The aim is to investigate the extent to which functional and morphological heterogeneity of gonadotropes underlies the patterns of FSH and LH secretion and their regulation by gonadal steroids, GnRH, and inhibin during the estrous cycle of the rat. The major questions to be addressed are whether there are sub-populations of gonadotropes in the pituitary gland, and whether these sub-populations change in functional capacity during the estrous cycle so as to account for the preovoluatory LH and FSH surges, and for the differential secretion of LH and FSH. I have developed a method for detecting hormone secretion by individual cells in mixed cultures which offers hope for resolution of this issue. It is the reverse hemolytic plaque assay and is based on complement mediated lysis of protein A coupled RBC incubated in a monolayer with pituitary cells and hormone antiserum. The resulting zones of RBC lysis (""""""""plaques"""""""") surrounding the pituitary cell are easily identified microscopically. Subsequent electron microscopy (or EM immunocytochemistry) on such functionally identified cells allows morphological and functional comparisons to be made.
The specific aims are: 1) LH secretion by individual rat gonadotropes will be assessed using the reverse hemolytic plaque assay, electron microscopy, and immunocytochemistry to answer the following questions: a. What fraction of the gonadotropes secrete LH in various physiologic states? b. Do the same gonadotropes show both the inhibitory and stimulatory effects of estrogen on LH secretion? 2) FSH secretion by individual rat gonadotropes will be assessed using the plaque assay, electronmicroscopy and immunocytochemistry to answer the following questions: a. What proportion of the AP cells are FSH secretors during various physiological and experimental states? b. During preferential FSH secretion, is FSH secreted by FSH-only gonadotropes or by FSH+LH gonadotropes? c. Which gonadotrope cell type does inhibin act on to preferentially inhibit FSH secretion?
| Smith, P F; Neill, J D (1987) Simultaneous measurement of hormone release and secretagogue binding by individual pituitary cells. Proc Natl Acad Sci U S A 84:5501-5 |
