Hyperosmolar stress acts in two ways on the implanting embryo and its major constituent, placental trophoblast stem cells (TSC). Stress causes homeostasis that slows development with lesser cell accumulation, increased cell cycle arrest, and apoptosis. Stress may also cause placental differentiation at implantation. To test for the homeostatic and differentiation-inducing consequences of stress, TSC were exposed to hyperosmolar stress for 24hr and tested using whole mouse genome arrays and Real-time quantitative (Q)PCR. At 0.5hr, all 31 highly changing mRNA (>1.5-fold compared with unstressed TSC) decreased, but by 24hr 158/288 genes were upregulated. Many genes upregulated at 24hr were near baseline levels in unstressed TSC, suggesting new transcription. Thus few genes change during the early stress response, but by 24hr TSC have adapted to start new transcription with large gene sets. Types of genes upregulated at 24hr included homeostatic genes regulating growth and DNA damage induced (GADD45β/γ), activator protein (AP)-1 (junB/junC/ATF3/4), heat shock proteins (HSP22/68), and cyclin-dependent kinase inhibitor [CDKI; p15, p21]. But, stress also induced transcription factors that mediate TSC differentiation to trophoblast giant cells (TGC)(Stra13, HES1, GATA-binding2), placental hormones [proliferin, placental lactogen (PL)1, prolactin-like peptide (PLP)M], and extracellular matrix genes (CCN1/2). Transcription factors for later placental cell lineages, spongiotrophoblast (MASH2, TPBPα) and syncytiotrophoblast (GCM1, TEF5) and placental hormones (PLPA, PLII), were not induced by 24hr stress. Thus stress induced the temporal and spatial placental differentiation normal after implantation. Although differentiation was induced, markers of TSC stemness such as inhibitor of differentiation (ID)2 remained at 100% of levels of unstressed TSC, suggesting that retained mRNA might mediate dedifferentiation were stress to subside.

In this review, we discuss the expression, regulation, downstream mechanisms, and function of stress-induced stress enzymes in mammalian oocytes, peri-implantation embryos, and the stem cells derived from those embryos. Recent reports suggest that stress enzymes mediate developmental functions during early mammalian development, in addition to the homeostatic functions shared with somatic cells. Stress-induced enzymes appear to insure that necessary developmental events occur: many of these events may occur at a slower rate, although some may occur more rapidly. Developmental events induced by stress may be mediated by a single dominant enzyme, but there are examples of responses that require the integration of more than one stress enzyme. The discussion focuses on the consequences of stress as a function of duration and magnitude, and this includes an emerging understanding of the threshold levels of duration and magnitude that lead to pathology. Other topics discussed are the reversibility of the developmental as well as homeostatic consequences of stress, the further problems with readaptation after stress subsides, and the mechanisms and functions of stress enzymes during early mammalian development. The analyses are done with specific concern for their practical impact in assisted reproductive technology (ART) and stem cell technologies.

Signaling by fibroblast growth factor (FGF) is essential is for trophoblast stem (TS) cells and preimplantation embryos. FGF4 provides essential signaling, but the expression of the complete set of 23 FGF family members has not been analyzed. Here, semi-quantitative RT-PCR and microarray analyses were used to define expression of all FGF ligand mRNA. RT-PCR was done for developmentally important FGF subfamilies, FGF10/FGF22 and FGF8/FGF17/FGF18 as well as FGF11. FGF4 and FGF18 are detected at highest levels by RT-PCR and microarrays. FGF10 was detected at low levels in both assays. FGF11 was detected at moderate levels by microarray, but not by RT-PCR. FGF17 was detected at low levels by array and moderate levels by RT-PCR. FGF8 and FGF22 were detected by RT-PCR, but not by microarrays during late cleavage divisions. FGF8, FGF5, and FGF9 were detected in the oocyte by microarray. FGF2, FGF3, and FGF7 were not detected by RT-PCR or microarrays and FGF13, FGF14, and FGF23 were not detected by microarray. Since a major role of FGF is to maintain TS cells, we tested human and mouse placental cell lines and early gestation human placenta for expression of FGF ligands. Expression in mouse TS cells was compared with preimplantation embryos, and human placental cell line expression was compared with human placenta, to infer which ligands are expressed in placental lineage vs. other cell lineages. The data suggest that human and mouse placenta share FGF18 and its high expression suggests preimplantation and early placental function.

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