We performed partial purification of low molecular weight luteinizing hormone binding inhibitor from porcine follicular fluids and examined its biological activities. Following ultrafiltration, gel filtration and anion exchange of the pooled porcine follicular fluids, low molecular weight fractions (500\p=n-\10,000 MW) inhibited [125I]hLH binding to porcine granulosa cells in a dose-dependent manner. The binding inhibition kinetics study revealed that the luteinizing hormone binding inhibitor may indicate a non\x=req-\ competitive inhibition with [125I]hLH binding. In vitro bioassay using adult mouse testicular interstitial cells revealed that the partially purified luteinizing hormone binding inhibitor reduced ovine LH\ x=req-\ stimulated testosterone and cAMP production in a dose-dependent manner, whereas the luteinizing hormone binding inhibitor did not affect basal production of testosterone and cAMP. The inhibitory activity was heat stable and did not disappear with activated charcoal adsorption. The results of the present study suggest that the luteinizing hormone binding inhibitor may play an important role as an ovarian non-steroidal regulator modulating the receptor binding of LH and LH-mediated steroidogenesis. Follicular development, ovulation, transformation of the follicle into the corpus luteum and maintenance and regression of the corpus luteum are highly integrated processes, probably under the control of a group of endocrine, paracrine and autocrine factors (1). Folliclestimulating hormone (FSH) and luteinizing hormone (LH) are primary regulators of ovarian function. It is clear that the biologic manifestations of gonadotropins are dependent on the presence of receptors on the cell membrane of ovarian target cells. Several studies have shown that many ovarian processes are accompanied by variations in the binding capacity of FSH and LH to ovarian target cells of various animal species (2-6). Moreover, it has been reported that the binding of gonadotropins to ovarian target cells might be regulated by FSH, LH, ovarian steroids and non-steroidal regu¬ lators (7).Previously, Yang et al. (8) reported the luteinizing hormone binding inhibitor (LHBI) in pseudopregnant and pregnant rat ovaries. We, too, demonstrated the existence of LHBÍ activity in the 30,000 x g aqueous extract of the human corpus luteum and the increase of LHBI activity in the luteal tissue as the human corpus luteum ages (9). As for porcine follicular fluids, Montz et al. (10) have shown that crude preparations of porcine follicular fluid proteins inhibited [125f]hCG binding to cultured porcine granulosa cells. Bhat and Moudgal (11) reported partially purified gonadotropin binding inhibi¬ tor from porcine follicular fluid, as a protein of approxi¬ mately molecular weight 80,000, which inhibited the binding of both [125I]hFSH and [,25I]hCG to rat ovarian receptor preparation. Thus the LHBI, which is consid¬ ered to act on the first step of LH action on gonadal tissue, has been demonstrated in various tissue and physiologi¬ cal fluids...

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We performed the purification of high-molecular-weight follicle-stimulating hormone binding inhibitor (FSHBI) from porcine follicular fluids. The FSHBI activities of high-molecular-weight fractions acquired by ultrafiltration of follicular fluids from small, medium and large follicles with Centriflo CF25 membrane cone were 277.2 +/- 24.6, 176.7 +/- 3.0 and 141.3 +/- 3.6 U, respectively. By affinity chromatography of CF25 retentate with a column of Blue Sepharose CL6B, 94.1 +/- 5.3% of FSHBI activity was recovered in the unretained fraction. The FSHBI in the unretained fraction was purified by anion-exchange chromatography with a column of Mono Q and gel filtration on Sephacryl S300HR. As a result of sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) of the final purified fraction, a single silver-stained band was observed at 310 kD under the non-reducing conditions. On the other hand, under the reducing conditions, SDS-PAGE revealed three bands at 178, 101 and 55 kD. A double reciprocal plot analysis of this substance showed competitive inhibition in FSH binding. The results of the present study suggest the existence of a 310 kD FSHBI composed of three subunits of 178, 101 and 55 kD in porcine follicular fluids.

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