Production and characterization of polyclonal antiserum to rat luteinizing hormone/chorionic gonadotropin receptor and its immunohistochemical application for studying receptor location and down-regulation.

A polyclonal antiserum against the affinity-purified nondenatured rat 90K LH/CG receptor polypeptide was raised in rabbits, characterized, and used to study the location of the LH/CG receptor in pseudopregnant rat luteal cells and the fate of the receptor-hCG complex together with the specific anti-hCG serum during hCG-induced down-regulation by immunochemical techniques. Even at a 1:3000 dilution, the antiserum recognized a single 90K polypeptide on Western blots of both the affinity-purified receptor and the initial detergent extract of the pseudopregnant rat ovarian membranes. It recognized sodium dodecyl sulfate-denatured and reduced, sodium dodecyl sulfate-denatured, and native forms of the receptor on dot blots; the immunoreaction was the most intense with the native receptor. The antiserum also contained antibodies that recognized the hormone-binding site, or a region near to it, and the occupied receptor. The majority of the LH/CG receptors were located on the luteal cells in pseudopregnant rat ovaries before the induction of down-regulation. The receptor content seemed to vary among the luteal cells, however, and on single cells, suggesting both functional heterogeneity and a functional polarization of the luteal cells. Upon induction of down-regulation with hCG both the receptors and the bound hormone disappeared from the luteal cell surfaces at a very slow rate, without any simultaneous appearance of receptor- or hCG-specific immunostaining in the luteal cell interior. No accumulation of receptor degradation products capable of [125I]iodo-hCG or antibody binding could be detected on Western blots of the tissue. The polyclonal LH/CG receptor antiserum described here is useful for studying the structure and function of this receptor, particularly for immunohistochemical investigations into receptor location and regulation.