Fangfei Wang

Shear Stress Induces Preimplantation Embryo Death That Is Delayed by the Zona Pellucida and Associated with Stress-Activated Protein Kinase-Mediated Apoptosis

Abstract In this study, we discovered that embryos sense shear stress and sought to characterize the kinetics and the enzymatic mechanisms underlying induction of embryonic lethality by shear stress. Using a rotating wall vessel programmed to produce 1.2 dynes/cm2 shear stress, it was found that shear stress caused lethality within 12 h for E3.5 blastocysts. Embryos developed an approximate 100% increase in mitogen-activated protein kinase 8/9 (formerly known as stress-activated protein kinase/junC kinase 1/2) phosphorylation by 6 h of shear stress that further increased to approximately 350% by 12 h. Terminal deoxynucleotidyltransferase dUTP nick end labeling/apoptosis was at baseline levels at 6 h and increased to approximately 500% of baseline at 12 h, when irreversible commitment to death occurred. A mitogen-activated protein kinase 8/9 phosphorylation inhibitor, D-JNKI1, was able to inhibit over 50% of the apoptosis, suggesting a causal role for mitogen-activated protein kinase 8/9 phosphorylation in the shear stress-induced lethality. The E2.5 (compacted eight-cell/early morula stage) embryo was more sensitive to shear stress than the E3.5 (early blastocyst stage) embryo. Additionally, zona pellucida removal significantly accelerated shear stress-induced lethality while having no lethal effect on embryos in the static control. In conclusion, preimplantation embryos sense shear stress, chronic shear stress is lethal, and the zona pellucida lessens the lethal and sublethal effects of shear stress. Embryos in vivo would not experience as high a sustained velocity or shear stress as induced experimentally here. Lower shear stresses might induce sufficient mitogen-activated protein kinase 8/9 phosphorylation that would slow growth or cause premature differentiation if the zona pellucida were not intact.

Entire mitogen activated protein kinase (MAPK) pathway is present in preimplantation mouse embryos

To understand how mitogenic signals are transduced into the trophoblasts in preimplantation embryos, the expression of mitogen‐activated protein kinase (MAPK) pathway molecules was tested. We used immunocytochemical means and reverse transcriptase‐polymerase chain reaction to test whether MAPK pathway molecule gene products exist at the protein and phosphoprotein level in the zygote and the RNA level in the egg and zygote. In addition, all antibodies detected the correct‐sized major band in Westerns of placental cell lines representing the most prevalent cell type in preimplantation embryos. A majority of mRNA transcripts of MAPK pathway genes were detected in unfertilized eggs, and all were expressed in the zygote. We found that the MAPK pathway protein set consisting of the following gene products was present: FRS2α, GRB2, GAB1, SOS1, Ha‐ras, Raf1/RafB, MEK1,2,5, MAPK/ERK1,2, MAPK/ERK5, and RSK1,2,3 (see abbreviations). These proteins were detected in trophoblasts in embryonic day (E) 3.5 embryos when they could mediate mitogenic fibroblast growth factor signals from the embryo or colony stimulating factor‐1 signals from the uterus. The phosphorylation state and position of the phosphoproteins in the cells suggested that they might function in mediating mitogenic signals. Interestingly, a subtle transition from maternal MAPK function to zygotic function was suggested by the localization for three MAPK pathway enzymes between E2.5 and E3.5, Raf1 phospho is largely cell membrane‐localized at E2.5 and E3.5, and MEK1,2 phospho accumulates in the nucleus on E2.5 and E3.5. However, MAPK phospho shifts from nuclear accumulation at E2.5 to cytoplasmic accumulation at E3.5. This finding is similar to the cytoplasmic MAPK phospho localization reported in fibroblast growth factor signaling fields in postimplantation embryos (Corson et al. [ 2003 ] Development 130:4527–4537). This spatial and temporal expression study lays a foundation to plan and analyze perturbation studies aimed at understanding the role of the major mitogenic pathway in preimplantation mouse embryos. Developmental Dynamics 231:72–87, 2004.

Hyperosmolar stress induces global mRNA responses in placental trophoblast stem cells that emulate early post-implantation differentiation.

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 24 h and tested using whole mouse genome arrays and Real-time quantitative (Q)PCR. At 0.5 h, all 31 highly changing mRNA (>1.5-fold compared with unstressed TSC) decreased, but by 24 h 158/288 genes were upregulated. Many genes upregulated at 24 h were near baseline levels in unstressed TSC, suggesting new transcription. Thus few genes change during the early stress response, but by 24 h TSC have adapted to start new transcription with large gene sets. Types of genes upregulated at 24 h included homeostatic genes regulating growth and DNA damage induced (GADD45beta/gamma), 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 protein (PLP)M], and extracellular matrix genes (CCN1/2). Transcription factors for later placental cell lineages, spongiotrophoblast (MASH2, TPBPalpha) and syncytiotrophoblast (GCM1, TEF5) and placental hormones (PLPA, PLII) were not induced by 24 h 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.

Use of Hyperosmolar Stress to Measure Stress-Activated Protein Kinase Activation and Function in Human HTR Cells and Mouse Trophoblast Stem Cells

Embryo growth is inversely correlated with hyperosmolar stress-induced stress-activated protein kinase/jun kinase (SAPK/JNK) induction. To examine whether stress has similar effects in stem cells derived from the embryo, the authors test trophoblast stem cells. The stress response of human placental and mouse trophoblast stem cell lines are tested here. Peak phosphorylated SAPK/JNK was induced by 400 mM sorbitol at 0.5 hours. At this dose, there is an SAPK/JNK-dependent decrease in mitogenic, phosphorylated cMyc at 0.5 hours preceding an SAPK/JNK-dependent decrease in cell cycle entrance at 24 hours. At 0.5 hours, SAPK/JNK decreases terminal deoxynucleotidyltransferase dUTP nick end labeling/apoptosis at sorbitol doses from 50 mM to 400 mM and induces phosphorylated cJun prior to an SAPK/JNK-dependent, approximate 8-fold increase in apoptosis by 24 hours at 400 mM. SAPK/JNK phosphorylation peaked at 0.5 to 4 hours and largely subsided by 12 hours. Thus, total SAPK/JNK exists before stress and mediates rapid, homeostatic molecular responses that become biologic consequences after phosphorylated SAPK/JNK ends. This suggests continuity in the homeostatic mechanisms and functions of SAPK/JNK in placental lineage cells during implantation, in which SAPK/JNK is completely responsible for cell cycle arrest and largely responsible for apoptosis.

Acquisition of essential somatic cell cycle regulatory protein expression and implied activity occurs at the second to third cell division in mouse preimplantation embryos

It is clear that G1–S phase control is exerted after the mouse embryo implants into the uterus 4.5 days after fertilization (E4.5); null mutants of genes that control cell cycle commitment such as max, rb (retinoblastoma), and dp1 are embryonic lethal after implantation with proliferation phenotypes. But, a number of studies of genes mediating proliferation control in the embryo after fertilization‐implantation have yielded confusing results. In order to understand when embryos might first exert G1–S phase regulatory control, we assayed preimplantation mouse embryos for the acquisition of expression of mRNA, protein, and phospho‐protein for max, Rb, and DP‐1, and for the proliferation‐promoting phospho‐protein forms of mycC (thr58/ser62) and Rb (ser795). The key findings are that: (1) DP‐1 protein was present in the nucleus as early as the four‐cell stage onwards, (2) max protein was in the nucleus, suggesting function from the four‐cell stage onwards, (3) both mycC and Rb all form protein was present at increasing quantities in the cytoplasm from the 2 cell and 4/8 cell stage, respectively, (4) the phosphorylated form of mycC phospho was present in the nucleus at high levels from the two‐cell stage through blastocyst‐stage, and (5) the phosphorylated form of Rb was detected at low levels in the two‐cell stage embryo and was highly expressed at the 4/8‐cell stage through the blastocyst stage. Taken together, these data suggest that activation of mycC phospho/max dimer pairs, (E2F)/DP‐1 dimer pairs, and repression of Rb inhibition of cell cycle progression via phosphorylation at ser795 occurs at the earliest stages of embryonic development. In addition, the presence of max, mycC phospho, DP‐1, and Rb phospho in the nuclei of embryonic and placental lineage cells in the blastocyst and in trophoblast stem cells suggests that a similar type of cell cycle regulation is present throughout preimplantation development and in both embryonic and extra‐embryonic cell lineages.