We used hyperosmolar stress to test blastocysts for their biologic and enzymatic responses to culture stress. Embryos mount dose- and time-dependent responses to hyperosmolar stress. Biological responses included slowed cavitation and cell accumulation and increased apoptosis at increasing doses. These responses were preceded by stress-activated protein kinase (SAPK) phosphorylation and nuclear translocation consistent with its causal role. For cavitation and new cell cycle initiation, 200 mM sorbitol caused stasis. Above 200 mM, sorbitol was ultimately lethal and below 200 mM, its embryos had milder effects. Phosphorylated SAPK was induced rapidly in embryos at 0.5 h in a dose-dependent manner from 0 to 600 mM sorbitol. Higher hyperosmolarity caused a biphasic peak of phosphorylated SAPK, but there was no return to baseline through 3 h. At 24 h, a dose-dependent response persisted that was linear from 0 to 200 mM sorbitol. Hyperosmolar stress rapidly induced, within 0.5 h, phosphorylated, nuclear c-Jun and decreased phosphorylated, nuclear c-Myc in a SAPK-dependent manner. The data suggest that SAPK is induced and functions on down-stream effector molecules in a temporal and quantitative manner consistent with its function in the embryonic homeostatic response to stress. The remarkable resistance of embryos to high concentrations of sorbitol suggests that part of its homeostatic response is different from that of somatic cells.

Stress reduces fertility, but the mechanisms mediating this are not understood. For a successful pregnancy, placental trophoblast stem cells (TSCs) in the implanting embryo proliferate and then a subpopulation differentiates to produce hormones. Normally, differentiation occurs when inhibitor of differentiation 2 (ID2) protein is lost in human and mouse placental stem cells. We hypothesize that stress enzyme-dependent differentiation occurs in association with insufficient TSC accumulation. We studied a well-defined model where TSC differentiation requires ID2 loss. The loss of ID2 derepresses the promoter of chorionic somatomammotropin hormone 1 (CSH1), the first hormone after implantation. Csh1 mRNA is known to be induced in stressed TSCs. In this study, we demonstrate that AMP-activated protein kinase (PRKAA1/2, aka AMPK) mediates the stress-induced proteasome-dependent loss of ID2 at high stress levels. At very low stress levels, PRKAA1/2 mediates metabolic adaptation exemplified by the inactivation of acetyl coA carboxylase by phosphorylation without ID2 loss. At the highest stress levels, irreversible TSC differentiation as defined by ID2 loss and slower cell accumulation occurs. However, lower stress levels lead to reversible differentiation accompanied by metabolic adaptation. These data support the hypothesis that PRKAA1/2 mediates preparation for differentiation that is induced by stress at levels where a significant decrease in cell accumulation occurs. This supports the interpretation that enzyme-mediated increases in differentiation may compensate when insufficient numbers of stem cells accumulate.

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.

Gene mutations in the phosphoinositide-metabolizing enzymes are linked to various human diseases. In mammals, PIKfyve synthesizes PtdIns(3,5)P 2 and PtdIns5P lipids that regulate endosomal trafficking and responses to extracellular stimuli. The consequence of pikfyve gene ablation in mammals is unknown. To clarify the importance of PIKfyve and PIKfyve lipid products, in this study, we have characterized the first mouse model with global deletion of the pikfyve gene using the Cre-loxP approach. We report that nearly all PIKfyve KO/KO mutant embryos died before the 32-64-cell stage. Cultured fibroblasts derived from PIKfyve flox/flox embryos and rendered pikfyve-null by Cre recombinase expression displayed severely reduced DNA synthesis, consistent with impaired cell division causing early embryo lethality. The heterozygous PIKfyve WT/KO mice were born at the expected Mendelian ratio and developed into adulthood. PIKfyve WT/KO mice were ostensibly normal by several other in vivo, ex vivo, and in vitro criteria despite the fact that their levels of the PIKfyve protein and in vitro enzymatic activity in cells and tissues were 50 -55% lower than those of wild-type mice. Consistently, steady-state levels of the PIKfyve products PtdIns(3,5)P 2 and PtdIns5P selectively decreased, but this reduction (35-40%) was 10 -15% less than that expected based on PIKfyve protein reduction. The nonlinear decrease of the PIKfyve protein versus PIKfyve lipid products, the potential mechanism(s) discussed herein, may explain how one functional allele in PIKfyve WT/KO mice is able to support the demands for PtdIns(3,5)P 2 /PtdIns5P synthesis during life. Our data also shed light on the known human disorder linked to PIKFYVE mutations.Reversible phosphorylation by kinases and phosphatases at positions 3, 4, and/or 5 of the inositol head group in phosphatidylinositol (PtdIns) 2 generates a family of seven phosphoinositide species (1-6). They all are now found to function as versatile membrane-anchored signals that control diverse and essential cellular processes. Consequently, mutations in the genes encoding the phosphoinositide-metabolizing enzymes are associated with an increasing number of human diseases (1-6). The mammalian enzyme that makes PtdIns(3,5)P 2 from PtdIns3P and PtdIns5P from PtdIns is PIKfyve (7,8). It is an evolutionarily conserved large protein of ϳ230 kDa, a product of a single gene in the animal kingdom, whose function is required for proper performance of the endosomal system and certain signaling pathways (9 -11). PIKfyve harbors a PtdIns3P-binding module, i.e. the FYVE finger domain that associates with the PtdIns3P-enriched endosomal membranes, assuring a rapid PIKfyve recruitment to this low abundance substrate of its catalytic activity (12). PIKfyve interacts physically or functionally with multiple partner proteins; the ones involved in PtdIns(3,5)P 2 homeostasis are the best studied (9). Thus, PIKfyve physically associates with the antagonistic enzyme, i.e. the PtdIns(3,5)P 2 -specific phosphatase Sac3, which ...

Shear stress at 1.2 dynes/cm(2) induces stress-activated protein kinase/jun kinase phosphorylation that precedes and causes apoptosis in embryos (Xie et al., 2006b, Biol Reprod). Pipetting embryos is necessary for many protocols, from in vitro fertilization to collecting embryos prior to analyzing gene expression by microarrays. We sought to determine if pipetting upregulates phosphorylated MAPK8/9 (formerly known as stress-activated protein kinase/jun kinase/SAPK/JNK1, 2). We found that phosphorylated MAPK8/9, a marker of MAPK8/9 activation, is upregulated in a dose-dependent manner by pipetting. Whereas embryos with the zona pellucida removed were more sensitive to stress-induced lethality mediated by 1.2 dynes/cm(2) shear force, phosphorylated MAPK8/9 was induced at lower numbers of pipet triturations in hatched embryos at E4.5. E4.5 embryos were more sensitive to induction of MAPK8/9 than unhatched embryos at E2.5 or E3.5. E3.5 embryos also showed a pipetting dose-dependent induction of FOS protein (formerly known as c-fos), a marker of shear stress in many cell types. Phosphorylated MAPK8/9 measured in ex vivo embryos from E1.5 to E4.5 were expressed at low levels. Embryos that had been pipetted sufficiently to induce phosphorylated MAPK8/9 and FOS had the same number of cells as untreated embryos 24 hr later. This suggests that rapid phosphorylation of MAPK8/9 due to transient shear stress does not mediate long-term negative biological outcomes. But, it is possible that techniques requiring multiple handling events would induce MAPK8/9 and cause biological outcomes or that other biological outcomes are affected by low amounts of transient shear stress. This study suggests that embryo handling prior to experimental measurement of signal transduction phosphoproteins, proteins and mRNA should be performed with care. Indeed, it is likely that shear stress may cause rapid transient changes in hundreds of proteins and mRNA.

Accumulating data suggest that 20% O2 causes human and mouse placental trophoblast stem cell (TSC) differentiation and suppresses proliferation. We tested the hypotheses that phosphorylated stress-activated protein kinase (pSAPK) levels report the optimal O2 level for TSC culture, and that pSAPK responds to contradictory signals. We tested the dose range of 0–20% O2 (0, 0.5, 2, and 20%) on five effects in cultured TSC. The results showed 1) TSC accumulation rates were highest at 2% O2, lower at 20% and lowest at 0–0.5%; 2) pSAPK protein levels were lowest at 2% O2, higher at 20%, and highest at 0–0.5%; 3) Cleaved caspase 3, an apoptosis marker, increased at 0.5% O2, and was highest at 0% O2. 4) Three markers for multipotency were highest at 2 and 20% and significantly decreased at 0.5%–0%. 5) In contrast three differentiation markers were lowest at 2% and highest at 0.5%–0%. Thus, 2% O2 is the optimum as defined by lowest pSAPK and differentiation markers and highest growth rate and multipotency markers, without appreciable apoptosis. In addition, two lines of evidence suggest that fibroblast growth factor (FGF)4 does not directly activate SAPK. SAPK activity increases transiently with FGF4 removal at 2% O2, but SAPK activity decreases when O2 is switched from 20% to 2% with FGF4 present. Thus, SAPK is activated by contradictory signals, but activity decreases when either signal is removed. Taken together, the findings suggest that pSAPK senses suboptimal signals during TSC culture and probably in vivo.

Stress-activated protein kinase/c-Jun kinase (SAPK/JNK) is thought to be necessary for preimplantation embryonic development (Maekawa et al., 2005). However, media increases SAPK/JNK phosphorylation and these levels negatively correlate with embryonic development (Wang et al., 2005). Culture-induced stress could confuse analysis of the role of SAPK in development. In this study, we tested how SAPK/JNK inhibitors influence embryonic development in optimal and non-optimal media and define the contribution of cell survival and proliferation to the embryonic response to these media. SAPK/JNK inhibitors retard embryonic development in suboptimal Ham's F10, but improve development in optimal potassium (K+) simplex optimized media (KSOM) +AA. In KSOM + amino acids (KSOM+AA), two SAPK/JNK inhibitors increase the rate of cavitation and hatching. These data suggest that (i) SAPK/JNK mediates the response to culture stress, not normal preimplantation embryonic development and (ii) SAPK/JNK inhibitors may be useful in ameliorating embryo stress caused by culture. To define the effects of media, we assayed the contribution of cell survival and proliferation and the differences in total cell number of cultured embryos. Embryos cultured from E3.5+24 h in the suboptimal medium (Ham's F10) induced significant but small increases in TdT (terminal deoxynucleotidyl transferase)-mediated dUDP nick-end labelling (TUNEL) positive cells. Bromodeoxyuridine (BrdU) incorporation in suboptimal Ham's F10 was significantly lower than in optimal KSOM+AA, suggesting that cell cycle arrest also contributes to slower increase in cell number in stressful media. This is the first report where TUNEL and BrdU were both assayed to define the relative contribution of cell cycle/S phase commitment and apoptosis to lessened cell number increase during embryo culture.

Eomesodermin (Eomes) is a transcription factor that is essential for trophoblast development. Stress stimuli activate stress-activated protein kinase (MAPK8/9) and modulate transcription factors in trophoblast stem cells (TSCs). In this study, we test the hypothesis that stress-induced Eomes upregulation and downstream trophoblast development are MAPK8/9-dependent. Immunocytochemical and immunoblot assays suggest that Eomes is induced by hyperosmolar stress in a dose- and time-dependent manner. Two MAPK8/9 inhibitors that work by different mechanisms, LJNKl1 and SP600125, block induction of Eomes protein by stress. During normal TSC differentiation, the transcription factor heart and neural crest derivatives expressed 1 (HAND1) is dependent on Eomes, and chorionic somatomammotropin hormone 1 (CSH1) expression is dependent on HAND1. Similar to Eomes, HAND1 and CSH1 induction by stress are MAPK8/9-dependent, and CSH1 is induced in nearly all stressed TSCs. CSH1 induction normally requires downregulation of the transcription factor inhibitor of differentiation 2 (ID2) as well as HAND1 upregulation. It was shown previously that hyperosmolar stress induces AMP-activated protein kinase (PRKAA1/2)-dependent ID2 loss in a MAPK8/9-independent manner. Inhibition of PRKAA1/2 with compound C and LJNKl1, more that MAPK8/9 inhibitors alone, inhibits the induction of CSH1 by stress. Taken together these data suggest that stress-induced MAPK8/9 and PRKAA1/2 regulate transcription factors Eomes/HAND1 and ID2, respectively. Together this network mediates induction of CSH1 by stress. Therefore, stress triggers a proportional increase in a normal early TSC differentiation event that could be adaptive in inducing CSH1. But the flexibility of TSCS to undergo stress-induced differentiation could lead to pathophysiological consequences if stress endured and TSC differentiation became unbalanced.