Detection of LH release from individual pituitary cells by the reverse hemolytic plaque assay: estrogen increases the fraction of gonadotropes responding to GnRH

Abstract

Gonadotropes that secrete LH were demonstrated microscopically among mixed anterior pituitary (AP) cells in culture with a reverse hemolytic plaque assay. LH released from a cell binds to adjacent RBCs bearing an LH Ab-protein A complex which results in a zone of complement-mediated hemolysis (a plaque) surrounding each gonadotrope. In untreated cultures a few, small plaques formed, but in the presence of hypothalamic GnRH (10(-7) M, a maximally stimulating dose) a 10-fold increase occurred in their number and size. Non-secretory gonadotropes were found in the pituitary of diestrous animals: 5-6% of all AP cells contained immunocytochemically detectable LH whereas only 2.5-3.0% formed plaques in the presence of GnRH (10(-7) M). Nearly all of the gonadotropes were secretory at proestrus. Estradiol treatment of diestrous cultures for 24 h increased the fraction of secretory gonadotropes to near the level observed in proestrous cultures. These results demonstrate the utility of the reverse hemolytic plaque assay for detection of LH secretion from individual gonadotropes, establish that not all cells containing LH can secrete it, and suggest that a previously unrecognized mode of estrogen action to evoke the preovulatory LH surge is mediated by increasing the fraction of secretory gonadotropes.