Active growth and differentiation of endometrial stromal cells during the decidualization of early pregnancy are followed by programmed cell death (apoptosis) in the antimesometrial region of the decidua. The location and temporal specificity of decidual cell apoptosis and the identification of transforming growth factor-beta 1 (TGF beta 1) and TGF beta 2 protein and mRNA in the decidua suggested that members of the TGF beta family control stromal apoptosis by autocrine or paracrine mechanisms. To examine this possibility, ovariectomized rats were pretreated sequentially with estradiol (2 days), no treatment (2 days), and medroxyprogesterone acetate 48 h before death. Endometrial stromal cells were selectively isolated by enzymatic digestion and cultured for 48 h before the experiments. The addition of TGF beta 1 (10(-9)-10(-12) M) stimulated a dose-dependent increase in 200-basepair nucleosomal fragmentation of DNA, indicating increased apoptosis. TGF beta 1 and TGF beta 2 had an equivalent effect in stimulating stromal apoptosis. If an autocrine or paracrine mechanism is responsible for the control of stromal apoptosis, increased apoptosis in cultures at high cell density and stromal secretion of TGF beta would be expected. Increasing the density of stromal cell cultures increased levels of nucleosomal DNA fragmentation. To examine stromal secretion of growth factor, cell cultures were incubated with defined medium, and concentrated media were subjected to immunoblot analysis using antibodies specific for TGF beta 1 and -beta 2. Only the antibody to TGF beta 2 detected secreted growth factor. The concentration of the secreted growth factor increased with increasing density of cell culture. To determine whether stromal secretion of TGF beta 2 is responsible for the increasing nucleosomal DNA fragmentation observed with increasing cell density, cells were cultured with and without the addition of neutralizing antibodies to TGF beta s. The addition of monoclonal antibodies to TGF beta 1,2,3 or TGF beta 2,3 inhibited apoptosis. These data provide evidence that TGF beta 2 controls apoptosis in endometrial stromal cells in vitro by an autocrine/paracrine mechanism and suggest that this mechanism controls apoptosis in these cells during early pregnancy.

During blastocyst implantation, cellular degeneration of decidual tissue spreads from the antimesometrial region to the mesometrial region to accommodate the developing conceptus. To identify the mechanism of decidual regression and its tissue localization, decidual formation was induced in either intact pseudopregnant rats or ovariectomized rats treated with steroids. Antimesometrial and mesometrial cells were separated by elutriation and cultured for 48 h before DNA analysis. Nucleosomal DNA fragmentation was observed in antimesometrial cells, whereas DNA fragmentation was less marked in mesometrial cells. To determine whether a similar pattern of apoptosis occurred during decidual development in vivo, decidua tissues from antimesometrial and mesometrial regions were obtained on days 8-14. Nucleosomal DNA fragmentation was first detected on day 10 in the antimesometrial region, with fragmentation in the mesometrial region delayed by at least 24 h. DNA fragmentation increased in both tissues with time, but was always more pronounced in antimesometrial cells. Sulfated glycoprotein-2 (SGP-2) has been associated with apoptosis, and its expression was examined by Northern analysis. Levels of SGP-2 messenger RNA (mRNA) were detected in the antimesometrial region on day 9 and increased markedly by day 10; mesometrial expression was delayed 24 h. Levels of SGP-2 mRNA in both regions decreased before the onset of DNA fragmentation. In contrast, cathepsin-D expression, detected by immunohistochemistry, was localized to few mesometrial and antimesometrial cells between days 9-12, with all cells showing extensive staining by day 14. To determine whether changes in progesterone control mechanisms initiated decidual apoptosis, plasma progesterone levels were measured by RIA, and progesterone receptor mRNA levels in decidual tissues were examined by reverse transcription-polymerase chain reaction. Both parameters remained unchanged during decidual growth and regression. These results provide biochemical evidence that decidual regression occurs by apoptosis initiated in the antimesometrial region and with progression to the mesometrial region. Apoptotic cell death occurs despite high levels of plasma progesterone and high levels of progesterone receptor message in decidual tissue.

The endometrium is a dynamic tissue that, in response to hormonal cues, undergoes cycles of growth and involution. Extracellular factors required for this remodeling are poorly understood. The potential role in endometrial turnover of apolipoprotein J (apoJ), a secretory glycoprotein that can bind lipids and membrane-active proteins, is proposed on the basis of its spatial and temporal patterns of expression during normal cycling, after ovariectomy, and in response to hormone manipulation. In the mouse, apoJ mRNA was expressed in uterine luminal and glandular epithelial cells coincident with the presence of apoJ protein. The apoJ gene was differentially expressed in the glandular and uterine luminal epithelial cells during the estrous cycle and following hormone depletion. Expression of apoJ was not induced in ovariectomized mice by estrogen, progesterone, or dexamethasone treatment alone. Progesterone administration after an initial estrogen pretreatment, however, resulted in dramatic induction of apoJ as the progesterone level declined. In contrast, apoJ was not induced when a long-lived progesterone analog, medroxyprogesterone, was substituted for progesterone. In the human menstrual cycle, apoJ was present in glandular lumens only during the late secretory phase. Declining progesterone levels, causing substantial tissue reorganization, are characteristic of the times of marked apoJ induction in uterine epithelial cells. These expression patterns are consistent with apoJ functioning as an extracellular cytoprotectant by mediating clearance of and/or neutralizing cytolytic tissue debris.

No abstract

During early pregnancy, the uterine endometrium responds to an implanting blastocyst with the extensive growth and differentiation of decidualization. This transformation of endometrial stromal cells begins in the antimesometrial side of the uterus to form a primary decidual zone, expands to form a secondary zone in the antimesometrium, and eventually transforms stromal cells in the mesometrial region. During pregnancy, both decidual zones regress by apoptosis, leaving decidual cells in the mesometrial region to form decidua basalis and the mature placenta. Molecular mechanisms controlling cell death during blastocyst implantation and decidualization are unknown. We examined the hypothesis that progesterone and estrogen control of endometrial differentiation and eventual apoptosis involves control of bcl-2 gene family expression. Ovariectomized rats were primed with estradiol and treated with progestin (medroxyprogesterone acetate, 3.5 mg) and estradiol (200 ng) before an intrauterine stimulus to initiate decidualization. Expression of the two bax messenger RNA transcripts, 1.0 and 1.5 kilobases, was examined by Northern blot analysis after hormone treatment and decidualization, and only the 1.0-kilobase transcript was induced. After the same treatments, the expression of bcl-2, a suppressor of apoptosis, decreased. In situ analysis revealed a cell type-specific increase in bax expression after the hormonal treatment and decidualization. This increase was first seen in luminal and glandular epithelial cells and then in the periluminal stroma 24 h after intrauterine stimulation, with eventual progressive expression throughout the stroma. Expression of bcl-2 decreased after hormone treatment and decidualization. Immunohistochemical studies of Bax showed that expression of Bax protein accompanied decidualization of the stroma. Bcl-2 protein was only seen in the luminal and glandular epithelia, and its level decreased as decidualization progressed. These data indicate that the balance between bax and bcl-2 expression is altered during stromal cell differentiation. Increased expression of bax precedes nucleosomal DNA fragmentation and eventual apoptosis, which plays a significant role in placental development.