Biology of Reproduction, Vol 25, 609-620, Copyright © 1981 by Society for the Study of Reproduction

Visualization of Binding and Internalization of a Horseradish Peroxidase-hCG Conjugate by Monkey Luteal Cells

¹ Pregnancy Research Branch and Endocrinology and Reproduction Research Branch, National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, Maryland 20205

We have prepared a conjugate of human chorionic gonadotropin (hCG) and horseradish peroxidase (HRP) that retained binding affinity for cell receptors and was biologically active. The hCG was covalently linked to dinitrophenylated HRP to produce a conjugate that contained HRP and hCG in a 1.6:1 molar ratio. The conjugate was separated from free hCG and HRP by gel chromatography. The isolated HRP:hCG had a biological activity that elicited dose-dependent enhancement of progesterone secretion by dispersed monkey luteal cells in vitro. The specificity of the conjugate to LH/hCG receptors was demonstrated by the insignificant nonspecific binding of HRP:hCG to dissociated cells that were previously saturated with native hCG. Electron microscopic studies revealed that the HRP:hCG was distributed uniformly on the surface of luteal cells previously fixed with aldehyde, and to some extent on the surface of those cells that were exposed to the conjugate at 4°C. In those cells labeled at 4°C, rinsed and warmed to 37°C, a rapid redistribution of HRP:hCG occurred into small clusters. When dissociated cells were incubated with the hCG conjugate at 37°C, it was localized in small clusters on the cell surface. Internalization of small aggregates of HRP:hCG occurred within 5 min at 37°C. This process occurred through invaginations of smooth segments of the plasma membrane. Once internalized, the conjugate was located within membrane bound vesicles of the cytoplasm. After 120 min at 37°C, the HRP:hCG was located in large cytoplasmic vesicles near the periphery of the cells, but they were not seen in association with Golgi complexes or lysosomes.