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Background Human uterine leiomyoma (fibroids; LYO) are the most common benign neoplasms in reproductive-aged women. Dysregulated extracellular matrix and irregular LYO reversion-inducing cysteine-rich protein with Kazal motifs (RECK) expression are thought to be mediated by aberrant microRNA (miR) expression. The relationship of miR-15b and RECK expression in LYO has not been studied. Methods The expression levels of miR-15b and RECK were determined by quantitative RT-PCR, Western blot, and immunohistochemistry in cultures derived from commercial primary leiomyoma (cpLYO) and myometrial (cpMYO) cell lines and leiomyoma (pLYO) and myometrium (pMYO) tissue from surgical samples respectively. The relationship between miR-15b and RECK expression in cpLYO and pLYO (compared to their respective myometrial controls) was evaluated following transfection of cell cultures with either miR-15b mimic or inhibitor. Results Elevated levels of miR-15b were observed in cpLYO (2.82-fold; p = 0.04) and pLYO cell (1.30-fold; p = 0.0001) cultures respectively compared to corresponding MYO cell controls. Following transfection with miR-15b mimic, cpLYO cells (0.62-fold; p

The microvasculature of the corpus luteum (CL), which comprises greater than 50% of the total number of cells in the CL, is thought to be the first structure to undergo degeneration via apoptosis during luteolysis. These studies compared the apoptotic potential of various cytokines (tumor necrosis factor α, TNFα; interferon gamma, IFNγ; soluble Fas ligand, sFasL), a FAS activating antibody (FasAb), and the luteolytic hormone prostaglandin F2α (PGF2α) on CL-derived endothelial (CLENDO) cells. Neither sFasL, FasAb nor PGF2α had any effect on CLENDO cell viability. Utilizing morphological and biochemical parameters it was evident that TNFα and IFNγ initiated apoptosis in long-term cultures. However, TNFα was the most potent stimulus for CLENDO cell apoptosis at early time points. Unlike many other studies described in non-reproductive cell types, TNFα induced apoptosis of CLENDO cells occurs in the absence of inhibitors of protein synthesis. TNFα-induced death is typically associated with acute activation of distinct intracellular signaling pathways (e.g. MAPK and sphingomyelin pathways). Treatment with TNFα for 5–30 min activated MAPKs (ERK, p38, and JNK), and increased ceramide accumulation. Ceramide, a product of sphingomyelin hydrolysis, can serve as an upstream activator of members of the MAPK family independently in numerous cell types, and is a well-established pro-apoptotic second messenger. Like TNFα, treatment of CLENDO cells with exogenous ceramide significantly induced endothelial apoptosis. Ceramide also activated the JNK pathway, but had no effect on ERK and p38 MAPKs. Pretreatment of CLENDO cells with glutathione (GSH), an intracellular reducing agent and known inhibitor of reactive oxygen species (ROS) or TNFα-induced apoptosis, significantly attenuated TNFα-induced apoptosis. It is hypothesized that TNFα kills CLENDO cells through elevation of reactive oxygen species, and intracellular signals that promote apoptosis.

The cyclic nature of the capillary bed in the corpus luteum offers a unique experimental model to examine the life cycle of endothelial cells, involving discrete physiologically regulated steps of angiogenesis, blood vessel maturation and blood vessel regression. The granulosa cells and theca cells of the developing antral follicle and the steroidogenic cells of the corpus luteum produce and respond to angiogenic factors and vasoactive peptides. Following ovulation the neovascularization during the early stages of corpus luteum development has been compared to the rapid angiogenesis observed during tumor formation. On the other end of the spectrum, the microvascular endothelial cells are the first cells to undergo apoptosis at the onset of corpus luteum regression. Important insights on the morphology and function of luteal endothelial cells have been gained from a combination of in vitro and in vivo studies on endothelial cells. Endothelial cells communicate with cells comprising the functional unit of the corpus luteum, i.e., other vascular cells, steroidogenic cells, and immune cells. This review is designed to provide an overview of the types of endothelial cells present in the corpus luteum and their involvement in corpus luteum development and regression. Available evidence indicates that microvascular endothelial cells of the corpus luteum are not alike, and may differ during the process of angiogenesis and angioregression. The contributions of vasoactive peptides generated by the luteal endothelin-1 and the renin-angiotensin systems are discussed in context with the function of endothelial cells during corpus luteum formation and regression. The ability of two cytokines, tumor necrosis factor alpha and interferon gamma, are evaluated as paracrine mediators of endothelial cell function during angioregression. Finally, chemokines are discussed as a vital endothelial cell secretory products that contribute to the recruitment of eosinophils and macrophages. The review highlights areas for future investigation of ovarian microvascular endothelial cells. The potential clinical applications of research directed on corpus luteum endothelial cells are intriguing considering reproductive processes in which vascular dysfunctions may play a role such as ovarian failure, polycystic ovary syndrome (PCOS), and ovarian hyperstimulation syndrome (OHSS).

The corpus luteum is a unique organ, which is transitory in nature. The development, maintenance and regression of the corpus luteum are regulated by endocrine, paracrine and autocrine signaling events. Defining the specific mediators of luteal development, maintenance and regression has been difficult and often perplexing due to the complexity that stems from the variety of cell types that make up the luteal tissue. Moreover, some regulators may serve dual functions as a luteotropic and luteolytic agent depending on the temporal and spatial environment in which they are expressed. As a result, some confusion is present in the interpretation of in vitro and in vivo studies. More recently investigators have utilized mutant mouse models to define the functional significance of specific gene products. The goal of this mini-review is to identify and discuss mutant mouse models that have luteal anomalies, which may provide some clues as to the significance of specific regulators of corpus luteum function.

We recently demonstrated that caspase-3 is important for apoptosis during spontaneous involution of the corpus luteum (CL). These studies tested if prostaglandin F2α (PGF2α) or FAS regulated luteal regression, utilize a caspase-3 dependent pathway to execute luteal cell apoptosis, and if the two receptors work via independent or potentially shared intracellular signaling components/pathways to activate caspase-3. Wild-type (WT) or caspase-3 deficient female mice, 25–26 days old, were given 10 IU equine chorionic gonadotropin (eCG) intraperitoneally (IP) followed by 10 IU human chorionic gonadotropin (hCG) IP 46 h later to synchronize ovulation. The animals were then injected with IgG (2 micrograms, i.v.), the FAS-activating antibody Jo2 (2 micrograms, i.v.), or PGF2α (10 micrograms, i.p.) at 24 or 48 h post-ovulation. Ovaries from each group were collected 8 h later for assessment of active caspase-3 enzyme and apoptosis (measured by the TUNEL assay) in the CL. Regardless of genotype or treatment, CL in ovaries collected from mice injected 24 h after ovulation showed no evidence of active caspase-3 or apoptosis. However, PGF2α or Jo2 at 48 h post-ovulation and collected 8 h later induced caspase-3 activation in 13.2 ± 1.8% and 13.7 ± 2.2 % of the cells, respectively and resulted in 16.35 ± 0.7% (PGF2α) and 14.3 ± 2.5% TUNEL-positive cells when compared to 1.48 ± 0.8% of cells CL in IgG treated controls. In contrast, CL in ovaries collected from caspase-3 deficient mice whether treated with PGF2α , Jo2, or control IgG at 48 h post-ovulation showed little evidence of active caspase-3 or apoptosis. CL of WT mice treated with Jo2 at 48 h post-ovulation had an 8-fold increase in the activity of caspase-8, an activator of caspase-3 that is coupled to the FAS death receptor. Somewhat unexpectedly, however, treatment of WT mice with PGF2α at 48 h post-ovulation resulted in a 22-fold increase in caspase-8 activity in the CL, despite the fact that the receptor for PGF2α has not been shown to be directly coupled to caspase-8 recruitment and activation. We hypothesize that PGF2α initiates luteolysis in vivo, at least in part, by increasing the bioactivity or bioavailability of cytokines, such as FasL and that multiple endocrine factors work in concert to activate caspase-3-driven apoptosis during luteolysis.

The corpus luteum is a dynamic tissue whose cellular composition is traced to the remnants of the ovarian follicle, neovascularization and infiltration of immune cells. The corpus luteum undergoes dramatic morphological and functional changes throughout its lifespan. Functionally the corpus luteum is responsible for the provision of progesterone in support of pregnancy. Depending on the species and the physiological state, the corpus luteum lifespan can last from less than a day to almost a year. During this time, whether it is during the development, maintenance, or regression stage the corpus luteum function is mediated by a number of influences including steroids, growth factors, gonadotropins, prostaglandins, cytokines, and peptide hormones. The variable expression of theses factors and their receptors control the fate of the corpus luteum.

The microvasculature of the corpus luteum (CL), which comprises greater than 50% of the total number of cells in the CL, is thought to be the first structure to undergo degeneration via apoptosis during luteolysis. These studies compared the apoptotic potential of various cytokines (tumor necrosis factor α, TNFα; interferon gamma, IFNγ; soluble Fas ligand, sFasL), a FAS activating antibody (FasAb), and the luteolytic hormone prostaglandin F2α (PGF2α) on CL-derived endothelial (CLENDO) cells. Neither sFasL, FasAb nor PGF2α had any effect on CLENDO cell viability. Utilizing morphological and biochemical parameters it was evident that TNFα and IFNγ initiated apoptosis in long-term cultures. However, TNFα was the most potent stimulus for CLENDO cell apoptosis at early time points. Unlike many other studies described in non-reproductive cell types, TNFα induced apoptosis of CLENDO cells occurs in the absence of inhibitors of protein synthesis. TNFα-induced death is typically associated with acute activation of distinct intracellular signaling pathways (e.g. MAPK and sphingomyelin pathways). Treatment with TNFα for 5–30 min activated MAPKs (ERK, p38, and JNK), and increased ceramide accumulation. Ceramide, a product of sphingomyelin hydrolysis, can serve as an upstream activator of members of the MAPK family independently in numerous cell types, and is a well-established pro-apoptotic second messenger. Like TNFα, treatment of CLENDO cells with exogenous ceramide significantly induced endothelial apoptosis. Ceramide also activated the JNK pathway, but had no effect on ERK and p38 MAPKs. Pretreatment of CLENDO cells with glutathione (GSH), an intracellular reducing agent and known inhibitor of reactive oxygen species (ROS) or TNFα-induced apoptosis, significantly attenuated TNFα-induced apoptosis. It is hypothesized that TNFα kills CLENDO cells through elevation of reactive oxygen species, and intracellular signals that promote apoptosis.

Background Recent data provide significant evidence to support the hypothesis that there are sub-populations of cells within solid tumors that have an increased tumor initiating potential relative to the total tumor population. CD133, a cell surface marker expressed on primitive cells of neural, hematopoietic, endothelial and epithelial lineages has been identified as a marker for tumor initiating cells in solid tumors of the brain, colon, pancreas, ovary and endometrium. Our objectives were to assess the relative level of CD133 expressing cells in primary human endometrial tumors, confirm their tumorigenic potential, and determine whether CD133 expression was epigenetically modified. Methods We assessed CD133 expression in primary human endometrial tumors by flow cytometry and analyzed the relative tumorigenicity of CD133+ and CD133- cells in an in vivo NOD/SCID mouse model. We assessed potential changes in CD133 expression over the course of serial transplantation by immunofluorescence and flow cytometry. We further examined CD133 promoter methylation and expression in normal endometrium and malignant tumors. Results As determined by flow cytometric analysis, the percentage of CD133+ cells in primary human endometrial cancer samples ranged from 5.7% to 27.4%. In addition, we confirmed the tumor initiating potential of CD133+ and CD133- cell fractions in NOD/SCID mice. Interestingly, the percentage of CD133+ cells in human endometrial tumor xenografts, as evidenced by immunofluorescence, increased with serial transplantation although this trend was not consistently detected by flow cytometry. We also determined that the relative levels of CD133 increased in endometrial cancer cell lines following treatment with 5-aza-2'-deoxycytidine suggesting a role for methylation in the regulation of CD133. To support this finding, we demonstrated that regions of the CD133 promoter were hypomethylated in malignant endometrial tissue relative to benign control endometrial tissue. Lastly, we determined that methylation of the CD133 promoter decreases over serial transplantation of an endometrial tumor xenograft. Conclusions These findings support the hypotheses that CD133 expression in endometrial cancer may be epigenetically regulated and that cell fractions enriched for CD133+ cells may well contribute to endometrial cancer tumorigenicity, pathology and recurrence.