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Uterine stromal cells undergo mitosis and differentiate into the decidua just prior to the expected time of implantation in humans and rodents. We have utilized a culture system that will be suitable for study of the molecular mechanisms regulating stromal cell proliferation. Stromal cells were isolated from the uteri of ovariectomized rats and were cultured in chemically defined medium. Cultured cells express the mesenchymal markers vimentin and desmin. They do not express the epithelial marker cytokeratin. Serum-starved stromal cells were stimulated to proliferate in a time frame consistent with the cell cycle through addition of a panel of growth factors (basic fibroblast growth factor [bFGF], epidermal growth factor, platelet-derived growth factor, transforming growth factor alpha, insulin-like growth factor I) and hormones to the culture medium. None of the growth factors tested significantly stimulated proliferation in the absence of progesterone. Furthermore, progesterone was the only steroid of those tested that stimulated mitosis in the presence of growth factors. Stromal cell proliferation in response to progesterone and bFGF was dose dependent and saturable. Addition of the progesterone receptor antagonist mifepristone (RU486) and an inhibitor of tyrosine kinase receptor activation (suramin) abolished stromal cell mitosis. Progesterone receptors and fibroblast growth factor receptor 1 (FGFR1) were identified by immunoblot analysis in proliferating stromal cells. Taken together, these results show that cultured stromal cells maintain progesterone-dependent cell cycle control that is mediated via progesterone receptors. Moreover, the data indicate that bFGF control of stromal cell proliferation is modulated via a specific isoform of FGFR1 containing the three-loop immunoglobulin-like domain.

Recent studies suggest that hormonal control of uterine cell proliferation may be moderated by polypeptide growth factors. It remains to be determined, however, whether growth factors cause or are the consequence of hormone action. Basic fibroblast growth factor (bFGF) has been shown to influence cell proliferation and differentiation of a variety of mesoderm-derived cells. To elucidate the regulatory mechanisms controlling stromal cell proliferation and differentiation required for embryo implantation further, immunohistochemical localization of the progesterone receptor and bFGF have been studied. The cell-specific distribution of these proteins was determined in the rat uterus during early pregnancy and after injection of the progesterone receptor antagonist mifepristone (RU 486) at days 1 and 2 post coitum (p.c.) to block implantation. Cell division was restricted to luminal and glandular epithelial cells in pregnant and RU 486-treated rats at day 3 p.c. At day 4 of pregnancy, cell proliferation switched from the epithelia to the stroma in pregnant rats, but after RU 486 treatment division of stromal cells was inhibited significantly (P < 0.05). Progesterone receptor distribution was altered and bFGF was absent in RU 486-blocked stromal cells. Expression of bFGF in luminal and glandular epithelial cells, however, was insensitive to the effects of progesterone receptor antagonism. bFGF content was stimulated in the luminal epithelium and in decidual cells by the implanting embryo. These results indicate that repression of progesterone receptor function in early pregnancy results in a cell-specific loss of bFGF from stromal cells and inhibition of their proliferation. The results further suggest that the regulation of endometrial cell bFGF content is modulated at the site of implantation by the embryo.

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Cellular proliferation and differentiation are critical components of uterine remodeling prior to embryonic implantation. Recent studies have shown that the ovarian hormones, estrogen and progesterone, modulate these cellular events through the production of growth factors. Basic fibroblast growth factor (bFGF) has been implicated in the control of cell proliferation, differentiation, and embryonic development. To clarify its role in uterine remodeling, the cellular distribution of bFGF was examined immunohistochemically in the rat uterus during early pregnancy (Days 2-6). Basic FGF localized intracellularly in stromal and epithelial cells and within the extracellular matrix at Days 2 and 3. It was distinctly evident at the apical surface of epithelial cells at Days 4 and 5 of pregnancy. Concurrent with this apical localization, bFGF was present in the uterine luminal fluid, suggesting release of this growth factor from epithelial cells. Embryonic implantation was accompanied by increased intracellular bFGF content in luminal epithelial and decidual cells. However, similar cells outside of the implantation site and in the artificially decidualized uterus did not express analogous bFGF levels, indicating that a unique signal from the embryo triggers bFGF expression. Changes in the cell-specific distribution of bFGF imply a multifunctional role for this growth factor in uterine cell proliferation, differentiation, and embryonic implantation. In addition, the apical release of bFGF from epithelial cells indicates utilization of a novel secretory pathway for bFGF export during early pregnancy.

Electron and fluorescence microscopic techniques have been used in a complementary fashion to study the patterns of follicle cell-oocyte interactions within cumulus-oocyte-complexes of various mammals. The principal findings are: (1) two distinct types of transzonal processes exist that are distinguishable on the basis of cytoskeletal composition; (2) in some of the species examined (pig, goat, primate), corkscrew-shaped processes rich in tubulin, traverse the zona pellucida and are invaginated into the oocyte cortex; (3) actin-rich processes either ramify as a network at the outer surface of the zona pellucida or penetrate the zona and make contact with the oolemma in a species specific manner. These results are discussed with respect both to the need to employ complementary optical methods in assessing connectivity patterns within COC and to the possible role that extracellular matrix-cell interactions play in the homeostatic control of oocyte growth and maturation.