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Keigo Nakamura, Kazuya Kusama, Atsushi Ideta, Kazuhiko Imakawa and Masatoshi Hori

Extracellular vesicles (EVs) present in uterine lumen are involved in conceptus-endometrial interactions during the pre-implantation period. Despite numerous studies conducted on interferon tau (IFNT), a major protein of maternal recognition of pregnancy, the effect of intrauterine EVs on the endometrium during pre-implantation periods has not been well-characterized. To characterize conceptus-derived intrauterine EVs independent of IFNT, transcripts found from RNA-seq analysis in RNAs extracted from primary bovine endometrial epithelial cells (EECs) treated with cyclic day 17 (C17) EVs, pregnant day 17 (P17) EVs or IFNT were analyzed. These analyses identified 82 transcripts uniquely induced by IFNT-independent P17 EVs, of which a large number of transcripts were associated with ‘the TNF signaling pathway’ and ‘Inflammatory response’. Moreover, high expression of CD40L, a member of the TNF superfamily, and its receptor CD40 were found in P17 EVs and in EECs, respectively. Furthermore, the expression of TNF signaling pathway-related genes was up-regulated by the treatment with P17 EVs, but these increases were down-regulated by NF-kB signaling inhibitor. These findings suggest that P17 EVs could induce a pro-inflammatory response in the endometrium, independent of IFNT, to regulate uterine receptivity, facilitating conceptus implantation.

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Paola Toschi, Emanuele Capra, Debora A Anzalone, Barbara Lazzari, Federica Turri, Flavia Pizzi, Pier A Scapolo, Alessandra Stella, John L Williams, Paolo Ajmone Marsan and Pasqualino Loi

The genotype of an organism is stable throughout its life; however, its epigenome is dynamic and can be altered in response to environmental factors, such as diet. Inheritance of acquired epigenetic modifications by the next generation occurs through the germline, although the precise mechanisms remain to be elucidated. Here, we used a sheep model to evaluate if modification of the maternal diet (CTR; control, UND: undernutrition; FA: undernutrition and folic acid supplementation) during the peri-conceptional period affects the genome-wide methylation status of the gametes of male offspring. Sperm DNA methylation, measured by Reduced Representation Bisulfite Sequencing (RRBS), identified Differentially Methylated Regions (DMR) in offspring that experienced in utero undernutrition, both in UND (244) and FA (240), compared with CTR. Gene ontology (GO) analysis identified DMRs in categories related to sperm function, therefore we investigated whether the fertilizing capacity of the semen from the three groups differed in an in vitro fertilization assay. Spermatozoa from the undernourished groups showed lower motility and sperm chromatin structure abnormalities, represented by a higher percentage of DNA fragmentation and an increased number of immature cells, compared with CTR. While good quality blastocysts were obtained from all three groups, the proportion of embryos reaching the blastocyst stage was reduced in the UND vs CTR, an effect partially rescued by the FA treatment. The data reported here show that nutritional stress during early pregnancy leads to epigenetic modifications in the semen of the resulting offspring, the effects of which in next generation remain to be elucidated.

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Inga Laezer, Sergio E Palma-Vera, Fan Liu, Marcus Frank, Nares Trakooljul, Andreas Vernunft, Jennifer Schoen and Shuai Chen

In mammals, around the time of ovulation, the hormonal profile dynamically changes in synchrony with reproductive events occurring in the oviduct, that is, sperm arrival, fertilization, and early embryo development. Extracellular vesicles (EVs) have been recently recognized as key components of the embryonic milieu; however, composition and function of oviductal EVs during this crucial period remains to be further explored. Therefore, we initially characterized EVs from porcine oviductal fluid specifically around the critical ovulation window: that is, estrus (E), late estrus (LE, day of expected ovulation), post ovulation (PO), and additionally diestrus (D). Total EV numbers gradually rose from D to E, LE and PO (P < 0.05), which corresponded to the total EV protein amount (P < 0.05). Strikingly, the mean size of EVs in PO was significantly smaller than in E and LE groups, which also had a lesser proportion of small EVs (P < 0.05). The EV protein cargoes during the periovulatory period were further analyzed by mass spectrometry. Qualitative analysis detected 1118 common proteins, which are most enriched in the cellular component of EVs/exosomes. Hierarchical clustering indicated similar protein profile within the biological replicates, but large discrepancy among stages. Further quantitative analysis discovered 34 and 4 differentially expressed proteins in the comparison between E and PO and in the comparison between E and LE, respectively. The dynamic EV protein profile together with the quick adaption in EV size and quantity suggests that porcine oviductal EV secretion are under the hormonal influence during the estrus cycle.

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Hafiza Khatun, Yasuhiko Wada, Toshihiro Konno, Hideki Tatemoto and Ken-ichi Yamanaka

We have previously reported that regulation of endoplasmic reticulum (ER) stress during in vitro culture acutely increases bovine embryo developmental rate and cryotolerance; these data indicate that ER stress is a critical factor reducing the quality of in vitro-produced embryos. In the current follow-up study, we examined whether ER stress attenuation during in vitro maturation influences meiotic maturation, oocyte quality, and subsequent embryonic development. Bovine cumulus oocyte complexes (COCs) derived from slaughterhouse ovaries were matured with or without tauroursodeoxycholic acid (TUDCA), a selective inhibitor of ER stress (0, 50, 100, and 200 µM) for 22 h followed by in vitro fertilization, and zygotes were cultured for 8 days. Of the different doses of TUDCA, 100 μM TUDCA significantly increased the maturation rate, and decreased reactive oxygen species in denuded oocytes, and appeared lower number of apoptotic cells in matured COCs. Subsequently, treatment of TUDCA (100 µM) decreased the localization and amount of GRP78/BIP protein level as well as ER stress (GRP78/BIP, PERK, IER1, ATF4, and XBP1) and apoptosis (CHOP and BAX)-related gene expression, while it increased the anti-apoptotic gene BCL2 level in matured COCs. Moreover, addition of TUDCA (100 µM) during IVM significantly improved the blastocyst formation rate (43.6 ± 1.8% vs 49.7 ± 1.3%) and decreased the number of apoptotic cells (7.7 ± 1.1% vs 5.03 ± 0.6%) in blastocysts. These findings suggest that the presence of ER stress during maturation impairs the developmental competence of bovine COCs and that this process can be reversed by TUDCA.

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Julieta Aylen Schander, Julieta Aisemberg, Fernando Correa, Manuel Luis Wolfson, Lorena Juriol, Cora Cymeryng, Federico Jensen and Ana María Franchi

Maternal lifestyle affects both mother health and pregnancy outcome in humans. Several studies have demonstrated that interventions oriented toward reducing stress and anxiety have positive effects on pregnancy complications such as preeclampsia, excessive gestational weight, gestational diabetes and preterm birth. In this work, we showed that the environmental enrichment (EE), defined as a noninvasive and biologically significant stimulus of the sensory pathway combined with voluntary physical activity, prevented preterm birth (PTB) rate by 40% in an inflammatory mouse model induced by the systemic administration of bacterial lipopolysaccharide (LPS). Furthermore, we found that EE modulates maternal metabolism and produces an anti-inflammatory environment that contributes to pregnancy maintenance. In pregnant mice uterus, EE reduces the expression of TLR4 and CD14 (the LPS receptor and its coactivator protein), preventing the LPS-induced increase in PGE2 and PGF2α release and nitric oxide synthase (NOS) activity. In cervical tissue, EE inhibits cervical ripening events, such as PGE2 release, matrix metalloproteinase (MMP)-9 increased activity and neutrophil recruitment, therefore conserving cervical function. It seems that EE exposure could mimic the stress and anxiety-reducing techniques mentioned above, explaining, at least partially, the beneficial effects of having a healthy lifestyle before and during gestation. Furthermore, we propose that designing an EE protocol for humans could be a noninvasive and preventive therapy for pregnancy complications, averting pre-term birth occurrence and dreaded sequelae that are present in the offspring born too soon.

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Y-F Liu, M-Y Li, Y-P Yan, W Wei, B Li, H-Y Pan, Z-M Yang and X-H Liang

Maintenance of a suitable uterine milieu is important for embryo development and subsequent implantation during early pregnancy. High estrogen level in proestrous and estrous stages is essential for uterine anti-bacterial activity during preimplantation period. Lipocalin-2 is an essential molecule which prevents bacterial infection by sequestering iron. In this study, the highest expression of lipocalin-2 is observed in the endometrial epithelium on day 1 of normal pregnancy and pseudopregnancy, which exhibit a similar hormone scenario. By injecting the agonists for estrogen receptor α and estrogen receptor β in ovariectomized mice, we found estrogen receptor α is the dominant member for estrogen regulation on lipocalin-2 expression. Estrogen treatment in estrogen receptor α-knockout mice further confirmed the role of estrogen receptor α. Using published data from whole-genome estrogen receptor α binding site assay, significant estrogen receptor α recruitment peaks are found at the downstream of lipocalin-2 gene after estrogen treatment. Furthermore, to study the anti-bacterial activity of lipocalin-2 in uterus, Escherichia coli is injected to mimic bacterial infection. Our results showed an obvious induction of lipocalin-2 in Escherichia coli-treated group. Taken together, this study indicates estrogen regulation of lipocalin-2 in uterine epithelium is mediated by estrogen receptor α, and lipocalin-2 may have anti-bacterial activity during early pregnancy.

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Elizabeth Soczewski, Esteban Grasso, Lucila Gallino, Vanesa Hauk, Laura Fernández, Soledad Gori, Daniel Paparini, Claudia Perez Leirós and Rosanna Ramhorst

Decidualization denotes the reprogramming of endometrial stromal cells that includes the secretion of different mediators like cytokines, chemokines, and the selective recruitment of immune cells. This physiological process involves changes in the secretome of the endometrial stromal cells leading to the production of immunomodulatory factors. The increased amount of protein secretion is associated with a physiological endoplasmic reticulum (ER) stress and the resulting unfolded protein response (UPR), allowing the expansion of ER and the machinery to assist the protein folding. Notably, the signaling pathways involved in the ER stress and the UPR are interconnected with the onset of a sterile inflammatory response, as well as with angiogenesis. Both of these processes have a key role in decidualization and placentation, therefore, alterations in them could lead to pregnancy complications. In this review, we will discuss how the induction of ER stress and the UPR processes that accompanies the decidualization are associated with embryo implantation and whether they might condition pregnancy outcome. The ER stress activates/triggers sensing proteins which, among others, induces kinase/RNAse-TXNIP expression, activating the NLRP3 inflammasome. This multiprotein system allows caspase-1 activation, which catalyzes the cleavage of the inactive IL-1β proform toward the mature secretory form, with pro-implantatory effects. However, the sterile inflammatory response should be later controlled in favor of a tolerogenic microenvironment to sustain pregnancy. In accordance, alterations of the ER stress and UPR processes can be reflected in recurrent implantation failures (RIF), recurrent pregnancy loss (RPL), or complications associated with deficient placentation, such as preeclampsia (PE).

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R John Aitken

Male and female germ lines are vulnerable to oxidative stress. In spermatozoa, such stress triggers a lipid peroxidation cascade that culminates in the generation of electrophilic lipid aldehydes that bind to DNA and a raft of proteins involved in the delivery of functionally competent cells. One set of targets for these aldehydes are the proteins of the mitochondrial electron transport chain. When this interaction occurs, mitochondrial ROS generation is enhanced leading to the sustained generation of oxidative damage in a self-perpetuating cycle. Such damage affects all aspects of sperm function including motility, sperm-egg recognition, acrosomal exocytosis and sperm-oocyte fusion. Oxidative stress in the male germ line also attacks the integrity of sperm DNA with potential impacts on the developmental capacity of embryos and the health and wellbeing of the offspring. Potential pathways of reactive oxygen species (ROS) generation in male germ cells could involve enhanced lipoxygenase activity, activation of NADPH oxidase and/or electron leakage from mitochondria. Similarly, in the female germ line, both the induction of oocyte senescence following ovulation and the deterioration of oocyte quality with maternal age appear to involve the generation of oxidative damage. In this case, the mitochondria appear to be a particularly important source of ROS compromising the viability and fertilizability of the oocyte and interfering with the normal segregation of chromosomes during meiosis. In light of these considerations, antioxidants should have some role to play in the preservation of reproductive function in both men and women; however, we still await appropriate trials to test this hypothesis.

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Luhan Yang, Claudia Baumann, Rabindranth De La Fuente and Maria M Viveiros

Accurate chromosome segregation relies on correct chromosome-microtubule interactions within a stable bipolar spindle apparatus. Thus, exposure to spindle disrupting compounds can impair meiotic division and genomic stability in oocytes. The endocrine disrupting activity of bisphenols such as bisphenol A (BPA) is well recognized, yet their damaging effects on spindle microtubules (MTs) is poorly understood. Here, we tested the effect(s) of acute exposure to BPA and bisphenol F (BPF) on assembled spindle stability in ovulated oocytes. Brief (4 h) exposure to increasing concentrations (5, 25, and 50 µg/mL) of BPA or BPF disrupted spindle organization in a dose-dependent manner, resulting in significantly shorter spindles with highly unfocused poles and fragmented pericentrin. The chromosomes remained congressed in an abnormally elongated metaphase-like configuration, yet normal end-on chromosome-MT attachments were reduced in BPF-treated oocytes. Live-cell imaging revealed a rapid onset of bisphenol-mediated spindle MT disruption that was reversed upon compound removal. Moreover, MT stability and regrowth were impaired in BPA-exposed oocytes, with few cold-stable MTs and formation of multipolar spindles upon MT regrowth. MT-associated kinesin-14 motor protein (HSET/KIFC1) labeling along the spindle was also lower in BPA-treated oocytes. Conversely, cold stable MTs and HSET labeling persisted after BPF exposure. Notably, inhibition of Aurora Kinase A limited bisphenol-mediated spindle pole widening, revealing a potential interaction. These results demonstrate rapid MT disrupting activity by bisphenols, which is highly detrimental to meiotic spindle stability and organization. Moreover, we identify an important link between these defects and altered distribution of key spindle associated factors as well as Aurora Kinase A activity.

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D Mattar, M Samir, M Laird and P G Knight

Angiogenesis plays an integral role in follicular and luteal development and is positively regulated by several intra-ovarian factors including vascular endothelial growth factor A (VEGFA) and fibroblast growth factor 2 (FGF2). Various transforming growth factor-β (TGF-β) superfamily members function as intra-ovarian regulators of follicle and luteal function, but their potential roles in modulating ovarian angiogenesis have received little attention. In this study, we used a bovine theca interna culture model (exhibiting characteristics of luteinization) to examine the effects of TGF-β1 and bone morphogenetic protein 6 (BMP6) on angiogenesis and steroidogenesis. VEGFA/FGF2 treatment promoted endothelial cell network formation but had little or no effect on progesterone and androstenedione secretion or expression of key steroidogenesis-related genes. TGF-β1 suppressed basal and VEGFA/FGF2-induced endothelial cell network formation and progesterone secretion, effects that were reversed by an activin receptor-like kinase 5 (ALK5) inhibitor (SB-431542). The ALK5 inhibitor alone raised androstenedione secretion and expression of several transcripts including CYP17A1. BMP6 also suppressed endothelial cell network formation under VEGFA/FGF2-stimulated conditions and inhibited progesterone secretion and expression of several steroidogenesis-related genes under basal and VEGFA/FGF2-stimulated conditions. These effects were reversed by an ALK1/2 inhibitor (K02288). Moreover, the ALK1/2 inhibitor alone augmented endothelial network formation, progesterone secretion, androstenedione secretion and expression of several steroidogenesis-related genes. The results indicate dual suppressive actions of both TGF-β1 and BMP6 on follicular angiogenesis and steroidogenesis. Further experiments are needed to unravel the complex interactions between TGF-β superfamily signalling and other regulatory factors controlling ovarian angiogenesis and steroidogenesis.