FUNCTIONAL-HETEROGENEITY OF GONADOTROPHS IN THE OVINE FETUS - ANALYSIS BY REVERSE HEMOLYTIC PLAQUE-ASSAY

Stimulation of sheep fetal gonadotrophs with 10(-7) M luteinizing horm one releasing hormone (LHRH) for 3 h in culture wells increased lutein izing hormone (LH) release over basal values. Using the reverse hemoly tic plaque assay (RHPA), we demonstrated that this increase was due to a recruitment of LH-secreting cells. During gestation, the percentage of LH-containing cells able to release and the mean size of plaques w ere the highest at around 100-130 days and were usually lower in femal es than in males. In an attempt to delineate the involvement of protei n kinase C (PKC) in LH release, cells were treated with an activator o f PKC, phorbol 12-myristate 13-acetate (PMA). Stimulation of cells wit h 10(-6) M PMA for 3 h enhanced LH release in culture wells 2- to 3-fo ld more than did 10(-7) M LHRH. This increase was a consequence of an enhanced number of LH-secreting cells and, in males only, of an enhanc ement of the mean plaque size. The percentage of LH-secreting cells am ong LH-containing cells and the plaque areas were maximal between 110 and 120 days of gestation in both sexes. They were usually lower in fe males than in males. Stimulation of cells with LHRH plus PMA enhanced LH release in culture wells in an additive manner when compared to eit her factor alone in both sexes and at all fetal ages. This additive ef fect reflected an increase in the number of secreting cells. Under the se conditions, plaque sizes were larger than the plaque sizes obtained with PMA alone in males and in females in late gestation. In conclusi on, our results show that LHRH stimulated LH secretion from sheep feta l cells by recruiting secreting cells when compared to controls. Both the percentage of LH-secreting cells and the secretory activity of eac h gonadotroph were maximal at around 100- 120 days of gestation and we re higher in males than in females. Results following treatment of cel ls with PMA, either alone or in combination with LHRH, suggest that th ese two secretagogues act on two different subpopulations of gonadotro phs and probably through different pathways.