The physiological responses to titanium dioxide nanoparticles exposure in pearl oysters (Pinctada fucata martensii).

To evaluate the physiological responses to titanium dioxide nanoparticles exposure in pearl oysters (Pinctada fucata martensii), pearl oysters were exposed for 14 days to different levels (0.05, 0.5, and 5 mg/L) of nano-TiO2 suspensions, while a control group did not undergo any nano-TiO2 treatment. And then recovery experiments were performed for 7 days without nano-TiO2 exposure. At days 1, 3, 7, 14, 17, and 21, hepatopancreatic tissue samples were collected and used to examine the activities of protease, amylase, lipase, catalase (CAT), glutathione peroxidase (GPx), superoxide dismutase (SOD), lysozyme (LYS), alkaline phosphatase (AKP), and acid phosphatase (ACP). The microstructure of the nacreous layer in shell was also analyzed by scanning electron microscopy. Results showed that pearl oysters exposed to 5 mg/L of TiO2 nanoparticles had significantly lower protease, amylase, and lipase activities and significantly higher CAT, SOD, GPx, LYS, ACP, and AKP activities than control pearl oysters did even after 7-day recovery (P-values <0.05). Pearl oysters exposed to 0.5 mg/L or 0.05 mg/L of TiO2 nanoparticles had lower protease, amylase, and lipase activities and higher CAT, SOD, GPx, LYS, ACP, and AKP activities than control pearl oysters did during the exposure period. After 7-day recovery, no significant differences in protease, lipase, SOD, GPx, CAT, ACP, AKP, or LYS activities were observed between pearl oysters exposed to 0.05 mg/L of TiO2 nanoparticles and control pearl oysters (P-values >0.05). In the period from day 7 to day 14, indistinct and irregular nacreous layer crystal structure in shell was observed. This study demonstrates that TiO2 nanoparticles exposure influences the levels of digestion, immune function, oxidative stress, and biomineralization in pearl oysters, which can be partially and weakly alleviated by short-term recovery. These findings contribute to understanding the mechanisms of action of TiO2 nanoparticles in bivalves. However, studies should evaluate whether a longer recovery period can restore to their normal levels in the future.

The relationship between VEGF-460(T>C) polymorphism and cancer risk: A systematic review and meta-analysis based on 46 reports.

BACKGROUND: Extensive studies on the link between single nucleotide polymorphisms (SNPs) in vascular endothelial growth factor (VEGF) and various malignancy risks produced conflicting results, notably for VEGF-460(T/C). To evaluate this correlation more comprehensively and accurately, we perform a meta-analysis. METHODS: Through retrieving 5 databases (Web of Science (WoS), Embase, Pubmed, Wanfang database (Wangfang), and China National Knowledge Infrastructure (CNKI)) and applying hand search, citation search, and gray literature search, 44 papers included 46 reports were enrolled. To evaluate the relationship between VEGF-460 and cancer risk, we pooled odds ratios (ORs) and 95% confidence intervals (CIs). RESULTS: Our results indicated that the VEGF-460 polymorphism is not related to malignancy susceptibility (dominant model, OR = 0.98, 95% CI = 0.87-1.09; recessive model, OR = 0.95, 95% CI = 0.82-1.10; heterozygous model, OR = 0.99, 95% CI = 0.90-1.10; homozygous model, OR = 0.92, 95% CI = 0.76-1.10; additive model, OR = 0.98, 95% CI = 0.90-1.07). While, in subgroup analysis, this SNP may reduce the risk of hepatocellular carcinoma. CONCLUSION: this meta-analysis indicated that VEGF-460 was irrelevant to overall malignancy risk, but it might be a protective factor for hepatocellular carcinoma.

Microbial community structure and diversity in fish-flower (mint) symbiosis.

The fish-flower symbiosis model is an eco-friendly sustainable farming technology combining plants, fish and microorganisms in a recirculating aquaculture system. However, there are few studies on the structure and diversity of microbial communities in fish intestines, culture water and plant roots during fish-flower symbiosis. Here, we cultured carp (Cyprinus carpio), crucian carp (Carassius auratus) and grass carp (Ctenopharyngodon idella) with mint (Mentha spicala L.) and extracted total genomic DNA from intestinal microorganisms, culture-water microorganisms and root microorganisms for each fish species for high-throughput sequencing of 16S rRNA genes. Analysis of microbial community structure and diversity revealed changes in abundance of microbial genera in the intestines and culture water of each fish species, including changes in the dominant taxa. Pirellula, Truepera, Aquincola, Cetobacterium and Luteolibacter were widespread in the fish intestine, culture water and mint root system. This study revealed the effects of mint feeding on the structure and diversity of microbial communities of fish, water bodies and the mint root system during fish-flower symbiosis, providing a theoretical reference for the promotion and application of fish-flower (mint) symbiosis technology and healthy fish culture technology.

The Correlation of Mouse Double Minute 4 (MDM4) Polymorphisms (rs4245739, rs1563828, rs11801299, rs10900598, and rs1380576) with Cancer Susceptibility: A Meta-Analysis.

BACKGROUND Mouse double minute 4 (MDM4) has been extensively investigated as a negative regulator of P53, its negative feedback loop, and the effect of its genetic polymorphisms on cancers. However, many studies showed varying and even conflicting results. Therefore, we employed meta-analysis to further assess the intensity of the connection between MDM4 polymorphisms and malignancies. MATERIAL AND METHODS We searched eligible articles in 5 databases (Cochrane Library, PubMed, Web of Science, Wan Fang Database, and China National Knowledge Infrastructure) up to August 2021. Odds ratios (ORs) and 95% confidence intervals (CIs) were utilized to probe the correlation of 5 MDM4 polymorphisms (rs4245739, rs1563828, rs11801299, rs10900598, and rs1380576) with carcinomas. We employed meta-regression and subgroup analysis to probe for sources of heterogeneity; Funnel plots, Begg's test, and Egger's test were used to evaluate publication bias. Sensitivity analysis was applied to assess the stability of the study. RESULTS Twenty-two studies, comprising 77 reports with 29 853 cases and 72 045 controls, were included in our meta-analysis. We found that rs4245739 polymorphism was a factor in reducing overall cancer susceptibility (dominant model, OR=0.85, 95% CI=0.76-0.95; heterozygous model, OR=0.86, 95% CI=0.78-0.96; additive model, OR=0.87, 95% CI=0.79-0.95), especially in Asian populations, and it also reduces the risk for esophageal squamous cell carcinoma (ESCC). The remaining 4 SNPs were not associated with cancers. CONCLUSIONS The rs4245739 polymorphism might reduce the risk of malignancies, especially in Asian populations, and it is a risk-reducing factor for ESCC incidence. However, rs1563828, rs11801299, rs10900598, and rs1380576 are not relevant to cancer susceptibility.

Preparation, bioactivity and mechanism of nano-hydroxyapatite/sodium alginate/chitosan bone repair material.

BACKGROUND: As the major inorganic component of natural bone, nano-hydroxyapatite (n-HA) on its own is limited in its use in bone repair, due to its brittleness. Chitosan (CS) and sodium alginate (SAL) are used to reduce its brittleness and tendency to degradation. However, the compressive strength of the composite is still low, and its biological performance needs further study. METHODS: Nano-hydroxyapatite/sodium alginate/chitosan (n-HA/SAL/CS) composite was prepared via an in situ synthesis method. Further, we prepared the n-HA/SAL/CS self-setting bone repair material by mixing n-HA/SAL/CS powder with a curing liquid (20 wt.% citric acid). In addition, the in vitro bioactivity and cell cytotoxicity were also explored. RESULTS: Transmission electron microscopy photos revealed that the n-HA crystals were uniformly distributed throughout the polymer matrix. Infrared IR spectroscopy indicated that the HA interacted with the COO- of SAL and NH2- of CS. The compressive strength of the n-HA/SAL/CS bone cement was 34.3 MPa and matched the demands of weight-bearing bones. Soaking in vitro in simulated body fluid demonstrated that the composite material had reasonably good bioactivity, while cytotoxicity tests indicated that the n-HA/SAL/CS cement could promote cell proliferation and was biocompatible. CONCLUSIONS: Compressive strength of n-HA/SAL/CS can satisfy the needs of cancellous bone, and in vitro bioactivity and cytotoxicity tests results indicated that the n-HA/SAL/CS composite could act as an optimal bone repair material.

Two-cell embryos are more sensitive than blastocysts to AMPK-dependent suppression of anabolism and stemness by commonly used fertility drugs, a diet supplement, and stress.

PURPOSE: This study tests whether metformin or diet supplement BR-DIM-induced AMP-activated protein kinase (AMPK) mediated effects on development are more pronounced in blastocysts or 2-cell mouse embryos. METHODS: Culture mouse zygotes to two-cell embryos and test effects after 0.5-1 h AMPK agonists' (e.g., Met, BR-DIM) exposure on AMPK-dependent ACCser79P phosphorylation and/or Oct4 by immunofluorescence. Culture morulae to blastocysts and test for increased ACCser79P, decreased Oct4 and for AMPK dependence by coculture with AMPK inhibitor compound C (CC). Test whether Met or BR-DIM decrease growth rates of morulae cultured to blastocyst by counting cells. RESULT(S): Aspirin, metformin, and hyperosmotic sorbitol increased pACC ser79P ~ 20-fold, and BR-DIM caused a ~ 30-fold increase over two-cell embryos cultured for 1 h in KSOMaa but only 3- to 6-fold increase in blastocysts. We previously showed that these stimuli decreased Oct4 40-85% in two-cell embryos that was ~ 60-90% reversible by coculture with AMPK inhibitor CC. However, Oct4 decreased only 30-50% in blastocysts, although reversibility of loss by CC was similar at both embryo stages. Met and BR-DIM previously caused a near-complete cell proliferation arrest in two-cell embryos and here Met caused lower CC-reversible growth decrease and AMPK-independent BR-DIM-induced blastocyst growth decrease. CONCLUSION: Inducing drug or diet supplements decreased anabolism, growth, and stemness have a greater impact on AMPK-dependent processes in two-cell embryos compared to blastocysts.

Using stem cell oxygen physiology to optimize blastocyst culture while minimizing hypoxic stress.

This review is a response to the Fellows Forum on testing 2% oxygen for best culture of human blastocysts (J Ass Reprod Gen 34:303-8, 1; J Ass Reprod Gen 34:309-14, 2) prior to embryo transfer. It is a general analysis in support of the position that an understanding of stem cell physiology and responses to oxygen are necessary for optimization of blastocyst culture in IVF and to enhance reproductive success in fertile women.

Synthesis and characterization of nano-hydroxyapatite/polyamide 66 biocomposites reinforced with multi-walled carbon nanotubes.

In this work, we investigate the enhanced mechanical properties of nano-hydroxyapatite/polyamide 66 (nHA/PA66) composites reinforced with multi-walled carbon nanotubes (MWCNTs) by means of the blending method. The MWCNTs-nHA/PA66 composites were characterized by various techniques, and the obtained results indicated that the MWCNTs were evenly distributed in the composite and that good interfacial bonding was formed between MWCNTs and PA66. The addition of MWCNTs improved the crystallinity of PA66, while it had little or no effect either on the composition or on the crystal structure of the composites. Moreover, the addition of MWCNTs in nHA/PA66 significantly improved the mechanical strength, and the tensile and compressive strengths attained maximum values of 90.3 and 126.8 MPa, respectively, with the addition of 0.1 wt% MWCNTs, whereas the bending strength attained a maximum value of 105.5 MPa with the addition of 0.05 wt% MWCNTs. Finally, L929 cells co-cultured with the MWCNTs-nHA/PA66 composite exhibited comparatively uninhibited cell growth, indicating that the addition of MWCNTs had negligible effect on the cytocompatibility of the original nHA/PA66 composite.

Commonly used fertility drugs, a diet supplement, and stress force AMPK-dependent block of stemness and development in cultured mammalian embryos.

PURPOSE: The purpose of the present study is to test whether metformin, aspirin, or diet supplement (DS) BioResponse-3,3'-Diindolylmethane (BR-DIM) can induce AMP-activated protein kinase (AMPK)-dependent potency loss in cultured embryos and whether metformin (Met) + Aspirin (Asa) or BR-DIM causes an AMPK-dependent decrease in embryonic development. METHODS: The methods used were as follows: culture post-thaw mouse zygotes to the two-cell embryo stage and test effects after 1-h AMPK agonists' (e.g., Met, Asa, BR-DIM, control hyperosmotic stress) exposure on AMPK-dependent loss of Oct4 and/or Rex1 nuclear potency factors, confirm AMPK dependence by reversing potency loss in two-cell-stage embryos with AMPK inhibitor compound C (CC), test whether Met + Asa (i.e., co-added) or DS BR-DIM decreases development of two-cell to blastocyst stage in an AMPK-dependent (CC-sensitive) manner, and evaluate the level of Rex1 and Oct4 nuclear fluorescence in two-cell-stage embryos and rate of two-cell-stage embryo development to blastocysts. RESULT(S): Met, Asa, BR-DIM, or hyperosmotic sorbitol stress induces rapid ~50-85 % Rex1 and/or Oct4 protein loss in two-cell embryos. This loss is ~60-90 % reversible by co-culture with AMPK inhibitor CC. Embryo development from two-cell to blastocyst stage is decreased in culture with either Met + Asa or BR-DIM, and this is either >90 or ~60 % reversible with CC, respectively. CONCLUSION: These experimental designs here showed that Met-, Asa-, BR-DIM-, or sorbitol stress-induced rapid potency loss in two-cell embryos is AMPK dependent as suggested by inhibition of Rex1 and/or Oct4 protein loss with an AMPK inhibitor. The DS BR-DIM or fertility drugs (e.g., Met + Asa) that are used to enhance maternal metabolism to support fertility can also chronically slow embryo growth and block development in an AMPK-dependent manner.

The Extent of Axillary Surgery Is Associated With Breast Cancer-specific Survival in T1-2 Breast Cancer Patients With 1 or 2 Positive Lymph Nodes: A SEER-Population Study.

This study aimed to compare the breast cancer-specific survival (BCSS) of a nonclinical trial population of T1-2 breast cancer patients with 1 to 2 positive lymph nodes who received breast-conserving surgery and either sentinel lymph node biopsy (SLNB) or axillary lymph node dissection (ALND).We used the Surveillance, Epidemiology and End Results (SEER) database to identify 17,028 patients with a median follow-up of 7.1 years. We assigned the patients into a SLNB-cohort (≤5 nodes) and an ALND-cohort (>5 nodes) based on the number of removed lymph nodes. We used Kaplan-Meier analysis to estimate the cumulative BCSS and used Cox-regression analysis to study the risk factors. We also performed subgroup analysis by the patients' age and hormonal receptor (HR) status.The cumulative BCSS and Overall Survival (OS) of the entire population were 94.4% and 91.4% at 5 years and 88.2% and 79.9% at 10 years, respectively. Axillary surgery (ALND vs SLNB) had no association with BCSS when adjusted for stage, HR status, tumor grade, or other factors. In subgroup analysis by age and HR status, ALND was associated with a significantly improved BCSS relative to SNLB (HR = 0.70, HR = 0.026, 95% confidence interval 0.51-0.96) only in patients younger than 50 years with HR- disease (N = 1281), but not in other subgroup of patients.In early-stage breast cancer patients with limited lymph node metastasis, ALND had better BCSS than SLNB only in patients younger than 50 years and with HR- disease. More studies are needed to confirm our findings.

Preparation, characterization and properties of nano-hydroxyapatite/polypropylene carbonate biocomposite.

The combination of nano-hydroxyapatite (n-HA) and polypropylene carbonate (PPC) was used to make a composite materials by a coprecipitation method. The physical and chemical properties of the composite were tested. Scanning electron microscope (SEM) observation indicated that the biomimetic n-HA crystals were uniformly distributed in the polymer matrix. As the n-HA content increased in the composite, the fracture mechanism of the composites changes from gliding fracture to gliding and brittle fracture. Furthermore, the chemical interaction between inorganic n-HA and polypropylene carbonate was also investigated and discussed in detail. The hydrogen bonds might be formed between -OH/CO3(2-) of n-HA crystal and the ester group (-COO-) of PPC. The tensile strength of n-HA/PPC (40/60) was similar to that of the cancellous bone, and reached ca 58 MPa. The osteoblasts were cultured for up to 7 days, and then the adhesion and proliferation of osteoblasts were measured by Methyl thiazolyl tetrazolium (MTT) colorimetry assay and SEM. The cells proliferated, grew normally in fusiform shape and well attached. The in vitro test confirmed that the n-HA/PPC composites were biocompatible and showed undetectable negative effect on osteoblasts. In vivo implantation of the composite in New Zealand white rabbits was performed. It can stimulate the growth of a new bone, and at the same time the material begins to degrade. These results suggested that the composite may be suitable for the reparation or replacement of bone defects and possessed the potential for clinical applications.

Hypoxic stress induces, but cannot sustain trophoblast stem cell differentiation to labyrinthine placenta due to mitochondrial insufficiency.

Dysfunctional stem cell differentiation into placental lineages is associated with gestational diseases. Of the differentiated lineages available to trophoblast stem cells (TSC), elevated O2 and mitochondrial function are necessary to placental lineages at the maternal-placental surface and important in the etiology of preeclampsia. TSC lineage imbalance leads to embryonic failure during uterine implantation. Stress at implantation exacerbates stem cell depletion by decreasing proliferation and increasing differentiation. In an implantation site O2 is normally ~2%. In culture, exposure to 2% O2 and fibroblast growth factor 4 (FGF4) enabled the highest mouse TSC multipotency and proliferation. In contrast, hypoxic stress (0.5% O2) initiated the most TSC differentiation after 24h despite exposure to FGF4. However, hypoxic stress supported differentiation poorly after 4-7 days, despite FGF4 removal. At all tested O2 levels, FGF4 maintained Warburg metabolism; mitochondrial inactivity and aerobic glycolysis. However, hypoxic stress suppressed mitochondrial membrane potential and maintained low mitochondrial cytochrome c oxidase (oxidative phosphorylation/OxPhos), and high pyruvate kinase M2 (glycolysis) despite FGF4 removal. Inhibiting OxPhos inhibited optimum differentiation at 20% O2. Moreover, adding differentiation-inducing hyperosmolar stress failed to induce differentiation during hypoxia. Thus, differentiation depended on OxPhos at 20% O2; hypoxic and hyperosmolar stresses did not induce differentiation at 0.5% O2. Hypoxia-limited differentiation and mitochondrial inhibition and activation suggest that differentiation into two lineages of the labyrinthine placenta requires O2>0.5-2% and mitochondrial function. Stress-activated protein kinase increases an early lineage and suppresses later lineages in proportion to the deviation from optimal O2 for multipotency, thus it is the first enzyme reported to prioritize differentiation.

Stress induces AMPK-dependent loss of potency factors Id2 and Cdx2 in early embryos and stem cells [corrected].

The AMP-activated protein kinase (AMPK) mediates rapid, stress-induced loss of the inhibitor of differentiation (Id)2 in blastocysts and trophoblast stem cells (TSC), and a lasting differentiation in TSC. However, it is not known if AMPK regulates other potency factors or regulates them before the blastocyst stage. The caudal-related homeodomain protein (Cdx)2 is a regulatory gene for determining TSC, the earliest placental lineage in the preimplantation mouse embryo, but is expressed in the oocyte and in early cleavage stage embryos before TSC arise. We assayed the expression of putative potency-maintaining phosphorylated Cdx2 ser60 in the oocyte, two-cell stage embryo, blastocyst, and in TSC. We studied the loss of Cdx2 phospho ser60 expression induced by hyperosmolar stress and its underlying mechanisms. Hyperosmolar stress caused rapid loss of nuclear Cdx2 phospho ser60 and Id2 in the two-cell stage embryo by 0.5 h. Stress-induced Cdx2 phospho ser60 and Id2 loss is reversed by the AMPK inhibitor compound C and is induced by the AMPK agonist 5-amino-1-ß-d-ribofuranosyl-imidazole-4-carboxamide in the absence of stress. In the two-cell stage embryo and TSC hyperosmolar, stress caused AMPK-mediated loss of Cdx2 phospho ser60 as detected by immunofluorescence and immunoblot. We propose that AMPK may be the master regulatory enzyme for mediating stress-induced loss of potency as AMPK is also required for stress-induced loss of Id2 in blastocysts and TSC. Since AMPK mediates potency loss in embryos and stem cells it will be important to measure, test mechanisms for, and manage the AMPK function to optimize the stem cell and embryo quality in vitro and in vivo.

Toxic stress prioritizes and imbalances stem cell differentiation: implications for new biomarkers and in vitro toxicology tests.

This hypothesis and review introduces rules of stem cell stress responses that provide biomarkers and alternative testing that replaces or reduces gestational tests using whole animals. These rules for the stress responses of cultured stem cells validate the organismal strategy of the stress response and show that it emulates what must happen if the conceptus implants during a response to stress in vivo. Specifically there is a profound threshold during a stress dose response where stem cell accumulation is significantly reduced. Below this threshold stress enzymes manage the stress response by converting anabolic to catabolic processes and by suppressing apoptosis, without affecting differentiation. However above this threshold the stem cell survival response converts to an organismal survival response where stress enzymes switch to new substrates and mediate loss of potency factors, gain of early essential differentiated lineages, and suppression of later essential lineages. Stressed stem cells 'compensate' for lower accumulation rates by differentiating a higher fraction of cells, and the organismal survival response further enhances adaptation by prioritizing the differentiation of early essential lineages. Thus compensatory and prioritized differentiation and the sets of markers produced are part of a response of cultured embryos and stem cells that emulate what must happen during implantation of a stressed gestation. Knowledge of these markers and use of stressed stem cell assays in culture should replace or reduce the number of animals needed for developmental toxicity and should produce biomarkers for stressed development in vitro and in vivo.

Adaptive and Pathogenic Responses to Stress by Stem Cells during Development.

Cellular stress is the basis of a dose-dependent continuum of responses leading to adaptive health or pathogenesis. For all cells, stress leads to reduction in macromolecular synthesis by shared pathways and tissue and stress-specific homeostatic mechanisms. For stem cells during embryonic, fetal, and placental development, higher exposures of stress lead to decreased anabolism, macromolecular synthesis and cell proliferation. Coupled with diminished stem cell proliferation is a stress-induced differentiation which generates minimal necessary function by producing more differentiated product/cell. This compensatory differentiation is accompanied by a second strategy to insure organismal survival as multipotent and pluripotent stem cells differentiate into the lineages in their repertoire. During stressed differentiation, the first lineage in the repertoire is increased and later lineages are suppressed, thus prioritized differentiation occurs. Compensatory and prioritized differentiation is regulated by at least two types of stress enzymes. AMP-activated protein kinase (AMPK) which mediates loss of nuclear potency factors and stress-activated protein kinase (SAPK) that does not. SAPK mediates an increase in the first essential lineage and decreases in later lineages in placental stem cells. The clinical significance of compensatory and prioritized differentiation is that stem cell pools are depleted and imbalanced differentiation leads to gestational diseases and long term postnatal pathologies.

The phosphoinositide kinase PIKfyve is vital in early embryonic development: preimplantation lethality of PIKfyve-/- embryos but normality of PIKfyve+/- mice.

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.

Benzopyrene and experimental stressors cause compensatory differentiation in placental trophoblast stem cells.

Stress causes decreased cell accumulation in early periimplantation embryos and the placental trophoblast stem cells derived from them. Benzopyrene and many other stressors activate stress enzymes that lead to suppressed stem cell accumulation through diminished proliferation and increased apoptosis. Trophoblast stem cells proliferate and a subpopulation of early postimplantation trophoblast cells differentiate to produce the first placental hormones that arise in the implanting conceptus. These hormones mediate antiluteolytic effects that enable the continuation of a successful implantation. The normal determination and differentiation of placental trophoblast stem cells is dependent upon a series of transcription factors. But, these transcription factors can also be modulated by stress through the activity of stress enzymes. This review enumerates and analyzes recent reports on the effects of benzopyrene on placental function in terms of the emerging paradigm that placental differentiation from stem cells can be regulated when insufficient production of stem cells is caused by stress. In addition, we review the other effects caused by benzopyrene throughout placental development.

Benzo(a)pyrene causes PRKAA1/2-dependent ID2 loss in trophoblast stem cells.

Benzo(a)pyrene (BaP), a cigarette smoke component, is metabolized to diol esters (BPDE) that bind to DNA and form mutagenic BPDE-DNA adducts. BaP activates stress enzymes including stress-activated protein kinase/jun kinase (MAPK8/9) in embryos, AMP-activated protein kinase alpha1/2 subunits (PRKAA1/2) in somatic cells, and inhibits the proliferation of trophoblast cell lineages. The loss of transcription factor inhibitor of differentiation (ID)2 is required for the initial differentiation of mouse trophoblast stem cells (TSC) in implanting mouse embryo to produce the first placental hormone, chorionic sommatomammotropin (CSH)1. Here we demonstrate that BaP activates PRKAA1/2 and causes ID2 protein loss in TSC in a time- and dose-dependent manner. Although PRKAA1/2 was activated at low BaP doses, PRKAA1/2-dependent ID2 protein loss occurred at a dose that was similar to the threshold that results in a significant decrease in TSC accumulation and decreased fraction of proliferating TSC. This suggests a possible relationship between stress-induced declines in cell accumulation and stem cell differentiation when BaP levels are high. The threshold BaP dose that induces significant ID2 loss is in the range of a 2-3 pack/day habit, suggesting that this mechanism may be involved with implantation failure in smoking women.

A major effect of simulated microgravity on several stages of preimplantation mouse development is lethality associated with elevated phosphorylated SAPK/JNK.

We tested whether microgravity affects mouse development during a period when gravity cues chick and frog embryo development. A rotating vessel developed approximately 0.1% simulated microgravity (MGS) for embryos. Microgravity simulation resulted in blocked cell accumulation in E2.5 embryos. E1.5 and E3.5 embryos showed lesser effects. For E1.5/2.5 embryos, cell accumulation block was followed by lethality at 48 hours after MGS. For E3.5 embryos, MGS blocked development without lethality but with apoptosis. E1.5-3.5 embryos from the rotational control developed lesser effects than MGS embryos. Embryonic stress-activated protein kinase (SAPK) was phosphorylated during MGS and mediated apoptosis. Increased pSAPK suggested that lethality is due to cellular stress induced by MGS, unlike the dysfunctional development after gravitational disorientation in frog and chick embryos. Thus, MGS causes lethality, a novel phenotype not often observed in microgravity or MGS. Embryonic lethality at E2.5 and apoptosis at E3.5 are associated with SAPK function, suggesting that MGS causes a general stress response that immediately affects many aspects of development. In addition, MGS and many aspects of In vitro fertilization/assisted reproductive technologies (IVF/ART) produce nonphysiological, nonevolutionary stresses that are mediated by SAPK, suggesting the primacy of this protein kinase in a wide range of mechanisms mediating negative reproductive outcomes in IVF/ART and potentially in spaceflight.