Novel high throughput screen reports that benzo(a)pyrene overrides mouse trophoblast stem cell multipotency, inducing SAPK activity, HAND1 and differentiated trophoblast giant cells.

INTRODUCTION: Cultured mouse trophoblast stem cells (mTSC) maintain proliferation/normal stemness (NS) under FGF4, which when removed, causes normal differentiation (ND). Hypoxic, or hyperosmotic stress forces trophoblast giant cells (TGC) differentiate. Hypoxic, hyperosmotic, and genotoxic benzo(a)pyrene (BaP), which is found in tobacco smoke, force down-regulation of inhibitor of differentiation (Id)2, enabling TGC differentiation. Hypoxic and hyperosmotic stress induce TGC by SAPK-dependent HAND1 increase. Here we test whether BaP forces mTSC-to-TGC while inducing SAPK and HAND1. METHODS: Hand1 and SAPK activity were assayed by immunoblot, mTSC-to-TGC growth and differentiation were assayed at Tfinal after 72hr exposure of BaP, NS, ND, Retinoic acid (RA), or sorbitol. Nuclear-stained cells were micrographed automatically by a live imager, and assayed by ImageJ/FIJI, Biotek Gen 5, AIVIA proprietary artificial intelligence (AI) software or open source, CellPose artificial intelligence/AI software. RESULTS: BaP (0.05-1µM) activated SAPK and HAND1 without diminishing growth. TSC-to-TGC differentiation was assayed with increasingly accuracy for 2-4 N cycling nuclei and >4 N differentiating TGC nuclei, using ImageJ/FIJI, Gen 5, AIVIA, or CellPose AI software. The AIVIA and Cellpose AI software matches human accuracy. The lowest BaP effects on SAPK activation/HAND1 increase are >10-fold more sensitive than similar effects for mESC. RA induces 44-47% 1st lineage TGC differentiation, but the same RA dose induces only 1% 1st lineage mESC differentiation. DISCUSSION: First, these pilot data suggest that mTSC can be used in high throughput screens (HTS) to predict toxicant exposures that force TGC differentiation. Second, mTSC differentiated more cells than mESC for similar stress exposures, Third, open source AI can replace human micrograph quantitation and enable a miscarriage-predicting HTS.

CoQ10 increases mitochondrial mass and polarization, ATP and Oct4 potency levels, and bovine oocyte MII during IVM while decreasing AMPK activity and oocyte death.

PURPOSE: We tested whether mitochondrial electron transport chain electron carrier coenzyme Q10 (CoQ10) increases ATP during bovine IVM and increases %M2 oocytes, mitochondrial polarization/mass, and Oct4, and decreases pAMPK and oocyte death. METHODS: Bovine oocytes were aspirated from ovaries and cultured in IVM media for 24 h with 0, 20, 40, or 60 µM CoQ10. Oocytes were assayed for ATP by luciferase-based luminescence. Oocyte micrographs were quantitated for Oct4, pAMPK (i.e., activity), polarization by JC1 staining, and mitochondrial mass by MitoTracker Green staining. RESULTS: CoQ10 at 40 µM was optimal. Oocytes at 40 µM enabled 1.9-fold more ATP than 0 µM CoQ10. There was 4.3-fold less oocyte death, 1.7-fold more mitochondrial charge polarization, and 3.1-fold more mitochondrial mass at 40 µM than at 0 µM CoQ10. Increased ATP was associated with 2.2-fold lower AMPK thr172P activation and 2.1-fold higher nuclear Oct4 stemness/potency protein at 40 µM than at 0 µM CoQ10. CoQ10 is hydrophobic, and at all doses, 50% was lost from media into oil by ~ 12 h. Replenishing CoQ10 at 12 h did not significantly diminish dead oocytes. CONCLUSIONS: The data suggest that CoQ10 improves mitochondrial function in IVM where unwanted stress, higher AMPK activity, and Oct4 potency loss are induced.

Blastocyst-Derived Stem Cell Populations under Stress: Impact of Nutrition and Metabolism on Stem Cell Potency Loss and Miscarriage.

Data from in vitro and in vivo models suggest that malnutrition and stress trigger adaptive responses, leading to small for gestational age (SGA) blastocysts with fewer cell numbers. These stress responses are initially adaptive, but become maladaptive with increasing stress exposures. The common stress responses of the blastocyst-derived stem cells, pluripotent embryonic and multipotent placental trophoblast stem cells (ESCs and TSCs), are decreased growth and potency, and increased, imbalanced and irreversible differentiation. SGA embryos may fail to produce sufficient antiluteolytic placental hormone to maintain corpus luteum progesterone secretion that provides nutrition at the implantation site. Myriad stress inputs for the stem cells in the embryo can occur in vitro during in vitro fertilization/assisted reproductive technology (IVF/ART) or in vivo. Paradoxically, stresses that diminish stem cell growth lead to a higher level of differentiation simultaneously which further decreases ESC or TSC numbers in an attempt to functionally compensate for fewer cells. In addition, prolonged or strong stress can cause irreversible differentiation. Resultant stem cell depletion is proposed as a cause of miscarriage via a "quiet" death of an ostensibly adaptive response of stem cells instead of a reactive, violent loss of stem cells or their differentiated progenies.

Stress responses at the endometrial-placental interface regulate labyrinthine placental differentiation from trophoblast stem cells.

Development can happen in one of two ways. Cells performing a necessary function can differentiate from stem cells before the need for it arises and stress does not develop. Or need arises before function, stress develops and stress signals are part of the normal stimuli that regulate developmental mechanisms. These mechanisms adjust stem cell differentiation to produce function in a timely and proportional manner. In this review, we will interpret data from studies of null lethal mutants for placental stress genes that suggest the latter possibility. Acknowledged stress pathways participate in stress-induced and -regulated differentiation in two ways. These pathways manage the homeostatic response to maintain stem cells during the stress. Stress pathways also direct stem cell differentiation to increase the first essential lineage and suppress later lineages when stem cell accumulation is diminished. This stress-induced differentiation maintains the conceptus during stress. Pathogenic outcomes arise because population sizes of normal stem cells are first depleted by decreased accumulation. The fraction of stem cells is further decreased by differentiation that is induced to compensate for smaller stem cell populations. Analysis of placental lethal null mutant genes known to mediate stress responses suggests that the labyrinthine placenta develops during, and is regulated by, hypoxic stress.

Eomesodermin, HAND1, and CSH1 proteins are induced by cellular stress in a stress-activated protein kinase-dependent manner.

Eomesodermin (Eomes) is a transcription factor essential for trophoblast development. Stress stimuli activate stress-activated protein kinase (MAPK8/9) and modulate transcription factors in trophoblast stem cells (TSC). 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 TSC. 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 than 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 TSC to undergo stress-induced differentiation could lead to pathophysiological consequences if stress endured and TSC differentiation became unbalanced.

Oxygen levels that optimize TSC culture are identified by maximizing growth rates and minimizing stress.

Accumulating data suggest that 20% O(2) 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 O(2) level for TSC culture, and that pSAPK responds to contradictory signals. We tested the dose range of 0-20% O(2) (0, 0.5, 2, and 20%) on five effects in cultured TSC. The results showed 1) TSC accumulation rates were highest at 2% O(2), lower at 20% and lowest at 0-0.5%; 2) pSAPK protein levels were lowest at 2% O(2), higher at 20%, and highest at 0-0.5%; 3) Cleaved caspase 3, an apoptosis marker, increased at 0.5% O(2), and was highest at 0% O(2); 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% O(2) 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% O(2), but SAPK activity decreases when O(2) 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.

Interpreting the stress response of early mammalian embryos and their stem cells.

This review analyzes and interprets the normal, pathogenic, and pathophysiological roles of stress and stress enzymes in mammalian development. Emerging data suggest that stem cells from early embryos are induced by stress to perform stress-enzyme-mediated responses that use the strategies of compensatory, prioritized, and reversible differentiation. These strategies have been optimized during evolution and in turn have aspects of energy conservation during stress that optimize and maximize the efficacy of the stress response. It is likely that different types of stem cells have varying degrees of flexibility in mediating compensatory and prioritized differentiation. The significance of this analysis and interpretation is that it will serve as a foundation for yielding tools for diagnosing, understanding normal and pathophysiological mechanisms, and providing methods for managing stress enzymes to improve short- and long-term reproductive outcomes.

Cellular stress causes reversible, PRKAA1/2-, and proteasome-dependent ID2 protein loss in trophoblast stem 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.

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.

Transient stress and stress enzyme responses have practical impacts on parameters of embryo development, from IVF to directed differentiation of stem cells.

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.

Pipetting causes shear stress and elevation of phosphorylated stress-activated protein kinase/jun kinase in preimplantation embryos.

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.

Using hyperosmolar stress to measure biologic and stress-activated protein kinase responses in preimplantation embryos.

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.

Impact of transient stress and stress enzymes on development.

Stress enzymes triggered by transient stress mediate reprioritization of developmental and homeostatic events to flexibly accomplish the next essential developmental event. This review analyzes recent studies on stress and stress enzyme function during early mammalian development and describes the diverse consequences that result from measurement, analysis of function, and management of stress and stress enzymes during development.

Effects of SAPK/JNK inhibitors on preimplantation mouse embryo development are influenced greatly by the amount of stress induced by the media.

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.

Shear stress induces preimplantation embryo death that is delayed by the zona pellucida and associated with stress-activated protein kinase-mediated apoptosis.

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.

FGF ligand family mRNA expression profile for mouse preimplantation embryos, early gestation human placenta, and mouse trophoblast stem cells.

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.

A role for hepatocyte growth factor during early postimplantation growth of the placental lineage in mice.

Hepatocyte growth factor (HGF) is implicated in placental development; hgfr and hgf null mutant embryos develop placental insufficiency and lethality at 11.5 days (E11.5) after fertilization. The function of HGF in placentation at implantation (E4.5) has not been studied. Using reverse transcription-polymerase chain reaction, we detected HGF receptor (HGFR) mRNA in preimplantation embryos and in cultured blastocyst outgrowths. HGFR protein was detected in trophoblast cells in blastocyst outgrowths. HGF mRNA was not detected at these stages but was detected in the uterus at E5.5. Using in situ hybridization, we detected HGF mRNA in the mesometrial uterus, near the embryo, from E6.5 through E8.5. At E8.5, HGFR mRNA was detected in the chorionic placenta, and HGF mRNA was detected in the allantois. The expression for HGF and HGFR suggested a maternal-to-embryonic communication before the development of the allantois. To test this, blastocyst outgrowths were cultured with HGF. HGF stimulated the outgrowth of trophoblasts in a time-dependent manner and stimulated the expression of proliferating cell nuclear antigen, but it did not scatter trophoblasts. HGF stimulated an increase in the trophoblast cell number, but caused a decrease in the total number of terminally differentiated trophoblasts expressing placental lactogen-1 protein. These data suggest that HGF stimulates the cell division, but not the differentiation, of trophoblast cells during implantation.

Expression of fibroblast growth factor receptors in peri-implantation mouse embryos.

FGF receptor (FGFR) function is essential during peri-implantation mouse development. To understand which receptors are functioning, we tested for the expression of all four FGF receptors in peri-implantation blastocysts. By RT-PCR, FGFR-3 and FGFR-4 were detected at high levels, FGFR-2 at lower levels, and FGFR-1 was detected at background levels compared to control tissues. Because FGFR-3 and FGFR-4 were detected at the highest levels, we studied these in detail. Between 3.5 days after fertilization (E3.5) and E6.0, FGFR-4 mRNA was detected ubiquitously in the peri-implantation embryo, restricted to the inner cell mass (ICM) and its derivatives and primitive endoderm by E6.0, and was not detected at E6.5. FGFR-3 mRNA was detected ubiquitously in the peri-implantation embryo with a tendency towards extraembryonic cells. We tested blastocyst outgrowths, a model for implantation, for FGFR-3 and FGFR-4 protein. FGFR-3 protein was detected in all cells early during the outgrowth. Later, FGFR-3 was detected in the extraembryonic endoderm and trophoblast giant cells (TGC), but not in the ICM. FGFR-4 protein was detected in all cells of the implanting embryo, but was restricted to the ICM/primitive endoderm in later stage outgrowths. The distribution of the receptor proteins in the blastocyst outgrowths is similar to the distribution of the mRNA detected by in situ hybridization of sections of embryos. The data suggest roles for FGFR-3 and FGFR-4 in peri-implantation development.

FGF is an essential regulator of the fifth cell division in preimplantation mouse embryos.

Fibroblast growth factor (FGF) signaling is required prior to gastrulation in the mouse embryo. To test for the spatial and temporal requirements of FGF signaling, a dominant negative FGF receptor (dnFGFR) was used to make transgenic mouse embryos. In mosaic embryos, cell division ceased at the fifth cell division in all cells that expressed the mutant receptor, but cell death did not increase. After the fifth cell division, the progeny of unaltered cells and cells expressing lacZ continued to accumulate at the same rate, suggesting that the FGF requirement is cell autonomous. In mosaic embryos, lacZ, but not dnFGFR expression was detected in mitotic trophoblasts adjacent to the ICM. Conversely, dnFGFR-expressing extraembryonic ectoderm cells were detected at the abembryonic pole in postmitotic cells. In blastocysts expressing the dnFGFR in all cells, the morphology appeared normal and inner cell masses (ICMs) formed, but resultant embryos had only one-third the number of cells as control embryos. In these blastocysts, cell division had also ceased at the fifth cell division, but cavitation, a concurrent morphogenetic event, initiated and progressed normally. To test for the continuing requirement of FGF, FGFR-3 was overexpressed in all cells and resulted in an increase in cell numbers after the fifth cell cycle. In a model for postimplantation development, addition of FGF-4 to blastocyst outgrowths increased the number of extraembryonic ectoderm cells, suggesting a continuing role for FGF. Thus, FGF signaling induces the cell division of embryonic and extraembryonic cells in the preimplantation mouse embryo starting at the fifth cell division. The signal requirement for FGF is cell autonomous, but is not required to prevent cell death. This provides the first evidence for the necessity of a growth factor before implantation.