Phillip B. C. Jones

Binding of gonadotropin releasing hormone agonist to rat ovarian granulosa cells

We have previously shown that gonadotropin releasing hormone (GnRH) and its agonists inhibit ovarian functions by a direct action on ovarian granulosa cells in vitro. A labeled GnRH agonist, [des-Gly10, D-Ser (TBu)6, Pro9-NHEt]GnRH, was used here to examine the possibility that these inhibitory actions of GnRH were mediated through specific receptors which recognize GnRH. Ovarian membrane fractions obtained from immature, hypophysectomized diethylstilbesterol-treated rats were incubated with the 125I-GnRH agonist and specific binding was determined by a filtration assay. Stereospecific, high affinity binding was detected in the ovarian membranes; the dissociation constant for the labeled GnRH agonist was determined to be 0.84 ± 0.33 × 10−10 M and the binding capacity was calculated to be 12.9 fmol/mg protein, or 0.142 fmol/μg DNA. The binding affinity for the GnRH decapeptide was 3.3 times lower than that of the GnRH agonist whereas two GnRH partial peptides did not compete for the 125I-agonist binding. After sequential treatment with FSH, LH and prolactin to the hypophysectomized female rats, the ovarian GnRH binding capacity increased per ovary, but decreased per mg ovarian protein. Furthermore, ovarian granulosa cells were isolated and their binding capacity was determined to be 25.2 fmol/mg protein, or 0.133 fmol/μg DNA, suggesting that the granulosa cells contain GnRH binding sites. Thus, this report demonstrates the presence of stereospecific, high affinity GnRH binding sites in the rat ovarian granulosa cells.

Modulation of prolactin receptors in cultured rat granulosa cells by FSH, LH and GnRH ☆

The hormonal modulation of prolactin (PRL)-binding capacity of rat granulosa cells was studied. Granulosa cells obtained from immature, hypophysectomized, estrogen-treated rats were cultured for 2 days in a serum-free medium in the presence of various hormones. FSH treatment in vitro stimulated granulosa cell PRL-binding capacity by approximately 4-6-fold in a dose-dependent manner. Concomitant treatment with 10-8 M GnRH inhibited the FSH-induced increase in PRL-binding capacity by 64%. In contrast, the inhibitory effect of GnRH was blocked by concomitant treatment with 10-6 M of a GNRH antagonists, [D-pGlu1, D-Phe2, D-Trp3,6]GnRH. PRL-binding capacity was also increased (approximately 2-fold) by in vitro treatment with cholera toxin (10 microgram/ml). In granulosa cells pre-treated with FSH in vitro for 2 days, hCG treatment for 2 additional days stimulated PRL-binding capacity in a dose-dependent manner (approximately 2-fold). Likewise, treatment with LH (100 ng/ml) also stimulated PRL-binding capacity by approximately 2-fold. These in vitro studies demonstrated that gonadotropins (FSH, LH and hCG) directly enhanced PRL binding by granulosa cells, whereas GnRH inhibited FSH action.

Regulation of ovarian 20α-hydroxysteroid dehydrogenase by gonadotropin releasing hormone and its antagonist in vitro and in vivo☆

Abstract Hypothalamic gonadotropin releasing hormone (GnRH) and its analogs have been shown to regulate progestin production by ovarian granulosa cells treated with FSH and prolactin. The present study examines the direct modulation of an ovarian progesterone metabolizing enzyme by GnRH and its analogs in vitro and in vivo . Granulosa cells from hypophyseclomized, diethylstilbestrol-treated rats were cultured with GnRH or its analogs. After 2 days, cells were homogenized and 20α-hydroxysteroid dehydrogenase (20α-OH-SDH) activity was measured using a direct enzyme assay. GnRH stimulated 20α-OH-SDH activity in a dose-dependent manner with an ED 50 of 8.95 × 10 −9 M and a GnRH aganist ([des-Gly 10 , d -Lcu 6 , (N 2 Me)Leu 7 ,Pro 9 NHEt]GnRH) was shown to be 5.6 fold more potent than GnRH. The stimulatory effect of GnRH was inhibited by concomitant treatment with a GnRH antagonist ([ d -pGlu 1 , d -Phe 2 d -Trp 3,6 ]GnRH) with a half maximal inhibitory dose ratio of 3 1 ([antagonist]/[GnRH]). Time course studies indicated that 12 h of GnRH treatment were required to stimulate a measurable increase in enzyme activity. Cycloheximide treatment inhibited the GnRH action, suggesting that GnRH induces the synthesis of 20α-OH-SDH. Furthermore, treatment with GnRH and its analogs in hypophysectomizcd rats in vivo stimulated enzyme activity without affecting the K m of the enzyme, whereas the stimulatory effect of GnRH was blocked by concomitant treatment with a potent GnRH antagonist. These studies demonstrated that GnRH and its analogs may directly regulate the synthesis of ovarian 20α-OH-SDH in vitro and in vivo .