Jing Dai

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.

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

PurposeThe 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)u2009+u2009Aspirin (Asa) or BR-DIM causes an AMPK-dependent decrease in embryonic development.MethodsThe 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 Metu2009+u2009Asa (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 ~50–85xa0% Rex1 and/or Oct4 protein loss in two-cell embryos. This loss is ~60–90xa0% reversible by co-culture with AMPK inhibitor CC. Embryo development from two-cell to blastocyst stage is decreased in culture with either Metu2009+u2009Asa or BR-DIM, and this is either >90 or ~60xa0% reversible with CC, respectively.ConclusionThese 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., Metu2009+u2009Asa) that are used to enhance maternal metabolism to support fertility can also chronically slow embryo growth and block development in an AMPK-dependent manner.

Comparison of 2, 5, and 20 % O2 on the development of post-thaw human embryos.

PurposeThe objective of this study is to investigate the effect of 2, 5, and 20xa0% O2 on post-thaw day 3 human embryo culture until blastocyst stage.MethodsOne hundred fifty-five day 3 human embryos were used. One hundred twenty out of 155 embryos were recovered after thawing. Surviving embryos were distributed into 2, 5, or 20xa0% O2 groups and cultured for 2.5xa0days. At the end of culture, blastocyst formation was assessed, and then, embryos were collected for RT-qPCR or immunofluorescence analysis.ResultsUsing visible blastocoel to define blastocyst formation, 58.7xa0% (27/46) of surviving day 3 embryos formed blastocyst at 2xa0% O2, 63.6xa0% (28/44) at 5xa0% O2, and 66.7xa0% (20/30) at 20xa0% O2. The difference in blastocyst formation rates was not significant. Average blastocyst cell number was 119.44u2009±u200911.64 at 2xa0% O2, 142.55u2009±u200922.47 at 5xa0% O2, and 97.29u2009±u200914.87 at 20xa0% O2. Average apoptotic rate was 4.7xa0%u2009±u20090.4xa0% for blastocyst formed at 2xa0% O2, 3.5xa0%u2009±u20090.7xa0% at 5xa0% O2, and 5.8xa0%u2009±u20091.1xa0% at 20xa0% O2. Apoptosis rate was significantly lower for blastocysts formed at 5xa0% O2 (pu2009<u20090.05). Compared with gene expression levels at 5xa0% O2, which were arbitrarily set as “1,” 20xa0% O2 is associated with significantly higher expression of BAX (2.14u2009±u20090.47), G6PD (2.92u2009±u20091.06), MnSOD (2.87u2009±u20090.88), and HSP70.1 (8.68u2009±u20094.19). For all genes tested, no significant differences were found between 2 and 5xa0% O2.ConclusionThe result suggests that development of cryopreserved human embryos from day 3 to blastocyst stage benefits from culture at 5xa0% O2.

Development and Validation of a Rex1-RFP Potency Activity Reporter Assay That Quantifies Stress-Forced Potency Loss in Mouse Embryonic Stem Cells.

Assays for embryonic stem cells (ESCs) of the blastocyst are needed to quantify stress-induced decreases of potent subpopulations. High-throughput screens (HTSs) of stressed ESCs quantify embryonic stress, diminishing laboratory animal needs. Normal or stress-induced ESC differentiation is marked by Rex1 potency factor loss. Potency reporter ESC assays were developed, using low-stress techniques to create transgenic ESCs. Rex1 and Oct4 promoters drove RFP and green fluorescent protein (GFP) expression, respectively. Lentivirus infection and fluorescence-activated cell sorting selection of ESCs obviated the need for stressful electroporation and antibiotic selection, respectively. We showed using immunoblots, microscopic analysis, flow cytometry, and fluorescence microplate reader that the response to stress of potency-reporter ESCs is similar to parental ESCs assayed by biochemical means. Stress caused a dose-dependent decrease in bright Rex1-RFP(+) ESCs and increase in Rex1 dim ESCs. At highest stress, ∼ 20% of bright Rex1-RFP cells are lost coinciding with a 2.8-fold increase in Rex1-RFP dim cells that approach 20%. This conversion of bright to dim cells tested by flow cytometry is commensurate with about 60% loss in fluorescence measured by microplate reader. Dose-dependent stress-induced Rex1-RFP and endogenous Rex1 protein decreases are similar. The data show that Rex1 reporter ESCs accurately report stress in a microplate reader-based HTS. The increasing dim Rex1 subpopulation size is balanced by the decreasing total ESC number during culture at multiple sorbitol doses. This is consistent with previous observations that stress forces potency decrease and differentiation increase to compensate for stress-induced diminished stem cell population growth.

Sildenafil Prevents Apoptosis of Human First-Trimester Trophoblast Cells Exposed to Oxidative Stress: Possible Role for Nitric Oxide Activation of 3′,5′-cyclic Guanosine Monophosphate Signaling

Human first-trimester trophoblast cells proliferate at low O2, but survival is compromised by oxidative stress, leading to uteroplacental insufficiency. The vasoactive drug, sildenafil citrate (Viagra, Sigma, St Louis, Missouri), has proven useful in reducing adverse pregnancy outcomes. An important biological function of this pharmaceutical is its action as an inhibitor of cyclic guanosine monophosphate (cGMP) phosphodiesterase type 5 activity, which suggests that it could have beneficial effects on trophoblast survival. To investigate whether sildenafil can prevent trophoblast cell death, human first-trimester villous explants and the HTR-8/SVneo cytotrophoblast cell line were exposed to hypoxia and reoxygenation (H/R) to generate oxidative stress, which induces apoptosis. Apoptosis was optimally inhibited during H/R by 350 ng/mL sildenafil. Sildenafil-mediated survival was reversed by l-NG-nitro-l-arginine methyl ester hydrochloride or cGMP antagonist, indicating a dependence on both nitric oxide (NO) and cGMP. Indeed, either a cGMP agonist or an NO generator was cytoprotective independent of sildenafil. These findings suggest a novel intervention route for patients with recurrent pregnancy loss or obstetrical placental disorders.

Sildenafil stimulates human trophoblast invasion through nitric oxide and guanosine 3′,5′-cyclic monophosphate signaling

OBJECTIVEnTo determine the effect of sildenafil, a phosphodiesterase type 5 inhibitor, on trophoblast invasiveness.nnnDESIGNnLaboratory investigation.nnnSETTINGnAcademic medical center.nnnPATIENT(S)nPlacental tissues discarded after first-trimester terminations were obtained from patients with informed consent.nnnINTERVENTION(S)nA cell line, HTR-8/SVneo, established from first-trimester cytotrophoblast, and villous explants, was treated with or without sildenafil, guanosine 3,5-cyclic monophosphate (cGMP) analog, cGMP inhibitor, or L-NAME (N(G)-nitro-L-arginine methyl ester hydrochloride) and cultured on fibronectin or Matrigel. Integrins α6β4 and α1β1 were detected by immunocytochemistry.nnnMAIN OUTCOME MEASURE(S)nTrophoblast outgrowth from villous tips, cytotrophoblast cell invasion, and integrin immunostaining were assessed in cytotrophoblast and explant cultures.nnnRESULT(S)nIntegrin expression in trophoblast cells ex vivo switched from α6 to α1, and invasiveness increased, when exposed to sildenafil or cGMP agonist. Either cGMP antagonist or L-NAME blocked integrin switching and invasion induced by sildenafil. Elevation of nitric oxide pharmacologically induced invasion, but not when cGMP antagonist was present.nnnCONCLUSION(S)nSildenafil altered trophoblast phenotype through a process dependent on nitric oxide availability and cGMP accumulation. In addition to its vasoactivity, sildenafil directly stimulates trophoblast extravillous differentiation, which would be favorable for implantation and reduce risk for adverse pregnancy outcomes.

Hypoxic Stress Forces Irreversible Differentiation of a Majority of Mouse Trophoblast Stem Cells Despite FGF4

ABSTRACT Hypoxic, hyperosmotic, and genotoxic stress slow mouse trophoblast stem cell (mTSC) proliferation, decrease potency/stemness, and increase differentiation. Previous reports suggest a period of reversibility in stress-induced mTSC differentiation. Here we show that hypoxic stress at 0.5% O2 decreased potency factor protein by ∼60%–90% and reduced growth to nil. Hypoxia caused a 35-fold increase in apoptosis at Day 3 and a 2-fold increase at Day 6 above baseline. The baseline apoptosis rate was only 0.3%. Total protein was never less than baseline during hypoxic treatment, suggesting 0.5% O2 is a robust, nonmorbid stressor. Hypoxic stress induced ∼50% of trophoblast giant cell (TGC) differentiation with a simultaneous 5- to 6-fold increase in the TGC product antiluteolytic prolactin family 3, subfamily d, member 1 (PRL3D1), despite the presence of fibroblast growth factor 4 (FGF4). Hypoxia-induced TGC differentiation was also supported by potency and differentiation mRNA marker analysis. FGF4 removal at 20% O2 committed cell fate towards irreversible differentiation at 2 days, with similar TGC percentages after an additional 3 days of culture under potency conditions when FGF4 was readded or under differentiation conditions without FGF4. However, hypoxic stress required 4 days to irreversibly differentiate cells. Runted stem cell growth, forced differentiation of fewer cells, and irreversible differentiation limit total available stem cell population. Were mTSCs to respond to stress in a similar mode in vivo, miscarriage might occur as a result, which should be tested in the future.

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

PurposeWe 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.MethodsBovine oocytes were aspirated from ovaries and cultured in IVM media for 24xa0h with 0, 20, 40, or 60xa0μM 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.ResultsCoQ10 at 40xa0μM was optimal. Oocytes at 40xa0μM enabled 1.9-fold more ATP than 0xa0μM CoQ10. There was 4.3-fold less oocyte death, 1.7-fold more mitochondrial charge polarization, and 3.1-fold more mitochondrial mass at 40xa0μM than at 0xa0μM CoQ10. Increased ATP was associated with 2.2-fold lower AMPK thr172P activation and 2.1-fold higher nuclear Oct4 stemness/potency protein at 40xa0μM than at 0xa0μM CoQ10. CoQ10 is hydrophobic, and at all doses, 50% was lost from media into oil by ~xa012xa0h. Replenishing CoQ10 at 12xa0h did not significantly diminish dead oocytes.ConclusionsThe data suggest that CoQ10 improves mitochondrial function in IVM where unwanted stress, higher AMPK activity, and Oct4 potency loss are induced.

Enhancement of trophoblast differentiation and survival by low molecular weight heparin requires heparin-binding EGF-like growth factor

STUDY QUESTIONnDoes low molecular weight heparin (LMWH) require heparin-binding epidermal growth factor (EGF)-like growth factor (HBEGF) signaling to induce extravillous trophoblast differentiation and decrease apoptosis during oxidative stress?nnnSUMMARY ANSWERnLMWH increased HBEGF expression and secretion, and HBEGF signaling was required to stimulate trophoblast extravillous differentiation, increase invasion in vitro and reduce trophoblast apoptosis during oxidative stress.nnnWHAT IS KNOWN ALREADYnAbnormal trophoblast differentiation and survival contribute to placental insufficiency syndromes, including preeclampsia and intrauterine growth restriction. Preeclampsia often manifests as a pro-thrombotic state, with unsuccessful transformation of the spiral arteries that reduces oxygen supply and can produce placental infarction. LMWH improves placental function by increasing blood flow. Recent data suggest that the actions of LMWH transcend its anti-coagulative properties, but the molecular mechanism is unknown. There is evidence that LMWH alters the expression of human HBEGF in trophoblast cells, which regulates human trophoblast pathophysiology. HBEGF, itself, is capable of increasing trophoblast survival and invasiveness.nnnSTUDY DESIGN, SIZE, DURATIONnFirst-trimester placental explants and the HTR-8/SVneo cell line, established using extravillous trophoblast outgrowths from first-trimester villous explants, were treated in vitro with LMWH to examine the effects on HBEGF signaling and trophoblast function under normal physiological and pathological conditions. A highly specific antagonist of HBEGF and other inhibitors of HBEGF downstream signaling were used to determine the relationship between LMWH treatment and HBEGF.nnnPARTICIPANTS/MATERIALS, SETTING, METHODSnPlacental tissues (n = 5) were obtained with IRB approval and patient consent from first-trimester terminations. Placental explants and HTR-8/SVneo cells were cultured on plastic or Matrigel™ and treated with a therapeutic dose of LMWH (Enoxaparin; 10 IU/ml), with or without CRM197, pan Erb-B2 Receptor Tyrosine Kinase (ERBB) inhibitor, anti-ERBB1 or ERBB4 blocking antibodies, or pretreatment of cells with heparitinase I. Extravillous differentiation was assessed by immunocytochemistry to determine the relative levels of integrins α6β4 and α1β1. Trophoblast invasiveness was assessed in villous explants by measuring outgrowth from villous tips cultured on Matrigel, and by invasion assays with HTR-8/SVneo cells cultured on Matrigel-coated transwell insert. Placental explants and HTR-8/SVneo cells were exposed to oxidative stress in a hypoxia-reoxygenation (H-R) model, measuring cell death by TUNEL assay, caspase 3 cleavage, and BCL-2α expression.nnnMAIN RESULTS AND THE ROLE OF CHANCEnLMWH induced extravillous differentiation, according to trophoblast invasion assays and integrin (α6β4-α1β1) switching. Treatment with LMWH rescued cytotrophoblasts and HTR-8/SVneo cells from apoptosis during exposure to reoxygenation injury, based on TUNEL, caspase 3 cleavage and BCL-2α expression. Experiments using CRM197, ERBB1 and ERBB4 blocking antibodies, pan-ERBB inhibitor and removal of cell surface heparin demonstrated that the effects of LMWH on trophoblast invasion and survival were dependent upon HBEGF signaling.nnnLARGE SCALE DATAnN/A.nnnLIMITATIONS, REASONS FOR CAUTIONnThe primary limitation of this study was the use of only in vitro experiments. Patient demographics from elective terminations were not available.nnnWIDER IMPLICATIONS OF THE FINDINGSnThese data provide new insights into the non-coagulation-related aspects of perinatal LMWH treatment in the management of placental insufficiency disorders.nnnSTUDY FUNDING/COMPETING INTEREST(S)nThis research was supported by grants from the National Institutes of Health (HD071408 and HL128628), the March of Dimes, and the W. K. Kellogg Foundation. There were no conflicts or competing interests.

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

PurposeThis 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.MethodsCulture mouse zygotes to two-cell embryos and test effects after 0.5–1xa0h 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 ~xa020-fold, and BR-DIM caused a ~xa030-fold increase over two-cell embryos cultured for 1xa0h 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 ~xa060–90% 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.ConclusionInducing drug or diet supplements decreased anabolism, growth, and stemness have a greater impact on AMPK-dependent processes in two-cell embryos compared to blastocysts.