Human ES cells (hESC) exposed to bone morphogenic protein 4 (BMP4) in the absence of FGF2 have become widely used for studying trophoblast development, but the soundness of this model has been challenged by others, who concluded that differentiation was primarily toward mesoderm rather than trophoblast. Here we confirm that hESC grown under the standard conditions on a medium conditioned by mouse embryonic fibroblasts in the presence of BMP4 and absence of FGF2 on a Matrigel substratum rapidly convert to an epithelium that is largely KRT7+ within 48 h, with minimal expression of mesoderm markers, including T (Brachyury). Instead, they begin to express a series of trophoblast markers, including HLA-G, demonstrate invasive properties that are independent of the continued presence of BMP4 in the medium, and, over time, produce extensive amounts of human chorionic gonadotropin, progesterone, placental growth factor, and placental lactogen. This process of differentiation is not dependent on conditioning of the medium by mouse embryonic fibroblasts and is accelerated in the presence of inhibitors of Activin and FGF2 signaling, which at day 2 provide colonies that are entirely KRT7 + and in which the majority of cells are transiently CDX2 + . Colonies grown on two chemically defined media, including the one in which BMP4 was reported to drive mesoderm formation, also differentiate at least partially to trophoblast in response to BMP4. The experiments demonstrate that the in vitro BMP4/hESC model is valid for studying the emergence and differentiation of trophoblasts.A popular model for examining the early commitment of cells to the trophoblast (TR) lineage is based on the initial observation of Xu et al. (1), who noted that a group of related factors in the TGF-β family, especially bone morphogenic protein 4 (BMP4), was capable of causing human ES cells (hESC) to differentiate efficiently to TRs. This differentiation occurred without extensive generation of mesoderm, endoderm, and ectoderm derivatives, as judged by microarray analysis of transcribed genes, although a low level of expression of genes characteristic of mesoderm and endoderm did occur. This model has become widely used (2-13) to study an aspect of early human development that is not easily addressed otherwise because of lack of access to human embryos. Over the course of these studies it was demonstrated that the key to obtaining differentiation primarily to TR rather than to other lineages when using BMP4 as the triggering agent was to exclude FGF2, a factor required for maintenance of hESC (14-17). When BMP4 is provided simultaneously with FGF2, the morphological transition of the cells is altered (10), and the colonies begin to form a range of mesoderm and endoderm derivatives in addition to TR (18). This effect is probably achieved by FGF2 signaling through the MEK/ERK pathway, thereby preserving NANOG expression (19,20). This body of work suggests that optimal differentiation to TR can be achieved best by maximizing BMP4 signaling while simultaneous...

Human embryonic stem cells (ESCs) readily commit to the trophoblast lineage after exposure to bone morphogenetic protein-4 (BMP-4) and two small compounds, an activin A signaling inhibitor and a FGF2 signaling inhibitor (BMP4/A83-01/PD173074; BAP treatment). During differentiation, areas emerge within the colonies with the biochemical and morphological features of syncytiotrophoblast (STB). Relatively pure fractions of mononucleated cytotrophoblast (CTB) and larger syncytial sheets displaying the expected markers of STB can be obtained by differential filtration of dispersed colonies through nylon strainers. RNA-seq analysis of these fractions has allowed them to be compared with cytotrophoblasts isolated from term placentas before and after such cells had formed syncytia. Although it is clear from extensive gene marker analysis that both ESC-and placenta-derived syncytial cells are trophoblast, each with the potential to transport a wide range of solutes and synthesize placental hormones, their transcriptome profiles are sufficiently dissimilar to suggest that the two cell types have distinct pedigrees and represent functionally different kinds of STB. We propose that the STB generated from human ESCs represents the primitive syncytium encountered in early pregnancy soon after the human trophoblast invades into the uterine wall.is encountered during at least two stages of human placental development (1-3). The first coincides with early implantation when a multinucleated syncytium forms, presumably by cell fusion events, ahead of proliferating, mononucleated, cytotrophoblast (CTB) cells originating from polar trophectoderm (3,4). This invasive syncytium emerges either during or soon after the trophoblast passes through the breached uterine epithelium and into the decidualized stromal layer beneath and appears to be responsible for hollowing out regions within the stroma to form lacunae (5), which become filled with fluid and cells from maternal blood and uterine glands and presumably provide a source of nutrients for the conceptus (3, 6). By about 12 d of gestation, soon after the blastocyst has sunk below the endometrial surface, strands of cytotrophoblast begin to form and penetrate through the primitive syncytium to form primary chorionic villi, which are subsequently invaded by extraembryonic mesoderm to form secondary and tertiary villi (villous trees) (2-4). The cytotrophoblast cells associated with the villi continue to divide and provide a progenitor cell population for the villous STB, which is the cell layer that covers the outer surface of the villi and forms the definitive interface involved in exchange of gases, nutrients, and excretory materials between the fetal placenta and maternal blood. Villous STB is also the major site for production of placental hormones. Cytotrophoblast cells at the tips of the anchoring villi proliferate and colonize the endometrium, thus expanding the placental bed and simultaneously remodeling maternal spiral arteries. The extent to which extravillous trophoblast becom...

We examined whether impairment of intracellular Ca(2+) homeostasis is related to poor embryo development in in vitro-aged oocytes. We found that in vitro aging of mouse oocytes affected the patterns of Ca(2+) oscillations at fertilization: these Ca(2+) oscillations were lower in amplitude and higher in frequency compared with oocytes without in vitro aging. We also observed that the intracellular Ca(2+) store was decreased in in vitro-aged oocytes. A decrease in the Ca(2+) store induced by thapsigargin, a specific endoplasmic reticulum (ER) membrane Ca(2+)-ATPase inhibitor, resulted in a lower fertilization rate and in poorer embryo development. The frequency of Ca(2+) oscillations was significantly increased at fertilization, whereas their amplitude was decreased in thapsigargin-treated oocytes. These results suggest that impairment of intracellular Ca(2+) homeostasis (such as a decrease in the ER Ca(2+) store) caused an alteration in Ca(2+) oscillations and the poor embryo development in in vitro-aged oocytes. Because embryo fragmentation is closely related to apoptosis, we examined expression of BAX (a proapototic protein) and BCL2 (an antiapoptotic protein) in in vitro-aged oocytes. Although BCL2 was strongly expressed in oocytes without in vitro aging, expression of BCL2 was significantly reduced in oocytes of other culture conditions and treatments such as those in in vitro aging and those that were pretreated with H(2)O(2) or thapsigargin. Acting together, alteration in Ca(2+) oscillations and decrease in BCL2 expression in in vitro-aged oocytes may lead to poor embryo development.

Oocyte quality is a key factor in determining embryo development; however, we have a poor understanding of what constitutes oocyte quality or the mechanisms governing it. Postovulatory aging of oocytes that have not been fertilized for a prolonged time after ovulation is known to significantly impair oocyte quality and subsequent embryo development after fertilization. Embryos derived from postovulatory-aged oocytes are prone to undergo apoptosis due to the decreased Bcl-2 expression. Postovulatory aging of oocytes changes the patterns of Ca 2+ oscillations at fertilization as a result of impaired Ca 2+ regulation in the endoplasmic reticulum. Moreover, postovulatory aging of oocytes impairs mitochondrial adenosine triphosphate production as a result of increasing oxidative stresses. Oxidative stresses also affect intracellular Ca 2+ regulation and impair embryo development after fertilization. Collectively, the mechanism of postovulatory oocyte aging might be involved in reactive oxygen species-induced mitochondrial injury followed by abnormal intracellular Ca 2+ regulation in the endoplasmic reticulum.

It is imperative to unveil the full range of differentiated cell types into which human pluripotent stem cells (hPSCs) can develop. The need is twofold: it will delimit the therapeutic utility of these stem cells and is necessary to place their position accurately in the developmental hierarchy of lineage potential. Accumulated evidence suggested that hPSC could develop in vitro into an extraembryonic lineage (trophoblast (TB)) that is typically inaccessible to pluripotent embryonic cells during embryogenesis. However, whether these differentiated cells are truly authentic TB has been challenged. In this debate, we present a case for and a case against TB differentiation from hPSCs. By analogy to other differentiation systems, our debate is broadly applicable, as it articulates higher and more challenging standards for judging whether a given cell type has been genuinely produced from hPSC differentiation.Reproduction (2014) 147 D1-D12

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We examined the molecular mechanisms of the antiestrogenic effects of clomiphene citrate (CC) in the endometrium using two types of cell lines, Ishikawa and EM-E6/E7/hTERT cells. CC or ICI182780 inhibited 17beta-estradiol (E2)-induced endometrial cell proliferation and transcriptional activation of the estrogen response element (ERE) gene. We directly visualized the ligand-estrogen receptor (ER)alpha interaction using green fluorescent protein (GFP)-tagged ER alpha in a single living cell. Whereas E2 changed the nuclear localization of GFP-ER alpha to a punctate distribution within 5 min, CC or ICI182780 changed the slower and less mobilization of GFP-ER alpha compared with E2. Pretreatment with CC or ICI182780 partly prevented the E2-induced nuclear redistribution of GFP-ER alpha. Fluorescence recovery after photobleaching revealed that GFP-ER alpha mobility treated with E2 was more rapid than that treated by CC or ICI182780. As coactivator recruitment to the ER is essential for ER-dependent transcription, we examined the interaction between ER alpha and steroid receptor coactivator-1 (SRC-1). The complex formation between ER alpha and SRC-1 was significantly increased by E2 but was prevented in the presence of CC or ICI182780 by coimmunoprecipitation. Moreover, the E2-induced colocalization of GFP-ER alpha and SRC-1 was prevented in the presence of CC or ICI182780 according to an immunofluorescence assay. We also observed that the reduction of SRC-1 using small interfering RNA for SRC-1 resulted in the inhibition of E2-induced cell proliferation and transcriptional activation of the ERE gene. Collectively, these results suggest that CC may inhibit E2-induced endometrial epithelial cell proliferation and ERE transactivation by inhibiting the recruitment of SRC-1 to ER alpha.

We recently reported that bezafibrate, a lipid-lowering drug of the fibrate class, administered in addition to clomiphene citrate (CC) successfully induced ovulation in CC-resistant polycystic ovary syndrome (PCOS) patients. We hypothesized that bezafibrate may directly affect ovarian follicle development. Insulin resistance and compensatory hyperinsulinemia are important for the pathogenesis of PCOS. In this study, we first examined the effects of tumor necrosis factor-alpha (TNF), which plays a role in insulin resistance, on follicle development by using the follicle culture system. TNF significantly inhibited follicle-stimulating hormone (FSH)-induced follicle development, 17beta-estradiol (E2) secretion, and ovulation rate in a dose-dependent manner. We then examined whether bezafibrate treatment could rescue the inhibition of FSH-induced follicle development and steroidogenesis by TNF. Bezafibrate treatment rescued inhibition of follicle development, secretion of E2, and ovulation rate by TNF. We examined the expression of peroxisome proliferator-activated receptor (PPAR) subtypes in mouse preantral follicles. As the protein expression of only PPARG was observed in mouse preantral follicles, we examined whether bezafibrate could affect follicle development and steroidogenesis through PPARG pathways. Treatment with GW1929, a selective PPARG agonist, restored inhibition of FSH-induced follicle development and steroidogenesis by TNF, whereas treatment with GW9662, a selective PPARG antagonist, canceled the restorative effects of bezafibrate. Collectively, the results in this study suggest that bezafibrate may directly exhibit a restorative effect on the inhibition of ovarian follicle development and steroidogenesis by TNF through the PPARG pathway.