Abstract
We developed a double immunofluorescence technique for detection of the rat luteinizing hormone/choriogonadotropin (LH/CG) receptor and bound hCG in the same rat ovarian section and used it in conjunction with confocal laser scanning microscopy (CLSM) to study the fate of the receptor-hormone complex in luteal cells during the hCG-induced down-regulation. Pseudopregnant immature females rats were perfusion-fixed before (0 hr) and 2, 6, 12, 24, or 36 hr after a down-regulating dose of hCG (500 IU IV). The cryosections were stained for the LH/CG receptor and bound hormone by sequential incubations with a polyclonal rabbit antiserum to purified rat LH/CG receptor and a mouse monoclonal antibody (MAb) to hCG, followed by sequential incubation with TRITC- and FITC-conjugated secondary antibodies to rabbit and mouse immunoglobulins, respectively. The results were semiquantitatively analyzed by a pseudo-three-dimensional (3D) plotting of the intensities of the receptor and hormone-specific fluorescence in luteal cells by CLSM. The analysis suggested that the majority of the LH/CG receptors are located on the luteal cells before induction of the down-regulation and that their content seem to vary not only among cells but also on the surface of single cells, thus supporting the previous concept of the functional heterogeneity among the cells and their functional compartmentation. At 2 hr after injection of the down-regulating dose of hCG, the LH/CG receptor-specific and hCG-specific fluorescences clearly co-localized on the luteal cells. Both the LH/CG receptor- and hCG-specific fluorescences disappeared from the luteal cell surfaces in a parallel fashion within 36 hr without a detectable accumulation of either fluorescence deep in the cell interior. These results suggest that the LH/CG receptor and bound hCG do not differ in their manner of in vivo processing in luteal cells. Therefore, the disappearance of the receptor and bound hormone occurs in a parallel fashion and without detectable internalization.