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Rhiannon E Lloyd, Raquel Romar, Carmen Matás, Alfonso Gutiérrez-Adán, William V Holt and Pilar Coy

In mammals, fertilization and early pre-implantation development occur in the oviduct. Previous results obtained in our laboratory have identified specific molecules in the oviduct that affect porcine sperm–egg interactions. The aim of the present study was to determine whether the contact between oocytes and oviductal fluid also affect embryo development, quality, and gene expression. In vitro matured porcine oocytes were exposed to bovine oviductal fluid (bOF) for 30 min prior to fertilization. Cleavage and blastocyst development rates were significantly higher from bOF-treated oocytes than from untreated oocytes. Blastocysts obtained from bOF-treated oocytes had significantly greater total cell numbers than those obtained from untreated oocytes. Using real-time PCR, grade 1 (very good morphological quality) and grade 2 (good morphological quality) blastocysts were analyzed for gene transcripts related to apoptosis (BAX, BCL2L1), mitochondrial DNA (mtDNA) transcription/replication (POLG, POLG2, and TFAM), blastomere connection and morula compaction (GJA1), and blastocyst formation and pluripotency (POU5F1). We found that the entire set of genes analyzed was differentially expressed between grade 1 and 2 blastocysts. Furthermore, bOF treatment reduced the ratio of BAX to BCL2L1 transcripts and enhanced the abundance of TFAM transcripts in grade 2 blastocysts. Not only do these findings demonstrate that factors within the bOF act on porcine oocytes both quickly and positively, but they also suggest that such factors could promote embryo development and quality by protecting them against adverse impacts on mtDNA transcription/replication and apoptosis induced by the culture environment.

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Serafín Pérez-Cerezales, Priscila Ramos-Ibeas, Dimitrios Rizos, Pat Lonergan, Pablo Bermejo-Alvarez and Alfonso Gutiérrez-Adán

Developmental plasticity enables the appearance of long-term effects in offspring caused by exposure to environmental stressors during embryonic and foetal life. These long-term effects can be traced to pre- and post-implantation development, and in both cases, the effects are usually sex specific. During preimplantation development, male and female embryos exhibit an extensive transcriptional dimorphism mainly driven by incomplete X chromosome inactivation. These early developmental stages are crucial for the establishment of epigenetic marks that will be conserved throughout development, making it a particularly susceptible period for the appearance of long-term epigenetic-based phenotypes. Later in development, gonadal formation generates hormonal differences between the sexes, and male and female placentae exhibit different responses to environmental stressors. The maternal environment, including hormones and environmental insults during pregnancy, contributes to sex-specific placental development that controls genetic and epigenetic programming during foetal development, regulating sex-specific differences, including sex-specific epigenetic responses to environmental hazards, leading to long-term effects. This review summarizes several human and animal studies examining sex-specific responses to environmental stressors during both the periconception period (caused by differences in sex chromosome dosage) and placental development (caused by both sex chromosomes and hormones). The identification of relevant sex-dependent trajectories caused by sex chromosomes and/or sex hormones is essential to define diagnostic markers and prevention/intervention protocols.

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Roldán-Olarte Mariela, Maillo Verónica, Sánchez-Calabuig María Jesús, Beltrán-Breña Paula, Rizos Dimitrios and Gutiérrez-Adán Alfonso

This study examines the impacts of the urokinase-type plasminogen activator (uPA) on the in vitro maturation (IVM) of bovine oocytes. Cumulus–oocyte complexes in IVM medium were treated with uPA, amiloride (an uPA inhibitor), dimethyl sulfoxide (DMSO) or left untreated (control group). After 24 h of IVM, oocytes were recovered for testing or were in vitro fertilized and cultured to the blastocyst stage. The factors examined in all groups were: (i) oocyte nuclear maturation (Hoëscht staining); (ii) oocyte cytoplasmic maturation (cortical granules, CGs, distribution assessed by LCA-FITC); (iii) oocyte and cumulus cell (CC) gene expression (RT-qPCR); and (iv) embryo development (cleavage rate and blastocyst yield). Oocytes subjected to uPA treatment showed rates of nuclear maturation and CG distribution patterns similar to controls (P > 0.05), whereas lower rates of oocyte maturation were recorded in the amiloride group (P < 0.05). Both in oocytes and CC, treatment with uPA did not affect the transcription of genes related to apoptosis, cell junctions, cell cycle or serpin protease inhibitors. In contrast, amiloride altered the expression of genes associated with cell junctions, cell cycle, oxidative stress and CC serpins. No differences were observed between the control and uPA group in cleavage rate or in blastocyst yield recorded on Days 7, 8 or 9 post-insemination. However, amiloride led to drastically reduced cleavage rate (28.5% vs 83.2%) and Day 9 embryo production (6.0% vs 21.0%) over the rates recorded for DMSO. These results indicate that the proteolytic activity of uPA is needed for successful oocyte maturation in bovine.

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Raúl Fernández-González, Juan de Dios Hourcade, Irene López-Vidriero, Alberto Benguría, Fernando Rodríguez De Fonseca and Alfonso Gutiérrez-Adán

We have reported that in vitro culture (IVC) of preimplantation mouse embryos in the presence of FCS produces long-term effects (LTE) on development, growth and behaviour of the offspring at adult age. To analyse the mechanisms underlying this phenomenon, we have examined development and global alterations in gene expression in the mouse blastocysts produced in the presence of FCS, conditions known to be suboptimal and that generate LTE. Embryos cultured in vitro in KSOM and in KSOM+FCS had a reduced number of cells in the inner cell mass at the blastocyst stage compared with in vivo derived embryos; however, only culture in KSOM+FCS leads to a reduction in the number of trophoblast cells. Gene expression levels were measured by comparison among three groups of blastocysts (in vivo, IVC in KSOM and IVC in KSOM+FCS). Different patterns of gene expression and development were found between embryos cultured in vitro or in vivo. Moreover, when we compared the embryos produced in KSOM versus KSOM+FCS, we observed that the presence of FCS affected the expression of 198 genes. Metabolism, proliferation, apoptosis and morphogenetic pathways were the most common processes affected by IVC. However, the presence of FCS during IVC preferentially affected genes associated with certain molecular and biological functions related to epigenetic mechanisms. These results suggest that culture-induced alterations in transcription at the blastocyst stage related to epigenetic mechanisms provide a foundation for understanding the molecular origin at the time of preimplantation development of the long-term consequences of IVC in mammals.

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Veronica Maillo, Maria Jesus Sánchez-Calabuig, Ricaurte Lopera-Vasquez, Meriem Hamdi, Alfonso Gutierrez-Adan, Patrick Lonergan and Dimitrios Rizos

The oviduct is a complex and organized thin tubular structure connecting the ovary with the uterus. It is the site of final sperm capacitation, oocyte fertilization and, in most species, the first 3–4days of early embryo development. The oviductal epithelium is made up of ciliary and secretory cells responsible for the secretion of proteins and other factors which contribute to the formation of the oviductal fluid. Despite significant research, most of the pathways and oviductal factors implicated in the crosstalk between gametes/early embryo and the oviduct remain unknown. Therefore, studying the oviductal environment is crucial to improve our understanding of the regulatory mechanisms controlling fertilization and embryo development. In vitro systems are a valuable tool to study in vivo pathways and mechanisms, particularly those in the oviducts which in livestock species are challenging to access. In studies of gamete and embryo interaction with the reproductive tract, oviductal epithelial cells, oviductal fluid and microvesicles co-cultured with gametes/embryos represent the most appropriate in vitro models to mimic the physiological conditions in vivo.

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Elina V García, Meriem Hamdi, Antonio D Barrera, María J Sánchez-Calabuig, Alfonso Gutiérrez-Adán and Dimitrios Rizos

Signaling components of bone morphogenetic proteins (BMPs) are expressed in an anatomically and temporally regulated fashion in bovine oviduct. However, a local response of this signaling to the presence of the embryo has yet to be elucidated. The aim of the present study was to evaluate if early embryo-oviduct interaction induces changes in the gene expression of BMP signaling components. For this purpose, we used an in vitro co-culture system to investigate the local interaction between bovine oviductal epithelial cells (BOEC) from the isthmus region with early embryos during two developmental periods: before (from the 2-cell to 8-cell stage) or during (from the 8-cell to 16-cell stage) the main phase of embryonic genome activation (EGA). Exposure to embryos, irrespective of the period, significantly reduced the relative abundance of BMPR1B, BMPR2, SMAD1, SMAD6 and ID2 mRNAs in BOEC. In contrast, embryos that interacted with BOEC before EGA showed a significant increase in the relative abundance of SMAD1 mRNA at the 8-cell stage compared to embryos cultured without BOEC. Moreover, embryos at the 16-cell stage that interacted with BOEC during EGA showed a significant increase in BMPR1B, BMPR2 and ID2 mRNA. These results demonstrate that embryo-oviduct interaction in vitro induces specific changes in the transcriptional levels of BMP signaling, causing a bidirectional response that reduces the expression levels of this signaling in the oviductal cells while increases them in the early embryo. This suggests that BMP signaling pathway could be involved in an early cross talk between the bovine embryo and the oviduct during the first stages of development.

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Enrique Gómez, Alfonso Gutiérrez-Adán, Carmen Díez, Pablo Bermejo-Alvarez, Marta Muñoz, Aida Rodriguez, Jesús Otero, María Alvarez-Viejo, David Martín, Susana Carrocera and José Néstor Caamaño

Parthenotes may represent an alternate ethical source of stem cells, once biological differences between parthenotes and embryos can be understood. In this study, we analyzed development, trophectoderm (TE) differentiation, apoptosis/necrosis, and ploidy in parthenotes and in vitro produced bovine embryos. Subsequently, using real-time PCR, we analyzed the expression of genes expected to underlie the observed differences at the blastocyst stage. In vitro matured oocytes were either fertilized or activated with ionomycin +6-DMAP and cultured in simple medium. Parthenotes showed enhanced blastocyst development and diploidy and reduced TE cell counts. Apoptotic and necrotic indexes did not vary, but parthenotes evidenced a higher relative proportion of apoptotic cells between inner cell mass and TE. The pluripotence-related POU5F1 and the methylation DNMT3A genes were downregulated in parthenotes. Among pregnancy recognition genes, TP-1 was upregulated in parthenotes, while PGRMC1 and PLAC8 did not change. Expression of p66 shc and BAX/BCL2 ratio were higher, and p53 lower, in parthenotes. Among metabolism genes, SLC2A1 was downregulated, while AKR1B1, PTGS2, H6PD, and TXN were upregulated in parthenotes, and SLC2A5 did not differ. Among genes involved in compaction/blastulation, GJA1 was downregulated in parthenotes, but no differences were detected within ATP1A1 and CDH1. Within parthenotes, the expression levels of SLC2A1, TP-1, and H6PD, and possibly AKR1B1, resemble patterns described in female embryos. The pro-apoptotic profile is more pronounced in parthenotes than in embryos, which may differ in their way to channel apoptotic stimuli, through p66 shc and p53 respectively, and in their mechanisms to control pluripotency and de novo methylation.

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Veronica Maillo, Celia de Frutos, Peadar O’Gaora, Niamh Forde, Gregory W Burns, Thomas E Spencer, Alfonso Gutierrez-Adan, Patrick Lonergan and Dimitrios Rizos

The aim of this study was to compare the transcriptome of the oviductal isthmus of pregnant heifers with that of cyclic heifers as well as to investigate spatial differences between the transcriptome of the isthmus and ampulla of the oviduct in pregnant heifers. After synchronizing crossbred beef heifers, those in standing oestrus (=Day 0) were randomly assigned to cyclic (non-bred, n=6) or pregnant (artificially inseminated, n=11) groups. They were slaughtered on Day 3 and both oviducts from each animal were isolated and cut in half to separate ampulla and isthmus. Each portion was flushed to confirm the presence of an oocyte/embryo and was then opened longitudinally and scraped to obtain epithelial cells which were snap-frozen. Oocytes and embryos were located in the isthmus of the oviduct ipsilateral to the corpus luteum. Microarray analysis of oviductal cells revealed that proximity to the corpus luteum did not affect the transcriptome of the isthmus, irrespective of pregnancy status. However, 2287 genes were differentially expressed (P<0.01) between the ampulla and isthmus of the oviduct ipsilateral to the corpus luteum in pregnant animals. Gene ontology revealed that the main biological processes overrepresented in the isthmus were synthesis of nitrogen, lipids, nucleotides, steroids and cholesterol as well as vesicle-mediated transport, cell cycle, apoptosis, endocytosis and exocytosis, whereas cell motion, motility and migration, DNA repair, calcium ion homeostasis, carbohydrate biosynthesis, and regulation of cilium movement and beat frequency were overrepresented in the ampulla. In conclusion, large differences in gene expression were observed between the isthmus and ampulla of pregnant animals at Day 3 after oestrus.

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Antonio D Barrera, Elina V García, Meriem Hamdi, María J Sánchez-Calabuig, Ángela P López-Cardona, Noelia Fonseca Balvís, Dimitrios Rizos and Alfonso Gutiérrez-Adán

During the transit through the oviduct, the early embryo initiates an extensive DNA methylation reprogramming of its genome. Given that these epigenetic modifications are susceptible to environmental factors, components present in the oviductal milieu could affect the DNA methylation marks of the developing embryo. The aim of this study was to examine if culture of bovine embryos with oviductal fluid (OF) can induce DNA methylation changes at specific genomic regions in the resulting blastocysts. In vitro produced zygotes were cultured in medium with 3 mg/mL bovine serum albumin (BSA) or 1.25% OF added at the one- to 16-cell stage (OF1–16), one- to 8-cell stage (OF1–8) or 8- to 16-cell stage (OF8–16), and then were cultured until Day 8 in medium with 3 mg/mL BSA. Genomic regions in four developmentally important genes (MTERF2, ABCA7, OLFM1, GMDS) and within LINE-1 retrotransposons were selected for methylation analysis by bisulfite sequencing on Day 7–8 blastocysts. Blastocysts derived from OF1–16 group showed lower CpG methylation levels in MTERF2 and ABCA7 compared with the BSA group. However, CpG sites within MTERF2, ABCA7 and OLFM1 showed higher methylation levels in groups OF1–8 and OF8–16 than in OF1–16. For LINE-1 elements, higher CpG methylation levels were observed in blastocysts from the OF1–16 group than in the other experimental groups. In correlation with the methylation changes observed, mRNA expression level of MTERF2 was increased, while LINE-1 showed a decreased expression in blastocysts from OF1–16 group. Our results suggest that embryos show transient sensitivity to OF at early stages, which is reflected by specific methylation changes at the blastocyst stage.

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Meriem Hamdi, María J Sánchez-Calabuig, Beatriz Rodríguez-Alonso, Sandra Bagés Arnal, Kalliopi Roussi, Roger Sturmey, Alfonso Gutiérrez-Adán, Patrick Lonergan and Dimitrios Rizos

During its journey through the oviduct, the bovine embryo may induce transcriptomic and metabolic responses, via direct or indirect contact, from bovine oviduct epithelial cells (BOECs). An in vitro model using polyester mesh was established, allowing the study of the local contact during 48 h between a BOEC monolayer and early embryos (2- or 8-cell stage) or their respective conditioned media (CM). The transcriptomic response of BOEC to early embryos was assessed by analyzing the transcript abundance of SMAD6, TDGF1, ROCK1, ROCK2, SOCS3, PRELP and AGR3 selected from previous in vivo studies and GPX4, NFE2L2, SCN9A, EPSTI1 and IGFBP3 selected from in vitro studies. Moreover, metabolic analyses were performed on the media obtained from the co-culture. Results revealed that presence of early embryos or their CM altered the BOEC expression of NFE2L2, GPX4, SMAD6, IGFBP3, ROCK2 and SCN9A. However, the response of BOEC to two-cell embryos or their CM was different from that observed to eight-cell embryos or their CM. Analysis of energy substrates and amino acids revealed that BOEC metabolism was not affected by the presence of early embryos or by their CM. Interestingly, embryo metabolism before embryo genome activation (EGA) seems to be independent of exogenous sources of energy. In conclusion, this study confirms that early embryos affect BOEC transcriptome and BOEC response was embryo stage specific. Moreover, embryo affects BOEC via a direct contact or via its secretions. However transcriptomic response of BOEC to the embryo did not manifest as an observable metabolic response.