Regulation of C-fos Protein in Gonadotrope Cells by Oxytocin and Gonadotropin-Releasing Hormone

Abstract

The integrated regulation of luteinizing hormone (LH) from the anterior pituitary gland is vital to the functioning of the ovulatory cycle in the female and consists of several components acting at different time points. The best-studied is the rapid release of LH elicited by gonadotropin-releasing hormone (GnRH). The so-called primary (immediate early) response genes (PRGs), including c-fos, regulate relatively long-term activities, such as mitosis, protein synthesis, protein release and cell differentiation. Regular ovulatory cycles occur as a result of interaction of several peptide factors including the primary factor, GnRH and oxytocin, although GnRH and oxytocin do not have identical activities. We wished to determine whether oxytocin could mediate changes in expression of c-fos protein and compare its effects with those of GnRH. Anterior pituitary glands were collected from female rats at proestrus and a single-cell suspension prepared. Cells were incubated with oxytocin or GnRH at selected concentrations for various times. C-fos protein was extracted and submitted to Western blot analysis. Other cells were stained immunohistochemically for c-fos and LH following incubation with the peptides and fixation. There was an increase in c-fos protein from 15 to 60 min in Western blots of cells from all incubations. After immunohistochemistry, it was observed that both oxytocin (100 n M) and GnRH (100 n M) increased the percentage of cells that expressed c-fos protein (p < 0.001) and of cells that expressed LH (p < 0.001). The responses to the peptides were concentration dependent. We found that neither all LH-containing cells expressed c-fos, nor all c-fos-containing cells immunostained for LH. The effects of the peptides were not the same. High concentrations of GnRH (1 µ M) induced the appearance of a higher percent of LH-containing cells having c-fos than did 10 n M GnRH (p < 0.01), whereas a lower percent of LH-containing cells with c-fos were observed when the oxytocin concentration was raised from 10 n M to 1 µ M (p < 0.02). It appears, therefore, that the two peptides have different regulatory effects on LH-containing cells, indicating the possibility of specialized function. The results emphasize the suggestion that stimulation of LH secretion is not the sole index of gonadotrope-directed activity by a peptide. Collectively, these results indicate that the peptides oxytocin and GnRH are able to modulate processes that are associated with longer-term activities of gonadotropes and also demonstrate that specific subpopulations of LH-containing gonadotropes are stimulated to express c-fos.