Antigonadotropic Effects of the Bovine Ovarian Gonadotropin-releasing Hormone-binding Inhibitor/Histone H2A in Rat Luteal and Granulosal Cells

R F Aten,H R Behrman

doi:10.1016/s0021-9258(18)60428-4

Abstract

A gonadotropin-releasing hormone (GnRH)-binding inhibitor (GnRH-BI) was purified from bovine ovaries and identified as histone H2A. In the present studies, the biological effects of partially purified and purified ovarian GnRH-BI, as well as calf thymus histone H2A, were examined in rat ovarian cells. Since GnRH has direct antigonadotropic actions on these cells, the effects on luteinizing hormone-stimulated cAMP accumulation in luteal cells and follicle stimulating hormone-induced cAMP and progesterone production in granulosal cells were evaluated. Antigonadotropic activity in both luteal and granulosal cells coeluted directly with GnRH-BI activity during purification from bovine ovaries, and the antigonadotropic effects were dose dependent and reversible. In contrast to GnRH, GnRH-BI maximally inhibited gonadotropin responses and the effects of GnRH-BI were not blocked by a GnRH antagonist. The purified ovarian GnRH-BI and calf thymus histone H2A had identical antigonadotropic properties, and the half-maximal concentrations for inhibiting the gonadotropin responses of granulosal and luteal cells was 2 and 5 microM, respectively. In conclusion, the ovarian GnRH-binding inhibitor, identified as histone H2A, not only inhibits the high affinity binding of GnRH to rat ovarian membranes but also evokes GnRH-like antigonadotropic responses in rat ovarian cells that do not appear to be mediated by binding to GnRH receptors.

Highlights

Antigonadotropic Effects of the Bovine Ovarian Gonadotropin-releasing Hormone-binding Inhibitor/Histone H2A in Rat Luteal and Granulosal Cells*
A GnRH-binding inhibitor (GnRH-BI)’ which reversibly inhibits the high affinity binding of GnRH to rat ovarian membranes is found in the ovaries of diverse speciesincluding the human, cow, sheep, and rat (1-3).In addition, granulosal phere of 95% air, 5% CO, in Dulbecco’s modified Eagle’smedium containing HEPESand glucose(380-2430, GIBCO) and supplemented with 0.1% bovine serum albumin, 0.01% bacitracin, and 1% Antibiotic-Antimycotic Solution (600-5240, GIBCO)
The GnRH-BI of bovine ovaries has been purified by a combination of reversed-phase HPLC, cation-exchange, and gel filtration chromatography (5)

Summary

THEJOURNAL OF BIOLOGICACLHEMISTRY

0 1989 by The American Society for Biochemistry and Molecular Biology, Inc. Vol 264, No., Issue of July 5, pp. 11072-11075,1989 Printed in U.S.A. 11072-11075,1989 Printed in U.S.A. Antigonadotropic Effects of the Bovine Ovarian Gonadotropin-releasing Hormone-binding Inhibitor/Histone H2A in Rat Luteal and Granulosal Cells*. GnRH-BI maximally inhibited gonadotropin responses The purification of the GnRH-BI from bovine ovaries and the andthe effects ofGnRH-BI were notblocked by a radioreceptor assay for GnRH-like activity are described in the comGnRH antagonist. A GnRH-binding inhibitor (GnRH-BI)’ which reversibly inhibits the high affinity binding of GnRH to rat ovarian membranes is found in the ovaries of diverse speciesincluding the human, cow, sheep, and rat (1-3).In addition, granulosal phere of 95% air, 5% CO, in Dulbecco’s modified Eagle’smedium containing HEPESand glucose(380-2430, GIBCO) and supplemented with 0.1% bovine serum albumin, 0.01% bacitracin, and 1% Antibiotic-Antimycotic Solution (600-5240, GIBCO). FSH follicle-stimulating hormone; GnRH, gonadotropin-releasing isolated and incubated in McCoy’s 5a medium containing 25 mM hormone; GnRH-A, [~-Ala~,des-Gly’~]GneRthHylamide; A-GnRH, HEPES (380-2330, GIBCO) and supplemented with 0.1% bovine. 4 100 ? Antigonadotropicactivity of bovine ovarian GnRH-BZ and GnRH in rut luteal cells in the absence and presence of a GnRH antagonist

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RESULTS

DISCUSSION