline, occupied less area on the growth surface, and contained cytoplasmic arrays of microtubules considerably less extensive than those of the controls and occupying areas defined by the more regular cellular perimeters. While progestin production in the absence or presence of a submaximally stimulatory concentration of A23187 was increased by both colchicine and nocodazole, the microtubule-depolymerizing agents had little or no effect on the production of the steroids by granulosa cells maximally stimulated by the ionophore. However, both basal and ionophore-induced progestin production were unaltered by taxol except at a concentration of 10 .mu.M in the presence of 0.25 .mu.g/ml A23187. These findings indicate that both microtubules and calcium are important in the regulation of granulosa cell steroidogenesis in vitro. Furthermore, calcium appears to alter the distribution of microtubules in cultured granulosa cells. However, it is possible that microtubules and calcium may act at distinct sites in the control of this biosynthetic process.