Biological Actions of Monoclonat Luteinizing Hormone/Human Chorionic Gonadotropin Receptor Antibodies1

Abstract

Several recent studies have elucidated the structure of the mammalian LH/hCG receptor; as reported in the present work, we have developed a series of monoclonal antibodies (mAbs) against the rat ovarian LH/hCG receptor using highly purified receptor as immunogen and by screening hybridomas with purified LH/hCG receptors. The mAbs were able to specifically immunoprecipitate LH/hCG receptors from solubilized preparations of rat ovarian membranes as well as from partially purified preparations. Western blotting with mAb P1B4 detected a probable receptor dimer and a receptor fragment in rat and porcine ovarian tissue but not in other tissues. This mAb also partially inhibited hCG binding to rat and porcine ovarian tissues. The receptor mAbs were able to inhibit hCG-induced progesterone synthesis in cultured human and porcine granulosa cells without affecting cAMP- and FSH-induced progesterone synthesis. The mAb P1B4 was used to demonstrate that the majority of ovarian receptors are internalized after hCG treatment and that in pseudopregnant rats receptors are present in the rough endoplasmic reticulum and in microvesicles. Bovine corpus luteal cells also contained P1B4 binding sites, as detected by immunohistochemical technique. Taken together, these results suggest that the mAbs are specific for the LH/hCG receptor, mAb P1B4 recognizes an epitope that is highly conserved among mammals, and this epitope is probably in the extracellular domain.

Homozygous mutation within the conserved Ala-Phe-Asn-Glu-Thr motif of exon 7 of the LH receptor causes male pseudohermaphroditism

“…The LH receptor monoclonal antibody P1B4 was a gift from Dr J Wimalasena (Department of Obstetrics and Gynecology, University of Tennesse, Knoxville, TN, USA). The antibody was raised against purified rat LH receptors, as described by Indrapichate et al (16), and has been shown to bind to LH receptors in the human.…”

Cytoskeletal Regulation of hCG-Induced Receptor Clustering in Transformed Leydig Cells

“…Samples were fixed in 0.5% paraformaldehyde, and hC'G receptors were visualized with the anti-LHR monoclonal antibody, PIB4. of the IgM class [30], fol lowed by fluorescein-conjugated rabbit anti-mouse IgM (Organon Teknica/Cappel). The percent capping was determined by counting a minimum of 100 cells, which were considered to have capped if the fluores cent surface label was concentrated in a polar area comprising half or less of the cell.…”

“…but exhibits only minimal cross-reactivity with the FSH receptor [30. 40], The available data show that the epitope is extracellular and presumably represents a well-conserved and LH/hCG-specific region of the N-terminal receptor domain [3], Earlier work demonstrated that the P1B4 antibody partially inhibits hCG binding and hCG-stimulated progesterone production in porcine granulosa cells [30]. Our immunofluorescence studies with the murine MA-10 cells did not directly address this issue, but the data suggest that the monoclonal antibody will recognize both the unoccupied and the hCG-bound forms of the receptor.…”

“…In this study, we used cytochemistry and fluorescence microscopy to demonstrate that capping of plasma membrane receptors, la beled by means of a monoclonal antibody [30], occur in cultured murine Leydig tumor cells (MA-10) after addition of hCG. Origi nally derived from a spontaneous Leydig cell tumor [31,32], these cells have retained many of the differentiated functions of normal Leydig cells and display a well-characterized re sponse to LH and hCG, with increased levels of intracellular cAMP and secreted progester one [33].…”

Steroid-mediated differentiation of neural/neuronal cells from epithelial ovarian precursors in vitro

“…Porcine granulosa cells were isolated from medium sized antral follicles (4-6 mm) as described previously, 28,29 and maintained as the human epithelial SC above, including propagation, freezing and thawing.…”