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Caitlyn Nguyen-Ngo, Carlos Salomon, Andrew Lai, Jane C Willcox and Martha Lappas

Spontaneous preterm birth is the leading cause of neonatal mortality and morbidity globally. Activation of the maternal immune system leads to a downstream cascade of pro-inflammatory events that culminate in the activation of spontaneous uterine contractions and the rupture of the fetal membranes. Anti-inflammatory agents may be a novel therapeutic approach to prevent inflammation-induced myometrial contractions and premature rupture of fetal membranes. The polyphenol gallic acid has been previously shown to exert potent anti-inflammatory effects. Thus, this study aimed to determine the effect of gallic acid on pro-inflammatory and pro-labor mediators in cytokine-stimulated gestational tissues in vitro. In primary human cells isolated from myometrium and fetal membranes (decidua, and amnion mesenchymal and epithelial cells), gallic acid treatment suppressed inflammation-induced expression of pro-inflammatory cytokines and chemokines and extracellular matrix-degrading and –remodelling enzymes. Gallic acid also significantly inhibited inflammation-induced myometrial activation as evidenced by decreased expression of contraction associated proteins, the uterotonic PGF2α and collagen cell contractility. Using a global proteomic approach, gallic acid may differentially regulate proteins associated with collagen synthesis, cell contractility and protein synthesis in primary myometrial and decidual cells. In summary, gallic acid inhibited inflammation-induced mediators involved in active labor in primary cells isolated from myometrium and fetal membranes. These in vitro studies suggest that the polyphenol gallic acid may be able to suppress production of pro-inflammatory and pro-labor mediators involved in myometrial contractions and rupture of fetal membranes. Future preclinical studies may elucidate the efficacy of gallic acid in preventing inflammation-driven preterm birth.

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D Randall Armant, Graham W Aberdeen, Brian A Kilburn, Gerald J Pepe and Eugene D Albrecht

Placental extravillous trophoblast remodeling of the uterine spiral arteries is important for promoting blood flow to the placenta and fetal development. Heparin-binding EGF-like growth factor (HB-EGF), an EGF family member, stimulates differentiation and invasive capacity of extravillous trophoblasts in vitro. Trophoblast expression and maternal levels of HB-EGF are reduced at term in women with preeclampsia, but it is uncertain whether HB-EGF is downregulated earlier when it may contribute to placental insufficiency. A nonhuman primate model has been established in which trophoblast remodeling of the uterine spiral arteries is suppressed by shifting the rise in estrogen from the second to the first trimester of baboon pregnancy. In the present study, we used this model to determine if placental HB-EGF is altered by prematurely elevating estrogen early in baboon gestation. Uterine spiral artery remodeling and placental expression of HB-EGF and other EGF family members were assessed on day 60 of gestation in baboons treated with estradiol (E2) daily between days 25 and 59 of gestation (term = 184 days). The percentages of spiral artery remodeling were 90, 84 and 70% lower (P < 0.01), respectively, for vessels of 26–50, 51–100 and >100 µm diameter in E2-treated compared with untreated baboons. HB-EGF protein quantified by immunocytochemical staining/image analysis was decreased three-fold (P < 0.01) in the placenta of E2-treated versus untreated baboons, while amphiregulin (AREG) and EGF expression was unaltered. Therefore, we propose that HB-EGF modulates the estrogen-sensitive remodeling of the uterine spiral arteries by the extravillous trophoblast in early baboon pregnancy.

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Seok Hee Lee, Hyun Ju Oh, Min Jung Kim and Byeong Chun Lee

Oviduct cells produce a favorable environment for the development of gametes by generating multiple growth factors. Particularly, in canine species, immature oocytes undergo serial maturation processes in the oviduct, while the other mammals already possess matured oocytes in ovulatory follicles. However, little is known about the potential effect exhibited by the components released from canine oviduct cells (OCs) for modulating the biological function of oocytes. Recently, exosomes are regarded as promising extracellular vesicles because they represent considerable data for molecular cargo. Therefore, we first investigated the effect of canine oviductal exosomes (OC-Exo) on oocyte development via EGFR/MAPK pathway. Our results showed that OC-Exo labeled with PHK67 are successfully incorporated with cumulus cells and oocytes during IVM. Also, OC-Exo markedly increased the proportion of cumulus-oocyte complexes (COCs) exhibiting cumulus expansion as well as cumulus cell proliferation and maturation rate of oocytes (p < 0.05). Furthermore, gene expression patterns related with EGFR/MAPK pathway including EGFR, PKA, TACE/ADAM17, MAPK1/3, MAPK14, PTGS2, TNFAIP6, GDF9, and BMP15 were positively modified in COCs cultured with OC-Exo (p < 0.05). In addition, OC-Exo significantly up-regulated the protein expression levels of p-EGFR, p-MAPK1/3, GDF9 and BMP15 in COCs (p < 0.05). Consequently, the current study provides a model for understanding the roles of OC-Exo as bioactive molecules for canine oocyte maturation via EGFR/MAPK pathway, which would open a new avenue for the application of exosomes to improve assisted reproductive technology in mammals, including humans.

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Daniela Weiser, Andrea Mietens, Beatrix Stadler, Davor Ježek, Gerhard Schuler and Ralf Middendorff

Contractions of the adult epididymal duct are well known in the context of sperm transport. Some reports also describe contractions of the epididymal duct during development, but data about their character, regulation and function are sparse. In the foetal human epididymis we found luminal cells and could identify them as exfoliated epithelial cells originating from the epididymis and not from testis by using antibodies against neutral endopeptidase as an epithelial epididymal duct marker. Exfoliated cells were also found in the epididymal duct after birth. Time-lapse imaging revealed directional transport of luminal cells in the neonatal rat epididymis interrupted by pendular movement. Spontaneous contractions were discovered in the neonatal epididymis and an association between these contractions and the transport of the luminal cells could be observed. Both, transport and spontaneous contractions, were affected significantly by substances known to contract (noradrenaline) or relax (the phosphodiesterase 5 inhibitor sildenafil) smooth muscle cells. Immunohistochemistry showed staining for the proliferation marker proliferating-cell-nuclear-antigen (PCNA) in cells of the ductal lumen of the neonatal rat epididymis indicating the extrusion of cells also during proliferation. Our data showed spontaneous contractions of the immature epididymal duct associated with the transport of exfoliated luminal cells before the first occurrence of sperm cells. Results suggest an important role including both (i) a mechanical place holder function of exfoliated luminal cells (ii) together with a novel idea of organized waste disposal of these cells during development.

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Malena Schanton, Julieta Lorena Maymó, María Fernanda Camisay, Antonio Pérez-Pérez, Roberto Casale, Victor Sanchez-Margalet, Alejandra Erlejman and Cecilia Varone

Pregnancy success requires a proper fetal maternal interaction at the establishment of implantation. Leptin has been described as a multitasking cytokine in pregnancy, particularly in the placenta, where it acts as an autocrine hormone. The expression of leptin in normal trophoblastic cells is regulated by different endogenous signals. We have previously reported that 17β-estradiol up-regulates placental leptin expression through genomic and non-genomic mechanisms. To improve the knowledge of estrogen receptor mechanisms in regulating leptin gene expression, we examined transcription nuclear factor kappa B (NFκB) effect on estradiol leptin induction in human BeWo cell line and human term placental explants. We demonstrated that estradiol induction effect on leptin expression is blocked by the inhibition of NFκB signaling. We also found that the overexpression of p65 subunit, the active form of NFκB, induces leptin expression. Moreover, the downregulation of estrogen receptor alpha (ERα through a specific siRNA, abolished NFκB effect on leptin expression. We also demonstrated that ERα enhanced NFκB signaling pathway activation in trophoblastic cells. Estradiol treatment significantly increased p65 expression and phosphorylation of the inhibitory protein κB alpha (IκBα). A reporter plasmid containing NFκB elements was also induced in response to estradiol stimulation. Localization experiments revealed that estradiol treatment induced nuclear localization of overexpressed p65. Moreover, the overexpression of ERα produced a complete displacement of p65 protein to the nucleus. Finally, immunoprecipitation experiments showed the presence of a complex containing ERαand NFκB. All these evidences suggest a cooperative behavior between ERαand NFκB transcription factors to induce leptin transcription.

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Tristan Frum and Amy Ralston

The mouse preimplantation embryo is a paradigm for discovery of the molecular principles governing formation of specific cell types during development. In this Point of View Article, we show that conditions commonly used for ex vivo culture of preimplantation development are themselves antagonistic to a pathway that is critical for blastocyst lineage commitment.

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Rongli Wang and Li Zou

A successful pregnancy crucially depends on well-regulated extravillous trophoblast migration and invasion. Maternally expressed gene 3 (MEG3) is a long noncoding RNA that plays an important role in regulating trophoblast cells cell function. As previously reported, the expression of MEG3 was reduced in preeclampsia, and downregulation of MEG3 could suppress trophoblast cells migration and promote its apoptosis. However, the downstream regulatory mechanism of MEG3 remains unknown. As reported, MEG3 could inhibit cell proliferation in endometrial carcinoma by regulating Notch signaling. Our previous studies have demonstrated that Notch1 is downregulated in preeclampsia and that inhibiting the expression of Notch1 could promote trophoblast cell apoptosis. Therefore, this study was designed to investigate the role of MEG3 and its the relationship with Notch1 in trophoblasts. In this study, the mRNA expression levels of both MEG3 and Notch1 were decreased in preeclampsia placenta (n = 15) compared to the normal samples (n = 15). Exogenous upregulation and downregulation of MEG3 in HTR8/SVneo cells were performed to investigate the role of MEG3 in cell biological behavior and its effects on Notch1 expression. The results showed that MEG3 enhancement promoted trophoblast cell migration and invasion and inhibited cell apoptosis. Downregulation of MEG3 elicited the opposite results. Associated factors, such as matrix metalloproteinases 2 (MMP2), BAX, and Bcl-2, were examined at the mRNA and protein levels. Our study demonstrated that MEG3 could regulate Notch1 expression to modulate trophoblast cell migration, invasion, and apoptosis, which may represent the molecular mechanism of poor placentation during preeclampsia.

Open access

Priscila Ramos-Ibeas, Ismael Lamas-Toranzo, Álvaro Martínez-Moro, Celia de Frutos, Alejandra C Quiroga, Esther Zurita and Pablo Bermejo-Alvarez

Failures during conceptus elongation are a major cause of pregnancy losses in ungulates, exerting a relevant economic impact on farming. The developmental events occurring during this period are poorly understood, mainly because this process cannot be recapitulated in vitro. Previous studies have established an in vitro post-hatching development (PHD) system that supports bovine embryo development beyond the blastocyst stage, based on agarose gel tunnels and serum and glucose-enriched medium. Unfortunately, under this system embryonic disc formation is not achieved and embryos show notorious signs of apoptosis and necrosis. The objective of this study has been to develop an in vitro system able to support embryonic disc formation. We first compared post-hatching development inside agarose tunnels or free-floating over an agarose-coated dish in serum and glucose-enriched medium (PHD medium). Culture inside agarose tunnels shaped embryo morphology by physical constriction, but it restricted embryo growth and did not provide any significant advantage in terms of development of hypoblast and epiblast lineages. In contrast to PHD medium, a chemically defined and enriched medium (N2B27) supported complete hypoblast migration and epiblast survival in vitro, even in the absence of agarose coating. Cells expressing the pluripotency marker SOX2 were observed in ~56 % of the embryos and ~25 % developed embryonic disc-like structures formed by SOX2+ cells. In summary, here we provide a culture system that supports trophectoderm proliferation, hypoblast migration and epiblast survival after the blastocyst stage.

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Maria Jose Ruiz Magaña, Jose Maria Puerta, Rocio Martínez-Aguilar, Tatiana Llorca, Osmany Blanco, Raquel Muñoz-Fernández, Enrique G Olivares and Carmen Ruiz-Ruiz

Endometrial stromal cells (EnSCs) and decidual stromal cells (DSCs) originate from fibroblastic precursors located around the vessels of the human nonpregnant endometrium and the pregnant endometrium (decidua), respectively. Under the effect of ovarian or pregnancy hormones, these precursors differentiate (decidualize), changing their morphology and secreting factors that appear to be essential for the normal development of pregnancy. However, the different physiological context – that is, non-pregnancy vs pregnancy – of those precursors (preEnSCs, preDSCs) might affect their phenotype and functions. In the present study, we established preEnSC and preDSC lines and compared the antigen phenotype and responses to decidualization factors in these two types of stromal cell line. Analyses with flow cytometry showed that preEnSCs and preDSCs exhibited a similar antigen phenotype compatible with that of bone marrow mesenchymal stem/stromal cells. The response to decidualization in cultures with progesterone and cAMP was evaluated by analyzing changes in cell morphology by microscopy, prolactin and IL-15 secretion by enzyme immunoassay and the induction of apoptosis by flow cytometry. In all four analyses, preDSCs showed a significantly higher response than preEnSCs. The expression of progesterone receptor (PR), protein kinase A (PKA) and FOXO1 was studied with Western blotting. Both types of cells showed similar levels of PR and PKA, but the increase in PKA RI subunit expression in response to decidualization was again significantly greater in preDSCs. We conclude that preEnSCs and preDSCs are equivalent cells but differ in their ability to decidualize. Functional differences between them probably derive from factors in their different milieus.

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Qi Li, Na Li, Hengwei Liu, Yu Du, Haitang He, Ling Zhang and Yi Liu

Endometriosis (EMs) is an estrogen (E2)-dependent inflammatory disorder. Although EMs is considered a benign disease, it presents with malignant characteristics, such as migration and invasion. An increasing number of studies have shown that aberrantly expressed circular RNAs (circRNAs) play an essential role in disease development and progression. However, the mechanisms by which circRNAs exert their pathological effects in EMs remain unclear. Hsa_circ_0001649, a novel cancer-associated circRNA, has been previously reported to be downregulated in several cancer types and related to cell migration and invasion. In the present study, real-time polymerase chain reaction (qRT-PCR) was carried out to measure hsa_circ_0001649 levels in human tissues, human primary endometrial stromal cells (ESCs) and a human endometrial stromal cell line (ThESCs). Matrix metalloproteinase 9 (MMP9) levels in ESCs and ThESCs were assessed by qRT-PCR and western blotting, and the migration and invasion capacities of ThESCs were evaluated by Transwell assay. As a result, hsa_circ_0001649 expression was significantly decreased in ectopic and eutopic endometrial samples compared with that in normal endometrial samples. E2 decreased hsa_circ_0001649 expression but increased MMP9 expression in ESCs and ThESCs. Furthermore, ThESCs were more invasive under E2 stimulation. However, these effects disappeared when ICI or hsa_circ_0001649 transfection was used. Collectively, our findings reveal that decreased hsa_circ_0001649 expression plays a role in E2-increased MMP9 expression through E2 receptors (ERs), which have critical functions in EMs.