pTr Cells Research Articles

Establishment and application of high throughput screening cell model for nutrient regulation of embryonic development

Embryo development exerts far-reaching influence on pregnancy outcome, postnatal development and lifelong health. Thereafter, to select functional nutrients to improve embryo development is of great importance. Herein, a stable porcine trophectoderm cell line expressing a luciferase reporter gene driven by a 1,009 bp PCNA gene promoter was constructed through lentiviral transduction and G418 selection. A high throughput screening assay was subsequently developed using the stable reporter cell line to screen a library of 225 nutrients. Seven nutrients with a minimum Z-score of 2.0 were initially identified to be capable of enhancing embryonic development. Among these nutrients, resveratrol, apigenin, and retinol palmitate were furtherly confirmed the beneficial effects for embryo development. Resveratrol significantly increased the expression of key genes involved in pTr cell proliferation and the number of S-phase cells. Resveratrol was furtherly confirmed to promote the expression of key genes in trophoblast development and increase embryo adhesion rate in vitro. Similarly, dietary 0.05% resveratrol supplementation significantly increased the number of embryo attachment and serum level of P4 and E2 in rats. Resveratrol could also improve maternal antioxidant levels and reduce intracellular ROS. Collectively, a high throughput screening cell model for nutrient regulation of embryonic development was established, which can be used to highly effectively select the potential candidates for embryo development. These findings have great implications for exploring optimal functional nutrients to improve embryo development, ultimately beneficial for pregnancy outcome, offspring postnatal development and lifelong health for human beings and mammalian animals.

Chlorpyrifos induces placental oxidative stress and barrier dysfunction by inducing mitochondrial apoptosis through the ERK/MAPK signaling pathway: In vitro and in vivo studies

Chlorpyrifos (CPF) is an organophosphorus pesticide that is widely used in agricultural production and residential environments worldwide. In this study, we determined the harmful effects and toxicological mechanism of CPF in porcine trophectoderm (pTr) cells and the placenta of female mice during pregnancy. The findings revealed that CPF significantly decreased cell viability and increased intracellular lactate dehydrogenase (LDH) release in pTr cells. Similarly, CPF induced reproductive toxicity in pregnant maternal mice, including decreased maternal, fetal, and placental weights. Moreover, following CPF treatment, pTr cells and the placenta of female mice showed significant apoptosis. JC-1 staining and flow cytometry analysis also revealed that the mitochondrial membrane potential (MMP) of pTr cells treated with CPF was significantly depolarized. Additionally, CPF can induce an increase in reactive oxygen species (ROS) and barrier dysfunction in pTr cells and the placenta of female mice. We further verified that CPF-induced mitochondrial apoptosis is mediated by the MAPK signaling pathway, as shown by using of small molecular inhibitors of related proteins. Also, CPF-induced oxidative stress, barrier dysfunction, and mitochondrial apoptosis in pTr cells were alleviated by U0126, an inhibitor of the ERK/MAPK signaling pathway. These findings suggested that exposure to CPF in early pregnancy might be a potential risk fator affecting placental formation and function in humans and animals.

Bensulide exposure causes cell division cycle arrest and apoptosis in porcine trophectoderm and uterine luminal epithelial cells

As the use of herbicides in agriculture has increased worldwide, the importance of identifying unexpected toxic effects on non-target organisms is emerging. Bensulide is used on various agricultural crops as an organophosphate herbicide; however, it can pose a high risk to non-target organisms because of its long half-life and accumulative potential. Despite its high risk, the hazardous effects of bensulide on implantation and mechanisms in cells have not been reported. Therefore, in this study, intracellular mechanisms and potential risk of implantation failure were identified in porcine trophectoderm (pTr) and uterine luminal epithelial (pLE) cells derived from pigs with human-like molecular mechanisms in implantation. The LC50 values of bensulide were 5.21 mg/L in pTr cells and 6.49 mg/L in pLE cells. Both cell lines were exposed to bensulide at concentrations <5 mg/L in subsequent experiments. Treatment with 5 mg/L bensulide activated ERK1/2 and JNK. Disrupted mitochondrial membrane potentials of both cell types were identified. In addition, mitochondrial Ca2+ concentration increased to 261.24% and 228.04% in pTr and pLE cells, respectively, and cytoplasmic Ca2+ concentrations decreased by approximately 50% in both cell types. The abnormal regulation of various intracellular environments by bensulide causes cell division cycle arrest and apoptosis. Finally, 5 mg/L bensulide inhibited transcription of implantation-related genes. Collectively, our results suggest that bensulide may interrupt implantation during early pregnancy by disrupting maternal-fetal interaction.

Oryzalin impairs maternal-fetal interaction during early pregnancy via ROS-mediated P38 MAPK/AKT and OXPHOS downregulation.

Oryzalin is a dinitroaniline pesticide for the control of weed growth via suppression of microtubule synthesis. There are studies about the deleterious effects of dinitroaniline pesticides on the reproductive system. Therefore, we attempted to demonstrate the toxic mechanisms of oryzalin on early pregnancy using porcine uterine epithelial cells (pLE) and trophectoderm (pTr) cells. According to our results, the viability and proliferation of pLE and pTr cells were suppressed in response to oryzalin exposure, and cell cycle progression was affected. Additionally, oryzalin induced apoptotic cell death and impaired mitochondrial membrane polarity in pLE and pTr cells. Moreover, we confirmed that oryzalin significantly downregulated adenosine triphosphate (ATP) production via the oxidative phosphorylation system and upregulated reactive oxygen species (ROS) generation in both pLE and pTr cells. The oryzalin-induced ROS generation was mitigated by N-acetylcysteine, a ROS scavenger, and further upregulation of phosphor-P38 MAPK/AKT/P70S6K protein expression was ameliorated in both pLE and pTr cells. We also confirmed that the suppression of migration and proliferation in oryzalin-treated pLE and pTr cells was restored upon oxidative stress mitigation. In summary, we revealed that the cytotoxic mechanisms of oryzalin-induced implantation failure were mediated by ROS-induced intracellular signaling regulation and migratory potential in pLE and pTr cells.

Zearalenone induces apoptosis and autophagy by regulating endoplasmic reticulum stress signalling in porcine trophectoderm cells.

Zearalenone (ZEA), a mycotoxin produced mainly by fungi belonging to Fusarium species in foods and feeds, causes a serious hazard to humans and animals. Numerous studies have revealed that ingesting ZEA can disrupt the reproductive function and impair the reproductive process in animals. This experiment was to investigate the toxicological effect and the mechanism of ZEA exposure on reproduction in pigs during early stages of pregnancy. In the present study, we treated with 0 to 80μmol/L ZEA for 12 or 24h in trophoblast ectoderm (pTr) cells. The results showed that ZEA had significantly decreased cell proliferation (P<0.05), which was accompanied by DNA damage-related cell cycle arrest at G2/M phase, activation of the apoptosis and endoplasmic reticulum (ER) stress, as well as impairment of barrier function (P<0.05). Western blot analysis and transmission electron microscopy (TEM) showed that exposure to ZEA can activation of autophagy in pTr cells. Importantly, pretreatment with chloroquine (CQ) or 3-methyladenine (3-MA) led to increased apoptosis in pTr cells. Interestingly, pTr cells pretreated with 4-phenylbutyric acid (4-PBA), an inhibitor of ER stress, resulted in reduced cell death in pTr cells, indicating a critical role for ER stress in the activation of autophagy. In conclusion, these results reveal that ZEA-triggered ER stress is critical for the cell fate decision of pTr cells during early porcine embryonic development. Application of small molecules with ability of blocking ER stress might be therapeutic option to reduce the deleterious effect of ZEA in pregnant animals.

DDIT3 regulates key enzymes in the methionine cycle and flux during embryonic development

One-carbon metabolism is a key metabolic network that integrates nutritional signals with embryonic development. However, the response of one-carbon metabolism to methionine status and the regulatory mechanisms are poorly understood. Herein, we found that methionine supplementation during pregnancy significantly increased fetal number and average fetal weight. In addition, methionine modulated one-carbon metabolism primarily through 2 metabolic enzymes, cystathionine β-synthase (CBS) and methionine adenosyltransferase 2A (MAT2A), which were significantly increased in fetal liver tissues and porcine trophoblast (pTr) cells in response to proper methionine supplementation. CBS and MAT2A overexpression enhanced the DNA synthesis in pTr cells. More importantly, we identified a transcription factor, DNA damage-inducible transcript 3 (DDIT3), that was the primary regulator of CBS and MAT2A, which bound directly to promoters and negatively regulated the expression of CBS and MAT2A. Taken together, our findings identified that DDIT3 targeting CBS and MAT2A was a novel regulatory pathway that mediated cellular one-carbon metabolism in response to methionine signal and provided promising targets to improve pregnancy health.

Progesterone Induces Apoptosis and Steroidogenesis in Porcine Placental Trophoblasts.

Simple SummaryThis study investigated the effects of progesterone treatment in vitro on apoptosis and steroidogenesis in porcine placental trophoblasts and the underlying molecular mechanisms. Trophoblasts were treated with different concentrations of progesterone for 48 h. Cell counts, steroidogenesis, and relevant gene and protein expression levels were measured. Progesterone inhibited trophoblast proliferation in a dose-dependent manner. High doses of progesterone significantly altered the expression levels of apoptosis-related and steroidogenesis-related genes and proteins, while low doses had a less pronounced effect. Thus, increased progesterone induces the apoptosis of porcine placental trophoblasts and induces abnormal steroidogenesis in the placenta. We believe that our study makes a significant contribution to the literature because it elucidates the effects of progesterone on porcine placental trophoblast functions.Placentation and placental steroidogenesis are important for pregnancy and maternal–fetal health. As pregnancy progresses, the main site of progesterone (P4) synthesis changes from the corpus luteum to the placenta, in which placental trophoblasts are the main cell type for P4 synthesis. Therefore, this study investigated the effects of P4 on apoptosis and steroidogenesis in porcine placental trophoblasts and the underlying molecular mechanisms. Porcine placental trophoblasts were treated with different concentrations of P4 for 48 h in a serum-free medium in vitro. Cell number, steroidogenesis, and relevant gene and protein expression levels were detected. A high dose of P4 (10.0 μM) significantly increased P4 (p < 0.01), androstenedione (p < 0.05), testosterone (p < 0.05), and estradiol (p < 0.05) production in porcine placental trophoblasts compared with that in control cells, while a low dose of P4 (1 × 10−3 μΜ) had no marked impact on steroid production. The mRNA expression of apoptosis-related genes (CASP3, CASP8, and Bax) (p < 0.05) and steroidogenesis-related genes (CYP11A1, CYP19A1, and StAR) (p < 0.01) was upregulated, and the expression of HSD3B and HSD17B4 was inhibited (p < 0.05) in the porcine placental trophoblasts treated with high doses of P4. Low doses of P4 had a lighter effect on gene expression than high doses. The expression of apoptosis-related proteins CASP3 (p < 0.05), and Bax (p < 0.01) and steroidogenesis-related proteins CYP19A1 (p < 0.05) and StAR (p < 0.01) was raised, but the proliferation-related protein CCND2 (p < 0.01) was downregulated in the pTr cells treated with high dose of P4. In comparison, a low dose of P4 inhibited the expression of Bax, CYP11A1 (all p < 0.01), and CCND2 (p < 0.05), but the expression of CASP3 (p < 0.05) and StAR (p < 0.01) was upregulated. In summary, excessive P4 can induce the apoptosis of porcine placental trophoblasts and lead to abnormal steroidogenesis in the placenta and hormone imbalance.

Ethalfluralin impairs implantation by aggravation of mitochondrial viability and function during early pregnancy

Ethalfluralin, a dinitroaniline-type herbicide, has been used for decades. As a result, its residues are detected on some farmlands. To determine the molecular mechanisms underlying the detrimental effects of ethalfluralin on early pregnancy, porcine luminal epithelium and trophectoderm cell lines were used. Ethalfluralin was found to inhibit the viability, proliferation, and migration of porcine luminal epithelial (pLE) and porcine trophectoderm (pTr) cells. Additionally, ethalfluralin induced apoptotic cell death by means of an imbalance in calcium homeostasis in both pLE and pTr cells. Ethalfluralin decreased mitochondrial membrane potential (ΔΨm) and impaired mitochondrial respiration by downregulating the mitochondrial respiratory complex-related genes. Ethalfluralin also activated endoplasmic reticulum stress signals and autophagy pathways, increased the phosphorylation of P38 MAPK and NF-κB, and suppressed the PI3K/AKT signaling pathway. Taken together, this study elucidated the molecular mechanisms by which ethalfluralin impedes the viability and mitochondrial function in fetal trophectoderm and maternal endometrial cells during early pregnancy.

Glucosamine alleviates zearalenone-induced damage to porcine trophectoderm cells by activating the PI3K/AKT signaling pathway.

As one of the mycotoxins commonly found in feed and food, zearalenone (ZEA) mainly harms the reproductive functions of humans and animals. In our study, we investigated the protective effects of glucosamine (GlcN) on ZEA-induced apoptosis and oxidative damage of porcine trophectoderm (pTr) cells. Our results showed that 0.5 mmol L-1 GlcN significantly alleviated ZEA-induced decline in pTr cell viability. In addition, GlcN also significantly reversed other toxicity of ZEA to pTr cells, including G2/M phase arrest, DNA damage, reactive oxygen species (ROS) production, and barrier function disruption. Further studies indicated that GlcN can reduce apoptosis and autophagy in pTr cells in response to ZEA treatment. These results were confirmed by flow cytometry, transmission electron microscopy (TEM) and western blotting to detect the expressions of apoptosis and autophagy related proteins. Notably, GlcN activated the expressions of the PI3K/AKT signaling pathway related proteins, suggesting that its protective effect on pTr cells may be mediated by the PI3K/AKT signaling pathway. To demonstrate this mechanism, we used the PI3K/AKT pathway inhibitor wortmannin to pretreat pTr cells, and showed that the protective effect of GlcN on ZEA-induced pTr cytotoxicity was eliminated. In conclusion, GlcN was able to alleviate ZEA-induced toxic effects in pTr cells. This study also provides a theoretical basis for GlcN alleviating the adverse effects of ZEA on early embryo development and proved its potential application prospects.

Glucosamine enhances proliferation, barrier, and anti-oxidative functions in porcine trophectoderm cells.

Trophectoderm (TE) is the first epithelium that appears during mammalian embryogenesis, and is a polarized transporting single cell layer that comprises the wall of the blastocyst. Previous studies have revealed the functional roles of glucose (Gluc), fructose (Fruc), and glutamine (Gln), which play a positive role in porcine trophectoderm (pTr) cell proliferation and migration, suggesting the importance of nutrients for normal development of the conceptus and implantation. This work was conducted to test the hypothesis that glucosamine (GlcN), which is synthesized from Gln and Fruc-6-phosphate through the hexosamine biosynthesis pathway (HBP), can stimulate proliferation and sustain the barrier and anti-oxidative functions of pTr cells. Cells were treated with 0, 0.25, or 0.5 mmol L-1 GlcN in the presence or absence of adiquat (DQ) for the indicated time points. The results showed that 0.25 or 0.5 mmol L-1 GlcN stimulated pTr cell viability and DNA replication compared to the control group. The addition of 0.25 mmol L-1 GlcN enhanced the phosphorylation of mTOR signaling proteins, which can be inhibited by the inhibitor of phosphatidylinositol 3-kinase (PI3K), LY294002. Transepithelial electrical resistance (TEER) was increased, and paracellular permeability was correspondingly reduced in GlcN treatment. GlcN attenuated DQ-induced cell death and reduced the level of reactive oxygen species (ROS). The decreased TEER values and increased paracellular permeability caused by DQ treatment were also inhibited by GlcN treatment. The addition of 0.5 mmol L-1 GlcN increased the protein expression of zonula occludens-3 (ZO-3), claudin-3, and claudin-4 in pTr cells, while inhibited the downregulation protein of claudin-1 and claudin-3 brought about by oxidative stress. Collectively, GlcN plays an important role in promoting proliferation and stimulating the mTOR cell signaling pathway, as well as ameliorating oxidative stress and augmenting barrier functions in pTr cells.

L-Tryptophan activates the aryl hydrocarbon receptor and induces cell cycle arrest in porcine trophectoderm cells

During implantation, the proliferation of trophectoderm cells (the outer epithelium of blastocysts) is related to conceptus elongation and placenta formation. Tryptophan (Trp) is a key regulator of embryogenesis and embryonic implantation during pregnancy. We sought to determine whether different concentrations of Trp alters porcine trophectoderm (pTr) cell proliferation. pTr cells were cultured in medium containing 40, 500, or 1000 μM Trp. The cell proliferation rate and the progression of the cells through the cell cycle were determined. To identify differentially expressed genes (DEGs) in the pTr cells, we compared mRNA transcriptomes by RNA-Seq after cell treatment with different concentrations of Trp. Some candidate DEGs were identified by quantitative reverse transcription PCR (qPCR). High L-Trp levels (500 and 1000 μM) inhibited cell proliferation and induced cell cycle arrest. We identified 19 DEGs between the 500 μM L-Trp and 40 μM L-Trp groups and 168 DEGs between the 1000 μM L-Trp and 40 μM L-Trp groups and subsequently used qPCR to validate some genes that were upregulated or downregulated. The functional gene networks in which the DEGs were most enriched included those associated with regulating DNA replication and the cell cycle, and the majority of the DEGs in both of these functional pathways was downregulated. The results showed that the addition of 500 and 1000 μM Trp significantly increased the abundance of proteins in the Aryl Hydrocarbon Receptor (AHR) signaling pathway. Collectively, these results indicate a novel and important role for Trp in mediating the proliferation of porcine placental cells largely via the AHR signaling pathway. Additionally, these findings help to explain the side effects of excessive Trp supplementation on placenta development and embryo growth in mammals.

Genomic Effects of Arginine on Cell Behavior of Conceptus Trophectoderm in Ungulates

Abstract Hatched ungulate (e.g., pigs, sheep and other ruminants) blastocysts undergo dramatic morphological transitions from spherical to tubular to filamentous forms to conceptuses (embryo/fetus and associated extraembryonic membranes) before implantation. L-Arginine (Arg), a conditionally essential amino acid, is required for this process to activate the mTOR cell signaling pathway to induce proliferation of both porcine and ovine conceptus trophectoderm cells. However, the genomic effects of arginine on trophectoderm cells is unknown. RNA-seq was used for a comparative transcriptome analysis of porcine and ovine trophectoderm cells to further understand effects of Arg on regulation of metabolism in trophectoderm cells. An established porcine trophectoderm (pTr) cell line isolated from D12 porcine conceptuses, as well as an established ovine trophectoderm (oTr) cell line isolated from D15 ovine conceptuses were used to determine response to Arg at the physiological concentration of 0.2 mM in a 48-h culture. In pTr cells, a total of 2,723 differentially expressed genes (DEG; 1,482 up and 1,241 down) were identified in response to Arg. In oTr cells, a total of 5,369 DEG (2,819 up and 2,550 down) were detected. Comparison analyses showed that the Arg-treated pTr and oTr transcriptomes share 873 common DEG (273 up and 342 down). Canonical pathway analyses identified the top enriched pathways in both pTr and oTr cells, including activation of actin cytoskeleton signaling, adrenomedullin signaling, and IGF-1 signaling; and inhibition of cell cycle G2/M checkpoint regulation, and p53 signaling. In response to Arg, pathways associated with cholesterol biosynthesis, and estrogen-mediated S-phase entry were exclusively activated in the pTr cells; whereas interferon signaling, ephrin receptor signaling, and integrin signaling were specifically activated in the oTr cells. Results from this study advance understanding of mechanisms responsible for elongation of ovine and porcine conceptuses and enable the rational design of future experiments.

Effects of N-carbamylglutamate on steroidogenesis and relative abundances of mRNA transcripts in pig placental trophoblasts

Supplementation of N-carbamylglutamate (NCG) improves gestation outcomes, with increased piglet within-litter uniformity of birth weight and reduced peripheral steroid concentrations in pregnant sows and ewes. It was hypothesized that the effect of NCG on placental function results from direct effects on the placental trophoblasts. There, therefore, was investigation of the effects of NCG on pig placental trophoblast (pTr) steroidogenesis, mRNA transcript abundance, and cell proliferation in vitro. The pTr were treated with NCG in serum-free medium for 24–48 h. Treatment with NCG inhibited pTr progesterone, androstenedione, testosterone (all P < 0.01), and estradiol (P < 0.05) production, whereas it promoted (P < 0.05) pTr proliferation. Treatment with NCG suppressed (P < 0.05) the relative abundances of CYP11A1, CYP19A1, and CASP3 and increased abundances of CCDN1 (P < 0.01) and CDK4 (P < 0.05) mRNA transcripts in pTr, whereas NCG treatment had no effect (P > 0.10) on relative abundances of StAR, HSD17B4, or HSD3B mRNA transcripts. Treatments with NCG can increase pTr cell numbers of sows through upregulating CCND1 and CDK4 and suppressing CASP3 mRNA transcript abundances, while modulating steroidogenesis through effects on CYP11A1 and CYP19A1 mRNA transcript abundances. It is concluded that NCG may have a direct action on pTr and may regulate placental function by suppressing pTr differentiation as a consequence of lesser steroid synthesis while promoting pTr proliferation and inhibiting apoptosis in sows.

Melatonin improves uterine-conceptus interaction via regulation of SIRT1 during early pregnancy.

Melatonin has been shown to improve in vitro fertilization and offspring survival after bacterial infection, but its role in regulating maternal-fetal communication during early pregnancy has not been investigated. Results of this study demonstrated expression of abundant melatonin receptors in conceptus and endometrium during early pregnancy. In gilts, expression of melatonin receptor 1A (MTNR1A or MT1) and melatonin receptor 1B (MTNR1B or MT2) increased in trophectoderm (Tr) and uterine luminal epithelium (LE) with advancing days during early pregnancy in a different manner. Melatonin increased proliferation and migration of porcine trophectoderm (pTr) cell, the percent pTr cells in the G2 phase of the cell cycle, and the expression of implantation-related genes by pTr cells and endometrial luminal epithelium (pLE). Melatonin also attenuated the production of LPS-induced pro-inflammatory cytokines and tunicamycin-induced endoplasmic reticulum (ER) stress-sensing proteins. The expression of sirtuin 1 (SIRT1) as a potential target of melatonin increased between Days 9 and 14 of gestation. Co-treatment with SIRT1 inhibitor EX527 and melatonin restored cell-cell interactions through PI3K and MAPK signaling. Knockdown of SIRT1 decreased the expression of implantation-related genes, as well as migration of pTr and pLE cells. The expression of microRNAs regulated by SIRT1 was suppressed in response to melatonin. Furthermore, melatonin significantly increased lipopolysaccharide (LPS)-reduced fertilization and embryogenesis in zebrafish model. These results suggest that melatonin may improve the uterine-conceptus interactions via the regulation of SIRT1 during early pregnancy.

Flufenoxuron disturbs early pregnancy in pigs via induction of cell death with ER-mitochondrial dysfunction

The use of pesticides can result in unintended side effects, such as environmental pollution and animal diseases; in serious cases, it may cause abortion. Flufenoxuron is an inhibitor of chitin synthesis that is used widely as a pesticide on farmland. It is difficult to break down and therefore accumulates in the body, and has also been detected in breast milk. Moreover, the effects of flufenoxuron in pregnancy remain elusive. Therefore, we investigated the effects of flufenoxuron on early pregnancy. Our results suggested that flufenoxuron inhibits cell development and cell cycle progression in porcine trophectoderm (pTr) cell and porcine endometrial luminal epithelial (pLE) cell lines through the repression of signal transduction pathways. Flufenoxuron induced programmed cell death through DNA fragmentation and apoptotic signals. In addition, flufenoxuron induced ROS production, ER stress, and mitochondrial malfunction; consequently, the cytosolic and mitochondrial calcium levels were increased. Expression of proteins on the ER-mitochondrial axis was increased by flufenoxuron. Cell migration was decreased by flufenoxuron treatment between pLE and pTr cells. In addition, the expression of pregnancy-related genes was decreased flufenoxuron. Collectively, our results indicated that flufenoxuron may be harmful to livestock and women in the early stages of pregnancy.

Function of CCL5 in maternal-fetal interface of pig during early pregnancy

Chemokines refer to chemoattractant cytokines, which have crucial functions in inflammation and immune responses in multiple cellular processes. In the present study, we described the potential role of porcine CCL5 in embryo implantation and fetal-maternal environment during early pregnancy. We first carried out phylogenetic analysis of porcine CCL5, and analyzed the cell specific localization of CCL5 and its receptor CCR3 in a kinetic approach within porcine estrous cycles and early gestation stage. In addition, CCL5 stimulated porcine uterine luminal epithelial (pLE) and porcine trophectoderm (pTr) cell proliferations, and cell cycle progressions via AKT and MAPK intracellular signaling tractions. Furthermore, CCL5 attenuated tunicamycin-induced endoplasmic reticulum (ER) stress signaling, and lipopolysaccharides-triggered inflammatory responses in pLE and pTr cells. Taken together, our study showed that CCL5 is involved in the placental development or promotes the placental development.

ROS-induced autophagy regulates porcine trophectoderm cell apoptosis, proliferation, and differentiation

Significant embryo loss remains a serious problem in pig production. Reactive oxygen species (ROS) play a critical role in embryonic implantation and placentation. However, the potential mechanism of ROS on porcine trophectoderm (pTr) cell fate during the peri-implantation period has not been investigated. This study aimed to elucidate the effects of ROS on pTr cell phenotypes and the regulatory role in cell attachment and differentiation. Herein, results showed that exogenous H2O2 inhibited pTr cell viability, arrested the cell cycle at S and G2/M phases, and increased cell apoptosis and autophagy protein light chain 3B and Beclin-1, whereas these effects were reversed by different concentrations of N-acetyl-l-cysteine (NAC) posttreatment. In addition, NAC abolished H2O2-induced autophagic flux, inhibited intracellular and mitochondrial ROS, and restored expression of genes important for mitochondrial DNA and biogenesis, cell attachment, and differentiation. NAC reversed H2O2-activated MAPK and Akt/mammalian target of rapamycin pathways in dose-dependent manners. Furthermore, analyses with pharmacological and RNA interference approaches suggested that autophagy regulated cell apoptosis and gene expression of caudal-related homeobox 2 and IL-1β. Collectively, these results provide new insights into the role of the ROS-induced autophagy in pTr cell apoptosis, attachment, and differentiation, indicating a promising target for decreasing porcine conceptus loss during the peri-implantation period.

Trichlorfon inhibits proliferation and promotes apoptosis of porcine trophectoderm and uterine luminal epithelial cells

Trichlorfon is an organophosphate insecticide widely used in agriculture. Additionally, it is applied to pigs for control of endo- and ectoparasites. Previous studies have shown the effects of trichlorfon in pigs during late stages of gestation; however, little is known about its effects during early pregnancy, including implantation and placentation. We investigated whether trichlorfon affects proliferation and apoptosis of porcine trophectoderm (pTr) and uterine luminal epithelial (pLE) cells. Trichlorfon inhibited the proliferation of pTr and pLE cells, as evidenced by cell cycle arrest, and altered the expression of proliferation-related proteins. In addition, trichlorfon induced cell death and apoptotic features, such as loss of mitochondrial membrane potential and DNA fragmentation, in pTr and pLE cells. Moreover, trichlorfon treatment decreased concentrations of Ca2+ in the cytoplasm in both cell lines and increased concentrations of Ca2+ in mitochondria of pTr cells. Trichlorfon inhibited the activation of phosphoinositide 3-kinase/AKT and mitogen-activated protein kinase signaling pathways in pTr and pLE cells. Therefore, we suggest that trichlorfon-treated pTr and pLE cells exhibited abnormal cell physiology which might lead to early pregnancy failure.

Hypoxia-dependent accumulation of hypoxia-inducible factor-1 alpha induces transient cell cycle arrest in porcine trophectoderm cells

In the uterine environment, the pre-implantation embryo adapts to low oxygen concentrations through intracellular responses including modification of gene expression, progression via the cell cycle and metabolism. In this study, we determined mechanisms underlying the adaptation of pig embryos to oxygen deficiency in the maternal-conceptus microenvironment in in vitro experiments using our established porcine trophectoderm (pTr) cells in culture. The transition from G1 to S phase in pTr cells was reduced in response to 2% oxygen during a short period (<24 h), and the hypoxia-induced G1 arrest was reversible during prolonged hypoxia exposure. Acute hypoxia up-regulated expression of transcription factors p21 and p27 and down-regulated cell cycle regulators associated with the G1/S phase transition including cyclin D1, cyclin E1 and E2F1 mRNAs and proteins. Furthermore, hypoxia exposure for 24 h markedly increased the abundance of HIF-1α protein. Even under acute hypoxia, by HIF-1α silencing reduced the hypoxia-induced transient G1 arrest and expression of p21 and p27 genes was restored. Contrary to acute hypoxia, the accumulation of HIF-1α protein decreased as the length of the hypoxic period increased. Overall results of the present study suggest that increases in HIF-1α are responsible for initial response to hypoxia that results in a transient cell cycle arrest in pTr cells and cell cycle progression is restored by increasing degradation of HIF-1α during prolonged hypoxia. These findings advance understating of cellular adaptation of developing pre-implantation porcine conceptuses to hypoxic stress.

Stem cell factor-induced AKT cell signaling pathway: Effects on porcine trophectoderm and uterine luminal epithelial cells

Stem cell factor (SCF) is a multipotent growth factor that elicits diverse biological actions in various aspects of embryogenesis and animal development. The aim of the present study was to assess SCF-induced intracellular signaling and cellular activities in porcine trophectoderm (pTr) and uterine luminal epithelial (pLE) cells which are well known as useful to elucidate developmental events. SCF induced abundances of p-AKT, p-P70RSK and RPS6 proteins in pTr cells reached to their maximum, and then returned to basal levels by 120min. In pLE cells, SCF induced protracted effect to increase AKT phosphorylation which was well correlated with the time course for P70RSK and RPS6 phosphorylation. LY294002 (an inhibitor of AKT) decreased SCF-induced p-AKT, p-P70RSK and p-RPS6 proteins. Also, immunofluorescence analyses revealed that p-RPS6 was abundant within the cytoplasm of SCF-treated cells, but p-RPS6 was present only at basal levels in cells treated with LY294002. In the presence of LY294002, both SCF-stimulated transient and sustained AKT phosphorylation were inhibited in pLE cells. Furthermore, SCF increased migration of pTr and pLE cells, but LY294002 significantly reduced this effect of SCF. In conclusion, results of the present study suggest that SCF secreted by the endometrium induces autocrine/paracrine signaling responses that stimulate migration of pTr and pLE cells through activation of the AKT cell signaling pathway. Those results support the hypothesis that SCF is a critical regulatory factor for conceptus development and implantation during pregnancy in pigs.