Stanislawa Stoklosowa

The effect of oxytocin on steroid hormone secretion by isolated porcine follicular cells in tissue culture

Abstract The effect of two doses (10 −7 M or 2 × 10 −7 M ) of oxytocin (OT) on progesterone, estradiol and androgen secretion by isolated follicular cell types: theca interna (T) and granulosa (G) cells in monolayer culture has been examined. Three culture types were grown: T alone, G alone, and coculture of granulosa and theca cells (GT). The direct action of oxytocin was shown by: 1. the decrease of progesterone secretion by G cells cultured alone under the influence of the higher dose (2 × 10 −7 M ), 2. the decline of estradiol and androgen secretion by cocultures, 3. the change of secretory pattern of estradiol and androgen secretion in coculture.

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

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.

Hormonal secretion of cultured rat ovarian follicles isolated at various hours of proestrus

SummaryRat ovarian follicles were isolated at nine different times during proestrus and cultured for 3 d. During the period of cultivation, estrogen and progestagen secretion was measured. Other follicles were dissected out at the same time periods and analyzed directly for these hormones. The most intense hormone release was observed during the first 24 h in culture. The concentration of steroids secreted in vitro by follicles previously collected on the morning of proestrus was relatively low. The secreted estrogen concentration increased gradually from follicles previously isolated at later times during proestrus, with a maximum at 1800. The peak secretion of progestagens in culture was from follicles isolated at 2000. There was a marked decrease in secretion of both hormones from follicles isolated past their optimal times. The same secretion pattern of estrogens and progestagens was shown by follicles in vitro at 24 h as with similar follicles in vivo, according to their time of isolation. From these results it is evident that the normal rhythm of steroid secretion, predetermined at the time of follicle excision, was maintained during 24 h in organ culture. After a longer period in culture, the dynamics of hormone synthesis became altered, although follicles appeared morphologically normal up to 48 h.

UItrastructure of Theca Interna Cells of Porcine Ovarian Follicle

The theca interna cells display organelles typical for steroid‐producing cells. This is more obvious in the preovulatory phase than in the luteal phase. Parallel to the growth of the follicle the amount of smooth endoplasmic reticulum, of lipid droplets and of lysosomes increases. Concurrently, the amount of rough endoplasmic reticulum decreases. Junctions of the “abutment nexus” type are more numerous in the preovulatory follicle.

The effect of microtubule-disrupting drugs on morphology, progesterone and prorenin secretion of bovine cultured ovarian theca cells.

The effect of three microtubule-disrupting drugs (vinblastine, colchicine and nocodazole) on basal and LH-stimulated secretion of progesterone and prorenin by bovine theca cells was studied. Microtubules were visualized immunocytochemically using a monoclonal antibody against the alpha-subunit of tubulin and a secondary antibody conjugated with rhodamine. Progesterone and prorenin secretion were detected by adequate radioimmunoassays. Theca cells treated with LH alone or with a combination of LH, colchicine and vinblastine, showed round shape and disorganization of microtubules which were more obvious than after treatment with the two disrupting drugs alone. Nocodazole or LH treatment alone resulted in the formation of numerous cell processes, conspicuously different in shape from those in the controls. All three drugs increased basal progesterone secretion independently on the dose, but were without effect on basal prorenin secretion. LH-stimulated progesterone secretion was significantly suppressed by each of the three drugs while LH-stimulated prorenin secretion was decreased only by high doses of vinblastine and nocodazole. Colchicine in all three doses used did not exert any significant effect. The results obtained suggest that microtubules are involved in progesterone but not in prorenin secretion.

Corpus luteum cells grown as monolayers

Die Fähigkeit der Corpus-luteum-Zellen als Monolayers zu wachsen wurde untersucht. Die von Schwein und Ratte entnommenen Gelbkörper wurden zerkleinert und mit einer 0,25% igen Trypsinlösung in eine Suspension übergeführt. Die Kulturen wurden bis zum 8. Tag beobachtet. Die wachsenden Luteinzellen zeigen die typische Form und auch die ausgeprägte Aktivität der Δ5-3β-OH Steroiddehydrogenase.