Mechanisms Subserving Calcium’s Modulation of Luteinizing Hormone Action in Isolated Swine Granulosa Cells*

Abstract

We studied the mechanism(s) by which calcium ions modulate progesterone biosynthesis by isolated swine granulosa cells incubated in chemically defined medium in vitro. In selectively calcium-deficient incubations, the capacity of 8-bromo-cAMP to stimulate pregnenolone synthesis from endogenous sterol substrate was significantly impeded. This effect of calcium ions was specific, because calcium ions did not influence basal pregnenolone production or alter progesterone production in response to exogenously supplied cholesterol substrate. Moreover, calcium ions did not modify other biosynthetic processes in granulosa cells, such as de novo synthesis of cholesterol from [14C]acetate or the aromatization of testosterone to 17 beta-estradiol. The possible role of calmodulin in mediating calcium's actions in pig granulosa cells was tested by measuring the calmodulin content of these cells and assessing the functional responses to classical calmodulin antagonists. By immunoassay, swine granulosa cells contained high concentrations of calmodulin, viz. 4.21-4.88 micrograms calmodulin/mg protein. Moreover, calmodulin antagonists inhibited LH-stimulated progesterone production with the following rank order of potencies [estimated by half-maximally inhibitory concentrations (ID50)]: penfluridol (1 microM), trifluoroperazine (9 microM), chlorpromazine (95 microM), and trifluoperazine sulfoxide (greater than 300 microM). In addition, the nonphenothiazine calmodulin antagonist W7 inhibited stimulated progesterone production with an ID50 of 16.7 microM. W5 was less active. None of these antagonists significantly suppressed LH-stimulated cAMP generation at the low concentrations capable of inhibiting progesterone production. The effects of calcium ions seemed to depend upon the availability of intracellular pools of calcium, because TMB-8, an inhibitor of intracellular calcium mobilization, effectively suppressed LH-stimulated progesterone production (ID50, 18 microM). However, even 100 microM TMB-8 failed to alter basal progesterone production or suppress LH-stimulated cAMP generation in these cells. In summary, the present studies indicate that calcium ions significantly modulate LH's stimulation of pregnenolone biosynthesis from endogenous cholesterol substrate in swine ovarian cells. Calcium does not influence basal pregnenolone production, estrogen synthesis from androgen substrate, de novo biosynthesis of cholesterol from [14C]acetate, or progesterone production from exogenously supplied sterol substrate.(ABSTRACT TRUNCATED AT 400 WORDS)

Effects of Cadmium on Ultrastructure and Steroidogenesis in Cultured Porcine Ovarian Granulosa Cells

“…Basal progesterone production of cadmium-treated cells remained unchanged (20 ng/ml) or was enhanced; this supports the theory of cadmium -calcium competition. Calcium has been reported not to influence basal pregnenolon production or estrogen synthesis from androgen substrates, or progesterone production from exogenously supplied sterol substrates in porcine granulosa cells whereas it is known that calcium ions influence the stimulatory action of LH and modulate the effects of cAMP on progesterone biosynthesis (Veldhuis et al 1984).…”

Ovarian Carbonyl Reductase Delayed Onset of Persistent Estrus in the Offspring of Parathyroidectomized Mother Rats

“…The results that the ovarian CR activity and its concentration in Px-F1 rats, which had regular cyclicity, were higher than those in normal rats, which had persistent estrus, indicate that the maternal parathyroidectomy may positively influence the hypothalamo-pituitary-ovarian axis in the female offspring. Furthermore, Veldhuis et al (21) have reported that an inhibitor of intracellular calcium mobilization and calmodulin antagonists inhibit LH-stimulated progesterone production and suggested that calcium ions modulate LH action in ovarian cells in part by a calciumcalmodulin-dependent mechanism. Accordingly, the altered calcium metabolism during the fetal life may cause the delayed onset of persistent estrus in Px-F1 female rats.…”

Effects of Age and Calcium Ion on Testis Carbonyl Reductase in Rats

“…It has been suggested that the stimulatory effects of PGE1 and hCG on adenylate cyclase activity in the Leydig cells of rats (30-40 days of age) are biphasically regulated by calcium ions (8) and that calcium ions modulate LH ac tion in swine granulosa cells (10). Wiltbank et al (23) sug gested that the luteolytic action of PGF2a in the large lu teal cells may be mediated by intracellular calcium by ac tivating protein kinase C, inactivating adenylate cyclase, decreasing cAMP phosphodiesterase activity and decreas ing various metabolic enzyme activities.…”

“…Calcium ions appear to play an important role in gonadotropin-induced steroidogenesis as well as the ac tion of cyclic AMP in ovary and testis (7)(8)(9)(10)(11). Janszen et al (7) have shown that calcium ion potentiates the effect of LH on testosterone production in a suspension of whole interstitial cells from rat testis.…”