The Effects of Gonadotropin-Releasing Hormone and Estradiol on Luteinizing Hormone Biosynthesis in Cultured Rat Anterior Pituitary Cells*

Abstract

This study investigated the effects of physiological concentrations of GnRH and estradiol (E2) on LH biosynthesis and release using cultured anterior pituitary cells. Pituitaries from female rats were enzymatically dispersed and cultured for 48 h in steroid-free alpha-Modified Eagle's Medium, followed by a 24-h culture in medium with or without E2. The cells were then incubated for a 4-h (Exp 1 and 2) or 8-h (Exp 3) period in medium containing radiolabeled precursors with or without GnRH. Radioactive precursor incorporation into LH was determined by immunoprecipitation, while immunoreactive LH (iLH) content was quantified by RIA. In the first experiment, all concentrations of E2 (10(-11)-10(-8) M) enhanced iLH release in response to 1 nM GnRH, confirming previous reports. GnRH increased [3H]glucosamine (3H-Gln) incorporation into LH, but had no effect on [35S]methionine (35S-Met) incorporation. The higher concentrations of E2 enhanced GnRH-stimulated 3H-Gln LH production. In the second experiment, the effects of GnRH (10(-9) M) and E2 (5 X 10(-10) M) on the incorporation of [3H]galactose, [3H]mannose, [3H]fucose, or [35S]sulfate into LH were investigated. Although all precursors were incorporated into LH, no specific effect of GnRH and/or E2 on incorporation of any of the precursors into LH was noted. In Exp 3, pituitary cells were cultured with or without 0.5 nM E2 followed by an 8-h incubation with varying physiological concentrations of GnRH (10(-11)-10(-9) M) and radiolabeled precursors (3H-Gln and 35S-Met). GnRH stimulated iLH release in a dose-dependent manner, and this response was enhanced by E2. GnRH also increased the incorporation of both 3H-Gln and 35S-Met into LH, but the dose of GnRH required for this response was dependent upon the estrogen environment. In the absence of E2, only 10(-9) M GnRH increased 3H-Gln LH and 35S-Met LH production, whereas in cells exposed to E2, all concentrations of GnRH (10(-11)-10(-9) M) increased 3H-Gln LH and 35S-Met LH production. In all experiments, the specific activity of radiolabeled LH released under basal conditions was greatly reduced by stimulation with GnRH. These results suggest that GnRH regulates both LH glycosylation and LH polypeptide synthesis and that E2 lowers the physiological concentration of GnRH necessary to stimulate this biosynthetic response. Moreover, estrogen's enhancement of GnRH-stimulated LH release appears to be due to its action on mechanisms regulating the release of previously synthesized stored hormone as well as the release of newly synthesized LH.

Attenuin: What It Is, How It Works and What It Does

“…Results showed that while progesterone by itself had no effect (control groups), saturation of PR with the cognate ligand reversed the inhibitory effect of attenuin on LH protein levels, although not on LH secretion (Gordon et al, 2010b). Apart from these results showing that attenuin probably inhibits LH synthesis at post-transcriptional level, little is known about the mechanism involved in that inhibition -probably by increasing LH degradation (Kitahara et al, 1990) and/or decreasing GnRH-induced LH polypeptide glycosylation (Ramey et al, 1987) except that, as indicated earlier, the inhibitory pathway in LH synthesis involves the gonadotrope PR. However, the detailed mechanism underlying the PR-mediated attenuininduced inhibition of LH protein levels requires further detailed investigation.…”

“…The preovulatory secretion of LH is the result not only of increased LH synthesis (Tse et al, 1993) but also of increased LH secretion by gonadotropes (Ramey et al, 1987). While the effects of attenuin on LH secretion have been exhaustively studied, it is not known whether attenuin has any effect on LH synthesis.…”

Oestradiol is a potent mitogen and modulator of GnRH signalling in alphaT3-1 cells: are these effects causally related?

“…The direct effects have been extensively studied in primary cultures of rat pituitary cells in which oestradiol can amplify GnRH-stimulated gonadotrophin secretion (Tang et al 1982) and can increase GnRH receptor number (Clayton 1989) and influence gonadotrophin subunit expression (Ramey et al 1987, Phillips et al 1988. In terms of GnRH signalling, oestradiol has been shown to influence the Ca 2+ response to GnRH (Leong & Thorner 1991) and the effects of GnRH on Ca 2+ mobilisation and Ins(1,4,5)P 3 generation are increased in cells from pro-oestrous rats and reduced in those from ovariectomised rats (Mitchell et al 1988).…”

Ο ωοθηκικός έλεγχος της υποφυσιακής ευαισθησίας στη GnRH στις γυναίκες με PCOS

“…Στην παρούσα µελέτη, µία παρόµοια αύξηση στις συγκεντρώσεις της Ε2 στον ορό βρέθηκε στις φυσιολογικές και στις γυναίκες µε PCOS. Αυτό το στεροειδές ευαισθητοποιεί την υπόφυση στη GnRH για την παραγωγή γοναδοτροφινών µέσω πολλαπλών µηχανισµών, συµπεριλαµβανοµένης και της αύξησης του αριθµού των υποδοχέων της GnRH (self-priming effect) (Ramey et al, 1987;Danforth et al, 1990;Laws et al, 1990).…”

“…Έχει βρεθεί ότι η βραχυπρόθεσµη (<12 ώρες) έκθεση των καλλιεργηµένων υποφυσιακών κυττάρων στην Ε2 αυξάνει τον αριθµό των υποδοχέων της GnRH (Gregg et al, 1990) και την από τη GnRH προκαλούµενη έκκριση της LH (Huang and Miller, 1980). Η µακροπρόθεσµη (24-48 ώρες) in vitro έκθεση των γοναδοτρόφων του επίµυος (Speight and Fink, 1981b) ή του προβάτου (Fowler et al, 1992(Fowler et al, , 1993) στην Ε2 έχει µικρή επίδραση στην έκκριση της LH από τη GnRH, αν και η συνεχής έκθεση στην Ε2 κατά τη διάρκεια µίας πρόκλησης µε GnRH ενισχύει την από τη GnRH προκαλούµενη σύνθεση και έκκριση της LH (Ramey et al, 1987). Εχει προταθεί ότι η αυξηµένη στον ορό LH που παρατηρείται περίπου στο 40% των ασθενών µε PCOS σχετίζεται µε τη συνεχή θετική δράση της Ε2 στην υπόφυση (Lobo et al, 1981;Waldstreicher et al, 1988).…”