The effect of microtubule and microfilament-disrupting drugs on prolactin-stimulated progesterone synthesis and secretion by cultured porcine theca cells

Abstract

The effect of microtubule- and microfilament-disrupting drugs (colchicine, cytochalasin B and D) on basal and prolactin (Prl)-stimulated progesterone synthesis and secretion was studied. Microtubules were visualized immunocytochemically using a monoclonal antibody against the alpha-subunits of tubulin, while microfilaments were detected using a polyclonal antibody against actin. The second antibody was conjugated with FITC. Progesterone, androgen and estradiol concentration were detected in tissue and culture medium by appropriate radioimmunoassays. In Prl-treated cultures microtubules formed a network with more radial organisation than in the controls. After colchicine-treatment the cells were round, regular in shape and microfilaments were disintegrated and replaced by punctate aggregates. Actin filaments formed typical stress fibers in theca cells (Tc). In Prl-treated cells some microfilaments were broken resulting in a diffuse immunofluorescent pattern. After treatment with cytochalasin B and D many of the stress fibers disappeared, the cells became rounded and diffuse microfilaments were seen. Prl added to the culture medium increased synthesis of all investigated steroids and additionally stimulated progesterone secretion. Exposure of theca cells to colchicine caused an increase of basal progesterone secretion into the incubation medium which simultaneously decreased the steroid content in the cells. Colchicine suppressed Prl-stimulated synthesis of all three steroids studied and did not have any effect on their secretion. Exposure of theca tissue to cytochalasin B increased basal progesterone and androgen synthesis but drastically decreased basal estradiol synthesis by these cells. The opposite effect was observed after the addition of cytochalasin D, when estradiol synthesis increased and progesterone and androgen synthesis by theca cells was not affected. Microtubule and microfilaments-disrupting drugs had no effect on the secretion of the investigated steroids in Prl-treated cells. Our results suggest that microtubules and microfilaments are involved in Prl-stimulated steroid synthesis but not in steroid secretion.