Our objectives are to demonstrate with morphological and biochemical evidence that progesterone actions modulate positive and negative growth factors, cell cycle regulators, and cytokines which in turn regulate the stromal cell cycle and cell survival/death during pregnancy in the rat. We hypothesize that protein kinase C plays a pivotal role in mediating these actions. The experimental designs take advantage of the in vivo change in the threshold of stromal cells for PKC-stimulated apoptosis in decidual basalis of the rat at day 10 of pregnancy when the threshold is high and at day 14 when the threshold is low. Our goal is to lower the threshold for apoptosis by ovariectomy (Ovx), administration of anti-progestins and phorbol esters by enhancing PKC activity. We hypothesize the down- stream effects include enhanced expression of cell cycle arrest proteins and effectors of apoptosis. The threshold for apoptosis will be increased by administering progesterone to Ovx pregnant rats and drugs that inhibit PKC activity. We will examine stromal cells for changes in progesterone receptor isoform expression (PR-A, -B, -C), regulators of cell cycle progression (D-cyclins, PCNA) and arrest (p21, p27). Signaling pathways leading to cell death are monitored by expression of Bcl2, Bax, PKC, caspase-3 by Western blot analysis and enzyme assay. The cell-types involved and the distribution of cytokine expression are evaluated by immunochemistry. The role of progesterone and downstream bioactive signals that confer cell cycle progression and resistance to cell death, thus, will be distinguished from anti-progestin/PKC mediated pathways that lead to apoptosis and cell death in a relatively natural context. We hypothesize that each element in the network functions as an active and reactive link in the highly integrated scheme of protein signals that regulate the stromal cell cycle and that the effects of regulatory agents depend upon the cellular context for modulation and interpretation of cytokine signals. The results from this project will lead to a better understanding of the role of progesterone in maintenance of pregnancy and stromal cell function. The results will also provide a physiologic basis for the clinical manipulation of apoptotic thresholds to change the natural progression of related proliferative diseases such as uterine leiomyomatas and endometriosis and aid in the design of cytotoxic chemotherapies and immunotherapeutic strategies for treatment of gynecologic cancers.