pLE Cells Research Articles

Meptyldinocap induces implantation failure by forcing cell cycle arrest, mitochondrial dysfunction, and endoplasmic reticulum stress in porcine trophectoderm and endometrial luminal epithelial cells

Meptyldinocap is a dinitrophenol fungicide used to control powdery mildew. Although other dinitrophenol pesticides have been found to exhibit reproductive toxicity, studies of meptyldinocaps are scarce. This study investigated the adverse effects of meptyldinocap on porcine trophectoderm (pTr) and porcine endometrial luminal epithelial (pLE) cells, which play crucial roles in implantation. We confirmed that meptyldinocap decreased cell viability, induced apoptosis, and inhibited proliferation by decreasing proliferation-related gene expression and inducing changes in the cell cycle. Furthermore, meptyldinocap treatment caused mitochondrial dysfunction, endoplasmic reticulum stress, and disruption of calcium homeostasis. Moreover, it induces alterations in mitogen-activated protein kinase signaling cascades and reduces the migration ability, leading to implantation failure. Our findings suggest that meptyldinocap reduces the cellular functions of pTr and pLE cells, which are important for the implantation process, and interferes with interactions between the two cell lines, potentially leading to implantation failure. We also propose a mechanism by which the understudied fungicide meptyldinocap exerts its cytotoxicity.

Bifenox compromises porcine trophectoderm and luminal epithelial cells in early pregnancy by arresting cell cycle progression and impairing mitochondrial and calcium homeostasis

Bifenox is a widely used herbicide that contains a diphenyl ether group. However its global usage, the cell physiological effects that induce toxicity have not been elucidated. In this study, the effect of bifenox was examined in porcine trophectoderm and uterine epithelial cells to investigate the potential toxicity of the implantation process. To uncover the toxic effects of bifenox, cell viability and apoptosis following treatment with bifenox were evaluated. To investigate the underlying cellular mechanisms, mitochondrial and calcium homeostasis were investigated in both cell lines. In addition, the dysregulation of cell signal transduction and transcriptional alterations were also demonstrated. Bifenox reduced cell viability and significantly increased the number of cells arrested at the sub-G1 stage. Moreover, bifenox depolarized the mitochondrial membrane and upregulated the calcium flux into the mitochondria in both cell lines. Cytosolic calcium flux increased in porcine trophectoderm (pTr) cells and decreased in porcine luminal epithelium (pLE) cells. In addition, bifenox activated the mitogen-activated protein kinase and phosphoinositide 3-kinase signaling pathways. Furthermore, bifenox inhibited the expression of retinoid receptor genes, such as RXRA, RXRB, and RXRG. Chemokine CCL8 was also downregulated at the mRNA level, whereas CCL5 expression remained unchanged. Overall, the results of this study suggest that bifenox deteriorates cell viability by arresting cell cycle progression, damaging mitochondria, and controlling calcium levels in pTr and pLE cells. The present study indicates the toxic potential of bifenox in the trophectoderm and luminal epithelial cells, which can lead to implantation disorders in early pregnancy.

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.

Pendimethalin exposure disrupts mitochondrial function and impairs processes related to implantation.

Pendimethalin as a dinitroaniline herbicide is used to eliminate weeds during the cultivation of various crops such as grains, fruits, and vegetables. This study reveals that pendimethalin exposure at various concentrations led to disruption in Ca2+ homeostasis and mitochondrial membrane potential, as well as dysregulation of the mitogen-activated protein kinase signaling pathway and implantation-related genes in porcine trophectoderm and uterine luminal epithelial cells. The use of herbicides is a major control method in agriculture. Pendimethalin (PDM) has been increasingly used as a herbicide for approximately 30 years. PDM has been reported to cause various reproductive problems, but its toxicity mechanism in the pre-implantation stage has not been investigated in detail. Herein, we studied the effects of PDM on porcine trophectoderm (pTr) and uterine luminal epithelial (pLE) cells and identified a PDM-mediated anti-proliferative effect in both cell types. PDM exposure generated intracellular reactive oxygen species, induced excessive Ca2+ influx into mitochondria, and activated mitogen-activated protein kinase signaling pathway. Ca2+ burden resulted in the dysfunction of mitochondria and eventual disruption of Ca2+ homeostasis. Further, PDM-exposed pTr and pLE cells showed cell cycle arrest and programmed cell death. In addition, a decrease in migration ability and dysregulated expression of genes related to the functioning of pTr and pLE cells was evaluated. This study provides insight into time-dependent transitions within the cell environment after PDM exposure and elucidates a detailed mechanism of induced adverse effects. These results imply that PDM exposure can potentially cause toxic effects on the implantation-related process in pigs. Moreover, to the best of our knowledge, this is the first study to describe the mechanism by which PDM induces these effects, enhancing our understanding of the toxicity of this herbicide.

Picolinafen exposure induces ROS accumulation and calcium depletion, leading to apoptosis in porcine embryonic trophectoderm and uterine luminal epithelial cells during the peri-implantation period

The global use of herbicides accounts for more than 48% of total pesticide usage. Picolinafen is a pyridine carboxylic acid herbicide that is predominantly used to control broadleaf weeds in wheat, barley, corn, and soybeans. Despite its widespread use in agriculture, its toxicity in mammals has rarely been studied. In this study, we first identified the cytotoxic effects of picolinafen on porcine trophectoderm (pTr) and luminal epithelial (pLE) cells, which are involved in the implantation process during early pregnancy. Picolinafen treatment significantly decreased the viability of pTr and pLE cells. Our results demonstrate that picolinafen increased the number of sub-G1 phase cells and early/late apoptosis. In addition, picolinafen disrupted mitochondrial function and resulted in the accumulation of intracellular ROS, leading to a reduction in calcium levels in both the mitochondria and cytoplasm of pTr and pLE cells. Moreover, picolinafen was found to significantly inhibit the migration of pTr. These responses were accompanied by the activation of the MAPK and PI3K signal transduction pathways by picolinafen. Our data suggest that the deleterious effects of picolinafen on the viability and migration of pTr and pLE cells might impair their implantation potential.

Aclonifen could induce implantation failure during early embryonic development through apoptosis of porcine trophectoderm and uterine luminal epithelial cells

Aclonifen is a diphenyl-ether herbicide that is used to control the growth of weeds while growing crops such as corn and wheat. Although the biochemical effects of aclonifen are well characterized, including its ability to inhibit protoporphyrinogen oxidase and carotenoid synthesis, the toxicity of aclonifen in embryonic implantation and development during early pregnancy, has not been reported. Thus, in this study, we investigated the potential interference of aclonifen in embryonic implantation using porcine trophectoderm (pTr) and uterine luminal epithelial (pLE) cells isolated during implantation period of early pregnancy. Cell viability in both pTr and pLE cells significantly decreased in a dose-dependent manner following aclonifen treatment. Moreover, the proportion of cells in the sub-G1 phase of the cell cycle gradually increased upon treatment with increasing concentrations of aclonifen, which in turn led to an increase in the number of apoptotic cells, as determined by annexin V and propidium iodide staining. Aclonifen treatment caused mitochondrial dysfunction by increasing the depolarization of the mitochondrial membrane potential and the mitochondrial calcium concentration. Aclonifen inhibited cell mobility by suppressing the expression of implantation-related genes in pTr and pLE cells. To explore the underlying mechanism, we evaluated the phosphorylation of PI3K and MAPK signaling molecules. The phosphorylation of AKT, S6, JNK, and ERK1/2 were significantly increased by aclonifen. Collectively, our results suggest that aclonifen may interrupt implantation during early pregnancy by disrupting maternal–fetal interaction.

Fluroxypyr-1-methylheptyl ester induced ROS production and mitochondrial apoptosis through the MAPK signaling cascade in porcine trophectoderm and uterine luminal epithelial cells

FPMH (Fluroxypyr-1-methylheptyl ester) is a synthetic auxin herbicide used in agriculture. The mechanism by which FPMH induces adverse effects in porcine trophectoderm (pTr) and porcine uterine luminal epithelial (pLE) cells, which are involved in porcine implantation, have not been studied yet. Therefore, the present study investigates the toxicological effects of FPMH on pTr and pLE cells. We confirmed that FPMH induced cytotoxic effects on the cells, including apoptosis induction, mitochondrial membrane potential (MMP) depolarization, and ROS production. The phosphorylation of the MAPK pathway (ERK1/2, JNK, and p38) was dysregulated by FPMH administration. In addition, FPMH could suppress cell-cell adhesion and migration abilities of pTr and pLE, which are crucial for implantation. Therefore, exposure to FPMH induced adverse effects in pTr and pLE cells and could result in implantation failure.

Exposure to iprodione induces ROS production and mitochondrial dysfunction in porcine trophectoderm and uterine luminal epithelial cells, leading to implantation defects during early pregnancy

Iprodione is a well-known fungicide used in the cultivation of strawberries, tomatoes, grapes, and green beans. In recent studies, neurotoxicity, cardiotoxicity, and endocrine toxicity of iprodione have been reported. Although reproductive toxicity of iprodione has been identified in animal studies, its effects are limited to male fertility. Also, the toxic effects of iprodione on pregnancy, especially the implantation process, have not been elucidated. This study demonstrated a series of cytotoxic responses of iprodione along with the alteration of implantation-related gene expression in porcine trophectoderm (pTr) and luminal epithelium (pLE) cells. In this study, iprodione suppressed cell viability, proliferation, and migration of these cells. Iprodione induced G1 phase arrest and attenuated spheroid formation by pTr and pLE cells. Furthermore, iprodione caused mitochondrial dysfunction and excessive reactive oxygen species generation, which resulted in an increase in mitochondrial calcium levels. Consequently, DNA damage and apoptotic cell death were induced by iprodione treatment in pTr and pLE cells. This stress-induced cell death was mediated by alterations in intracellular signal transduction, including the PI3K/AKT and MAPK signaling pathways. This finding suggests the potential of iprodione to impair the implantation capacity by exerting cytotoxic effects on fetal and maternal cells.

Dinitramine induces implantation failure by cell cycle arrest and mitochondrial dysfunction in porcine trophectoderm and luminal epithelial cells

The herbicide market is growing rapidly, as weed control is a significant challenge in agriculture. Many studies have reported the toxicity of herbicides to non-target organisms. Dinitramine is a dinitroaniline herbicide that is particularly toxic to aquatic organisms. However, little is known about the effects of dinitramine on the female reproductive system. Therefore, in the present study, we utilized porcine trophectoderm (pTr) cells and porcine endometrial luminal epithelial (pLE) cells to verify the reproductive toxicity of dinitramine. Dinitramine reduced the viability of both cell types, by triggering cell cycle arrest, especially at the sub-G1 phase, and increasing apoptosis, inhibiting DNA replication. Dinitramine disrupted intracellular calcium homeostasis and induced oxidative stress by producing reactive oxygen species, leading to the loss of mitochondrial membrane potential and alteration of mitochondrial respiration. Mitogen-activated protein kinase pathways were altered, and migration decreased in pTr and pLE cells after dinitramine treatment; the expression of pregnancy-related genes in these cells was decreased. Thus, dinitramine reduced the viability and migratory capacity of both cell types, and this could interrupt the early stages of pregnancy.

Reproductive toxicity of folpet through deregulation of calcium homeostasis in porcine trophectoderm and luminal epithelial cells during early pregnancy

Folpet, a fungicide, is utilized even in cosmetics and pharmaceuticals. The LD50 of folpet in mammals, birds, and fish is relatively high. Recently, several negative effects of folpet on the respiratory system and cornea have been reported. However, there is no study on the negative effects of folpet on maternal–fetus interactions. In the present study, we used porcine trophectoderm (pTr) cells and porcine luminal epithelial (pLE) cells to investigate the toxic effects of folpet during implantation. Folpet treatment decreased cell proliferation and promoted apoptosis with cell cycle arrest. In addition, the ERK, JNK, and AKT signal pathways were activated by folpet treatment. Folpet treatment induced calcium overload in pTr and pLE cells mediating antimigratory and antiadhesive effects in both cell lines. Co-treatment with calcium chelates decreased the anti-implantation effect of folpet. Overall, our results demonstrated potential reproductive toxicity of folpet in pig.

Exposure to fipronil induces cell cycle arrest, DNA damage, and apoptosis in porcine trophectoderm and endometrial epithelium, leading to implantation defects during early pregnancy

Fipronil, a phenyl-pyrazole insecticide, has a wide range of uses, from agriculture to veterinary medicine. Due to its large-scale applications, the risk of environmental and occupational exposure and bioaccumulation raises concerns. Moreover, relatively little is known about the intracellular mechanisms of fipronil in trophoblasts and the endometrium involved in implantation. Here, we demonstrated that fipronil reduced the viability of porcine trophectoderm and luminal epithelial cells. Fipronil induced cell cycle arrest at the sub-G1 phase and apoptotic cell death through DNA fragmentation and inhibition of DNA replication. These reactions were accompanied by homeostatic changes, including mitochondrial depolarization and cytosolic calcium depletion. In addition, we found that exposure to fipronil compromised the migration and implantation ability of pTr and pLE cells. Moreover, alterations in PI3K-AKT and MAPK-ERK1/2 signal transduction were observed in fipronil-treated pTr and pLE cells. Finally, the antiproliferative and apoptotic effects of fipronil were also demonstrated in 3D cell culture conditions. In summary, our results suggest that fipronil impairs implantation potentials in fetal trophectoderm and maternal endometrial cells during early pregnancy.

Bifenthrin reduces pregnancy potential via induction of oxidative stress in porcine trophectoderm and uterine luminal epithelial cells

Exposure to pesticides has become a serious concern for the environment and human health. Bifenthrin, a synthetic pyrethroid pesticide, is one of the most frequently used pesticides worldwide. Despite the toxic potential of bifenthrin, no studies have elucidated the cytotoxic response of bifenthrin in maternal and fetal cells that are involved in the implantation process. In this study, the cytotoxic effect of bifenthrin was investigated using porcine trophectoderm (pTr) and uterine luminal epithelial (pLE) cells. The results showed that bifenthrin suppressed cell proliferation and viability in pTr and pLE cells. In particular, bifenthrin induced cell cycle arrest, resulting in apoptosis in both cell lines. We found that bifenthrin damaged the mitochondria and induced the production of reactive oxygen species, causing endoplasmic reticulum stress and calcium dysregulation in pTr and pLE cells. Finally, bifenthrin altered the MAPK/PI3K signaling pathway and pregnancy-related gene expression. Collectively, our results suggest that bifenthrin reduces the implantation potential of embryos and may help elucidate the mechanisms underlying toxin-derived cytotoxicity in maternal and fetal cells.

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.

Exposure to etoxazole induces mitochondria-mediated apoptosis in porcine trophectoderm and uterine luminal epithelial cells

Etoxazole is an organofluorine insecticide widely used in agriculture. Exposure to insecticides is a serious environmental problem owing to their cytotoxic effects in humans and animals. Reproductive toxicity of various organofluorine insecticides have been shown in previous studies. However, few studies have evaluated the toxicity of etoxazole in mammals. We aimed to examine the toxic effects of etoxazole in porcine trophectoderm (pTr) and uterine luminal epithelial (pLE) cells. To estimate the effects of etoxazole, we conducted assays after treatment with multiple concentration of etoxazole (0, 2, 4, 6 and 9 μM) to pTr and pLE cells for 0–72 h. Etoxazole decreased the cell proliferation, viability, and migration of pTr and pLE cells. Further, etoxazole induced apoptosis via cell cycle arrest and disruption of mitochondrial membrane potential. We also found that pro-apoptotic proteins and endoplasmic reticulum (ER) stress-response proteins were activated in response to etoxazole. Finally, we observed that etoxazole altered the PI3K/AKT and MAPK signaling pathways and the mRNA expression of genes associated with implantation. Collectively, these results suggest that etoxazole disrupts normal cellular physiology and might cause early implantation failure.

Ivermectin induces apoptosis of porcine trophectoderm and uterine luminal epithelial cells through loss of mitochondrial membrane potential, mitochondrial calcium ion overload, and reactive oxygen species generation

Ivermectin is a pesticide that has been used for over 30 years in livestock. Although there are a number of studies on the therapeutic potential of ivermectin, little is known about the effects of the drug during the early stage of pregnancy. In this study, we investigated the detrimental effects of ivermectin on porcine trophectoderm (pTr) and uterine luminal epithelial (pLE) cells. Ivermectin not only inhibited the proliferation of both cells via the regulation of cell cycle-associated genes, but also induced apoptosis in pTr and pLE cells. We also verified its effect on mitochondrial dysfunction as shown by loss of mitochondrial membrane potential, mitochondrial Ca2+ overload, and reactive oxygen species (ROS) generation in pTr and pLE cells. As a mechanistic approach, we evaluated ivermectin-mediated cell signaling interactions including PI3K, AKT and MAPK pathways. Overall, our results suggest that constant exposure to and accumulation of ivermectin may cause abnormal fetal morphogenesis and placentation during the early stages of pregnancy. Our results may further provide a comprehensive understanding of the detrimental effects of ivermectin during pregnancy and will contribute to the establishment of a complete safety profile for ivermectin and its association with environmental pollution and public health in humans and livestock.

Oxibendazole induces apoptotic cell death in proliferating porcine trophectoderm and uterine luminal epithelial cells via mitochondria-mediated calcium disruption and breakdown of mitochondrial membrane potential

The well-known and effective anthelmintic oxibendazole was recently shown to have a broad spectrum of biological abilities, such as anti-cancer and anti-inflammation activities. In contrast, the mechanism of oxibendazole's anti-proliferative effect via cell signaling pathways and its role in pre-implantation has not been studied. Therefore, in this study we demonstrated the effects of oxibendazole on the proliferation of porcine trophectoderm (pTr) cells and porcine luminal epithelial (pLE) cells, a well-known in vitro model system of the fetal-maternal interface. Cell proliferation decreased in both pTr and pLE cells in response to oxibendazole, and we determined that this was modulated through intracellular cell signal transduction. Phosphorylation of ERK1/2, P90RSK, and S6 were downregulated by exposure to a 200 nM dose of oxibendazole in both types of cells, while the expression of phosphorylated JNK, AKT, and P70S6K was upregulated. Pre-treatment with a PI3K/AKT inhibitor (Wortmannin), ERK1/2 inhibitor (U0126), and JNK inhibitor (SP600125) induced the signaling interactions of these molecules, and oxibendazole co-treatment with each inhibitor resulted in even greater decreases in cell proliferation. Furthermore, intracellular and mitochondrial calcium ion accumulation was observed, which would mean that calcium ion homeostasis was disrupted, causing damage to the mitochondrial membrane potential. These deteriorated conditions ultimately led to apoptotic cell death. Taken together, the results of the present study identified that the apoptotic effect of oxibendazole on pTr and pLE cells is regulated by cell signaling pathways, and thus oxibendazole could influence the connection between the conceptus and the maternal uterus.

Mitigation of ER-stress and inflammation by chemokine (C-C motif) ligand 21 during early pregnancy

The immune system plays an important role in pregnancy. Chemokines recruit leukocytes at the maternal-fetal interface during early pregnancy. However, the role of the chemokine, C-C motif chemokine ligand 21 (CCL21), is less known. The aim of this study was to identify the expression of CCL21 and its receptor, CCR7, in the endometrium during estrous cycle and early pregnancy, and to investigate the functional effects of CCL21 on porcine trophectoderm (pTr) and porcine uterine luminal epithelial (pLE) cells. Our results indicated that CCL21 and CCR7 are increased in the glandular (GE) and luminal epithelium (LE) of the endometrium during early pregnancy, compared to estrous pigs. Recombinant CCL21 improved pTr and pLE cell proliferation through activation of the PI3K and MAPK pathways and suppression of tunicamycin-induced endoplasmic reticulum (ER) stress or LPS-induced inflammation. Collectively, these results provide novel insights into CCL21-mediated signaling mechanisms at the maternal-fetal interface during early pregnancy.

Activation of CCL20 and its receptor CCR6 promotes endometrium preparation for implantation and placenta development during the early pregnancy period in pigs

The success of implantation and placentation during early pregnancy depends on the intrauterine environment of diverse histotrophs, including nutrients, growth factors, cytokines, and chemokines. Here, we determined the functional role and signal transduction of CC-motif chemokine 20 (CCL20) in the porcine endometrium during pregnancy. The expression of CCL20 and its receptor CCR6 was abundant in the glandular epithelium (GE) and luminal epithelium (LE) of the porcine endometrium during early pregnancy. Administration of CCL20 to porcine endometrial LE cells increased cellular proliferation with strong PCNA expression by activation of PI3K and MAPK signaling. Blocking PI3K and MAPK decreased the CCL20-mediated elevated proliferation of pLE cells. Moreover, the proliferation of pLE cells was enhanced by inhibiting the LPS-induced cytokines and tunicamycin-induced endoplasmic reticulum (ER) stress response proteins. Overall, these results suggest that CCL20 may improve the endometrial receptivity through inducing proliferative signal transduction in the porcine endometrium during the early gestational 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.

Characterization of C-C motif chemokine ligand 4 in the porcine endometrium during the presence of the maternal–fetal interface

Chemokines and their receptors play a crucial role in embryo implantation at the maternal–fetal interface during pregnancy. In this study, we investigated the role of CCL4 in development of the porcine endometrium in the early gestational period. Porcine CCL4 showed high similarity with the human counterpart, and mRNA expression of CCL4 and its receptor (CCR5) was predominantly present in the endometrium during early pregnancy. Treatment with CCL4 increased proliferation of porcine uterine luminal epithelial (pLE) cells by activation of PI3K and MAPK signal transduction. In addition, CCL4 recovered the endoplasmic-reticulum stress–reduced proliferation and decreased the unfolded protein response in pLE cells. Besides, the lipopolysaccharide-activated NF-κB pathway was suppressed in response to CCL4 in pLE cells. Inhibition of CCR5 decreased the proliferation of pLE cells and activation of the PI3K and MAPK pathways by CCL4. Furthermore, CCL4 enhanced conceptus-maternal interactions between porcine trophectoderm (pTr) cells and pLE cells during early pregnancy by activating expression of migration and implantation-related genes. Collectively, the results suggest that CCL4 may improve successful implantation in early pregnancy in pigs.