We report studies of preimplantation human embryo development that correlate time-lapse image analysis and gene expression profiling. By examining a large set of zygotes from in vitro fertilization (IVF), we find that success in progression to the blastocyst stage can be predicted with >93% sensitivity and specificity by measuring three dynamic, noninvasive imaging parameters by day 2 after fertilization, before embryonic genome activation (EGA). These parameters can be reliably monitored by automated image analysis, confirming that successful development follows a set of carefully orchestrated and predictable events. Moreover, we show that imaging phenotypes reflect molecular programs of the embryo and of individual blastomeres. Single-cell gene expression analysis reveals that blastomeres develop cell autonomously, with some cells advancing to EGA and others arresting. These studies indicate that success and failure in human embryo development is largely determined before EGA. Our methods and algorithms may provide an approach for early diagnosis of embryo potential in assisted reproduction.
The in vivo studies presented here demonstrate that epidermal growth factor (EGF) is an important autocrine and/or paracrine mediator of estrogen-induced growth and differentiation in mouse uterus and vagina. An antibody specific for EGF significantly inhibited estrogeninduced uterine and vaginal growth, thereby implicating EGF involvement in estrogen action. Furthermore, EGF administered via slow-release pellets in ovariectomized mice acted as a potent uterine and vaginal mitogen as well as an inducer of vaginal keratinization. Experiments with ovariectomized, adrenalectomized, hypophysectomized mice indicated that EGF mitogenesis does not require pituitary or adrenal hormones. Treatment with EGF also mimicked estrogen in the induction of uterine lactoferrin (a major estrogen-inducible secretory protein) mRNA and protein. These data suggest that EGF has estrogen-like effects in the promotion of cell growth in the reproductive tract and that EGF may serve as an important mediator of estrogen action in vivo.Estrogen-induced growth of various target tissues is mediated, in part, by the production of polypeptide growth factors, which may act in an autocrine or paracrine fashion to stimulate proliferation (1, 2). Two polypeptide growth factors, insulin-like growth factor I and epidermal growth factor (EGF), have been associated with the induction of uterine growth by estrogen (2, 3). Our laboratory and others have demonstrated that immunoreactive EGF precursor is present in the mouse uterus (3, 4), that estrogen treatment enhances the expression of EGF (3, 4) and EGF receptor (5) in the uterus, and that EGF is a potent mitogen in mouse uterine cell cultures (6). These studies provide evidence suggesting that EGF plays an important role in estrogen-induced uterine growth.Previous studies (3, 6) suggesting that EGF plays a role in uterine growth led us to further evaluate the role of EGF as a potential regulator of reproductive-tract growth in vivo. In this study, we demonstrate that estrogen action is inhibited by an EGF-specific antibody and that in vivo exposure to EGF alone mimics estrogen in the induction of uterine and vaginal growth and differentiation. Our results strongly suggest that EGF acts as an estrogen-inducible physiological mediator of mouse reproductive-tract growth in vivo. MATERIALS AND METHODS Quantitation of Uterine and Vaginal Growth FollowingExposure to an EGF-Specific Antibody. To determine whether an antibody specific for EGF could inhibit estrogen-induced growth, slow-release cholesterol-based pellets containing an antibody against EGF (whole antiserum; 2.5 mg per pellet; Collaborative Research) were prepared by Innovative Research of America. These pellets were divided into fourths and one quarter was implanted under the kidney capsule of 8-week-old female CD-1 mice that had been ovariectomized for at least 3 weeks. Appropriate control pellets containing normal whole serum (rabbit; 2.9 mg per pellet; Calbiochem) were also implanted. Three days following pellet implantation, the anim...
A map of human embryo development that combines imaging, molecular, genetic and epigenetic data for comparisons to other species and across pathologies would be greatly beneficial for basic science and clinical applications. Here, we compared mRNA and protein expression of key mediators of DNA methylation and histone modifications between mouse and human embryos, embryos from fertile/infertile couples, and following growth factor supplementation. We observed that individual mouse and human embryos are characterized by similarities and distinct differences in DNA methylation and histone modification patterns especially at the single-cell level. In particular, while mouse embryos first exhibited sub-compartmentalization of different histone modifications between blastomeres at the morula stage and cell sub-populations in blastocysts, differential histone modification expression was detected between blastomeres earlier in human embryos at the four- to eight-cell stage. Likewise, differences in epigenetic mediator expression were also observed between embryos from fertile and infertile couples, which were largely equalized in response to growth factor supplementation, suggesting that select growth factors might prevent alterations in epigenetic profiles during prolonged embryo culture. Finally, we determined that reduced expression via morpholino technologies of a single histone-modifying enzyme, Rps6ka4/Msk2, resulted in cleavage-stage arrest as assessed by time-lapse imaging and was associated with aneuploidy generation. Taken together, data document differences in epigenetic patterns between species with implications for fertility and suggest functional roles for individual epigenetic factors during pre-implantation development.
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