Stimulatory effect of gonadotropins on the synthesis of adenosine 3′ :5′-cyclic monophosphate and progesterone by suspensions of rat ovarian interstitial cells

Abstract

A cell suspension was prepared from immature rat ovaries by treatment with trypsin and collagenase. The isolated cells were capable of converting [8-'4C]adenine to cyclic ['4C]AMP and the rate of this conversion was stimulated in vitro by luteinizing hormone and human chorionic gonadotropine, but not by prolactin, norepinephrine, dopamine or albumin. The accumulation of progesterone was also measured in these cells by radioimmunoassay. In vitro addition of luteinizing hormone stimulated the accumulation of radioimmunoassayable progesterone. The conversion of [8-' 4 C] adenine to cyclic [, 4 C] AMP showed a rapid increase during the first 30 min of the incubation period when luteinizing hormone was added to the incubation medium. Progesterone accumulation in response to the same dose of luteinizing hormone showed a lag period for the first 30 min of incubation after which there was an increase up to 2 h. The luteinizing hormone-induced progesterone accumulation was sensitive to puromycin, but there was no effect on the luteinizing hormone-induced increase in cyclic ['4C]AMP formation from [8-'4C]adenine. Actinomycin D also inhibited the luteinizing hormone-induced progesterone accumulation, with no effect on cyclic AMP formation. The results suggest that the luteinizing hormone-induced progesterone accumulation in rat ovarian interstitial cell suspension is preceded by an increased accumulation of cyclic AMP and that the accumulation of steroid under the influence of luteinizing hormone involve processes sensitive to puromycin and antinomycin D.

Receptor mediated gonadotropin action in the ovary

“…Previously we have shown that collagenase dis¬ persed cells from ovaries of 26 day old rats respond to LH/hCG with increased accumulation of cAMP (Kawano et al 1975;Clark & Menon 1976;Azhar & Menon 1979a;Sen et al 1979), activation of protein kinase Clark et al 1976;Azhar & Menon 1978) and increased steroidogene¬ sis (Kawano et al 1975;Clark & Menon 1976;Azhar et al 1978b;Azhar & Menon 1979b;Sen et al 1979). Employing this system we have attempted to clarify the role of RNA and protein synthesis in LH/hCG stimulated progesterone production.…”

“…In addition, the role of RNA synthesis in LH induced steroidogenesis in the ovary is still inconclusive (Marsh 1976). In vitro studies with bovine corpus luteum slices (Savard et al 1965), rat ovarian cells (Kawano et al 1975) and isolated Graafian follicles (Lieberman et al 1975), actinomycin D has been demonstated to suppress the LH/hCG stimulated steroidogenesis. In contrast, Gorski & Padnos (1966) observed no effect of actinomycin D on gonadotrophin stimulated steroi¬ dogenesis in rabbit ovary.…”

“…The intracellular mechanism bv which LH stimulates steroidogenesis and the role of macromolecules in this process are not fully understood. Although a requirement for macromolecular synthesis, particularly protein svnthesis, has often been proposed (Marsh 1968(Marsh , 1976Savard et al 1965;Hermier et al 1971;Kawano et al 1975;Lieberman et al 1975;Gorski & Padnos 1966), the exact nature of this requirement is still unknown. In most of these studies only a single inhibitor of protein synthesis, predominantly puromvcine or cycloheximide, was utilized to block the stimulatory effect of LH on steroidogenesis.…”

“…Dispersed ovarian cells were prepared by diges¬ tion with collagenase and DNAse as described previously (Clark 8c Menon 1976). Cell viability was usually over 959r as determined by Trypan Blue exclusion (Kawano et al 1975;Clark 8c Menon 1976). Cells were suspended in medium 109 containing 0.17c BSA (w/v) and adjusted to a concentration of approximately 1-2 x 107 cells/ml for the evaluation of cAMP accumulation and progeste¬ rone production and macromolecular synthesis.…”

Modulation of Progesterone Synthesis and Cytochrome P450 Levels in Rat Luteal Cells during Human Chorionic Gonadotropin-Induced Desensitized State*

“…At the end of the incubation, the reaction was terminated by placing the tubes in a boiling water bath for 5 min, followed by the addition of 0.6 ml water and 10 /nl [ 3 H]progesterone (10,000 cpm) to monitor recovery. The tubes were then kept overnight at 4 C, after which the samples were extracted twice with petroleum ether and subsequently assayed for progesterone by RIA (9). In some experiments, as described in Results, cells were preincubated with aminoglutethimide (75 ng/ral) for 30 min and then further incubated in the presence of LDL or saline for 1 h at 37 C. Mitochondria were subsequently isolated, and pregnenolone production and EPR measurements were carried out.…”