Ovarian Gonadotropin-Releasing Hormone-Like Protein(s): Demonstration and Characterization*

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Abstract
Both GnRH and prostaglandin F2.alpha. inhibit LH-stimulated cAMP accumulation and progesterone secretion in isolated luteal cells. Moreover, since ovarian GnRH receptors have been demonstrated, this study was conducted to determine if GnRH-like substances were present in the luteinized rat ovary. Detection of GnRH-like activity was based on a sensitive and specific ovarian membrane GnRN radioreceptor assay and an immunoassay specific for GnRH. Ovaries were extracted with an aqueous medium containing formic acid, HCl, trifluoroacetic acid, and NaCl. Material present in the supernatant fraction which absorbed to Waters C18 Sep-Paks was subsequently eluted with acetonitrile and lyophilized. The redissolved ovarian extract showed substantial radioreceptor activity, but very little immunnoassayable activity. The GnRH-like activity of the ovary was sensitive to proteolytic enzyme digestion and to incubations at 50 C for as little as 5 min, had an apparent mol wt greater than 1,000 but less than 10,000, and was not soluble in ether. Extracts of plasma did not radioreceptor or immunoreactive activity, whereas hypothalamic extracts exhibited both radioreceptor and immunoreactive activities. Liver and kidney extracts showed less radioreceptor activity than ovarian extracts and very little immunoreactive activity. Two peaks of radioreceptor activity appeared when the ovarian extract was further fractionated by reverse phase HPLC. The two peaks of GnRH-like activity were clearly separated from GnRH or [D-Ala6,des-Gly10] GnRH ethylamide, an analog used in this study, when these were included in ovarian extracts. It is concluded that rat ovaries contain a GnRH-like protein(s) with membrane binding properties similar to those of GnRH but with other characteristics distinctly different from those of GnRH. The ovarian GnRH-like protein is immunologically different from GnRH, sensitive to elevated temperatures which do not effect GnRH, and chromatographically different frm GnRH during reverse phase HPLC.