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Bovine endometrium consists of epithelial and stromal cells that respond to conceptus interferon tau (IFNT), the maternal recognition of pregnancy (MRP) signal, by increasing expression of IFN-stimulated genes (ISGs). Endometrial epithelial and stromal-cell-specific ISGs are largely unknown but hypothesized to have essential functions during pregnancy establishment. Bovine endometrial epithelial cells were cultured in inserts above stromal fibroblast (SF) cells for 6 h in medium alone or with IFNT. The epithelial and SF transcriptomic response was analyzed separately using RNA sequencing and compared to a list of 369 DEGs recently identified in intact bovine endometrium in response to elongating bovine conceptuses and IFNT. Bovine endometrial epithelial and SF shared 223 and 70 DEGs in common with the list of 369 endometrial DEGs. Well-known ISGs identified in the epithelial and SF were ISG15, MX1, MX2, and OAS2. DEGs identified in the epithelial but not SF included a number of IRF molecules (IRF1, IRF2, IRF3, and IRF8), mitochondria SLC transporters (SLC25A19, SLC25A28, and SLC25A30), and a ghrelin receptor. Expression of ZC3HAV1, an anti-retroviral gene, increased specifically within the SF. Gene ontology analysis identified the type I IFN signaling pathway and activation of nuclear factor kappa B transcription factors as biological processes associated with the epithelial cell DEGs. This study has identified biologically relevant IFNT-stimulated genes within specific endometrial cell types. The findings provide critical information regarding the effects of conceptus IFNT on specific endometrial compartments during early developmental processes in cattle.Summary sentenceConceptus-induced, interferon-tau-dependent genes were identified within bovine endometrial epithelial and stromal fibroblast cells providing critical information regarding conceptus-induced modifications to specific endometrial compartments in cattle.

Background In post-partum dairy cows, the energy needs to satisfy high milk production induces a status of more or less pronounced Negative Energy Balance (NEB). NEB associated with fat mobilization impairs reproductive function. In a companion paper, we described constitutive gene expression in the three main endometrial cell types (stromal, glandular and luminal epithelial cells) isolated by laser capture micro-dissection (LCM) showing the specificities of their transcriptomic profiles. This study investigates the specific impact of NEB on gene expression in these cells around 80 days after parturition at day 15 of the oestrus cycle and describes their specific response to NEB. Results Following the description of their constitutive expression, the transcriptome profiles obtained by RNA sequencing of the three cells types revealed that differences related to the severity of NEB altered mainly specific patterns of expression related to individual cell types. Number of differentially expressed genes between severe NEB (SNEB) and mild NEB (MNEB) cows was higher in ST than in LE and GE, respectively. SNEB was associated with differential expression of genes coding for proteins involved in metabolic processes and embryo-maternal interactions in ST. Under-expression of genes encoding proteins with functions related to cell structure was found in GE whereas genes encoding proteins participating in pro-inflammatory pathways were over-expressed. Genes associated to adaptive immunity were under-expressed in LE. Conclusion The severity of NEB after calving is associated with changes in gene expression around 80 days after parturition corresponding to the time of breeding. Specific alterations in GEs are associated with activation of pro-inflammatory mechanisms. Concomitantly, changes in the expression of genes encoding proteins involved in cell interactions and maternal recognition of pregnancy takes place in ST. The combination of these effects possibly altering the uterine environment and embryo maternal interactions may negatively influence the establishment of pregnancy.

Background A number of studies have examined mRNA expression profiles of bovine endometrium at estrus and around the peri-implantation period of pregnancy. However, to date, these studies have been performed on the whole endometrium which is a complex tissue. Consequently, the knowledge of cell-specific gene expression, when analysis performed with whole endometrium, is still weak and obviously limits the relevance of the results of gene expression studies. Thus, the aim of this study was to characterize specific transcriptome of the three main cell-types of the bovine endometrium at day-15 of the estrus cycle. Results In the RNA-Seq analysis, the number of expressed genes detected over 10 transcripts per million was 6622, 7814 and 8242 for LE, GE and ST respectively. ST expressed exclusively 1236 genes while only 551 transcripts were specific to the GE and 330 specific to LE. For ST, over-represented biological processes included many regulation processes and response to stimulus, cell communication and cell adhesion, extracellular matrix organization as well as developmental process. For GE, cilium organization, cilium movement, protein localization to cilium and microtubule-based process were the only four main biological processes enriched. For LE, over-represented biological processes were enzyme linked receptor protein signaling pathway, cell-substrate adhesion and circulatory system process. Conclusion The data show that each endometrial cell-type has a distinct molecular signature and provide a significantly improved overview on the biological process supported by specific cell-types. The most interesting result is that stromal cells express more genes than the two epithelial types and are associated with a greater number of pathways and ontology terms.

The dissection of the veins is the trickiest step of Uterine transplantation (UTx). Performing the anastomosis of a single uterine vein could bring a therapeutic benefit and simplification of surgery and serve for managing unilateral venous thromboses. The objectives of this project were to evaluate the expression of early markers of ischemia-reperfusion and to compare findings following one or two vein anastomoses. Orthotopic uterine auto-transplantations were performed on an ovine model with anastomosis of either two (group 1) or one utero-ovarian veins (group 2). Blood gases, histology and ischemia- reperfusion markers transcripts (PTGS2, IL6, IL8, SOD2, C3, BAX/BCL2 and TLR4) were analyzed as well as PTGS2 protein expression using Western Blot and fluorescence immunolocalization on endometrial biopsies after 3h of reperfusion. Ten ewes were included in the experimentation, 4 were in group1, 3 in group 2, the others being sham operated controls. No significant differences were observed between the two phenotypes. Based on these results, the anastomosis of one single uterine vein appears to be an approach consistent with short-term graft survival. Further experiments will be needed to confirm the reliability of this approach, especially the long-term follow-up of the uterine graft including its ability to support gestation to term.

Background: In postpartum dairy cows, the energy needs to satisfy high milk production induces a more or less pronounced Negative Energy Balance (NEB) status. NEB associated with fat mobilization impairs reproductive function. This study investigated the specific impact of NEB on gene expression in the three main types of endometrial cells at time planned for insemination and implantation. Endometrial cell types (stromal, glandular and luminal epithelial cells) were isolated by laser micro-dissection allowing the study of constitutive gene expression and their specific response to NEB. Methods: Nine Swedish Red cows receiving a control diet or a mild restricted diet to induce differences of energy balance were categorized into mild (MNEB, n = 5) and severe negative energy balance (SNEB, n = 4). The three endometrial cell types: luminal (LE), glandular (GE) epithelium and stroma (ST) were collected by laser microdissection from endometrial biopsies performed at 80 days postpartum. Results: Transcriptome profiles obtained by RNA sequencing revealed differences in constitutive gene expression between the three cells types and also differences in specific responses related to the severity of NEB. Number of differentially expressed genes between SNEB and MNEB cows was higher in ST than in LE and GE, respectively. SNEB was associated with differential expression of genes related to metabolic processes and embryo-maternal interactions in ST. Under-expression of genes related to cell structure was found in GE whereas genes related to pro-inflammatory pathways were over-expressed. Genes associated to adaptive immunity were under-expressed in LE. Conclusion: The three different main cells types of the endometrium, have very different patterns of gene expression. The severity of NEB after calving is associated with changes in gene expression at time of breeding. Specific alterations in GEs are associated with activation of pro-inflammatory mechanisms. Concomitantly, changes in the expression of genes related to cell to cell interactions and maternal recognition of pregnancy takes place in ST. The combination of these effects possibly altering the uterine environment and embryo maternal interactions may negatively influence the establishment of pregnancy.

Over the last decades, fertility of dairy cows has declined due to selection strategies focusing on milk yield. To study the effect of genetic merit for fertility on the proteome of the bovine uterine luminal fluid, Holstein heifers with low- and two groups of heifers with high-fertility index (high-fertility Holstein and Montbéliarde) were investigated. To focus on the maternal effect, heifers from all groups were synchronized and received on Day 7 high-quality embryos. Uterine luminal fluid from Day 19 pregnant heifers was analyzed in a holistic proteomic approach using nano-LC-MS/MS analysis combined with a label-free quantification approach. In total, 1737 proteins were identified, of which 597 differed significantly in abundance between the three groups. The vast majority of proteome differences was found comparing both high-fertility groups to the low-fertility Holstein group, showing that the genetic predisposition for fertility is prevalent regarding the uterine luminal fluid proteome. Evaluation of this dataset using bioinformatic tools revealed an assignment of higher abundant proteins in low-fertility Holstein to several metabolic processes, such as vitamin metabolic process, which comprises folate receptor alpha (FOLR1) and retinol-binding protein, indicating an involvement of disturbed metabolic processes in decreased fertility. Moreover, immune system-related proteins — lactotransferrin and chromogranin A — were enriched in low-fertility cows together with interferon tau 3 h and interferon tau-2. Our results indicate that the genetic merit for fertility leads to substantial quantitative differences at the level of proteins in uterine fluid of pregnant animals, thus altering the microenvironment for the early conceptus.

This study investigated bovine conceptus-induced modifications to the endometrial transcriptome related to effects of interferon tau (IFNT), conceptus origin (in vivo vs. in vitro), and conceptus sex. In vitro (IVF) or in vivo (superovulation and artificial insemination, AI) produced blastocysts were transferred into recipient heifers on day 7 of the estrous cycle. On day 15, IVF- or AI-derived conceptuses were obtained by uterine flushing and individually placed on endometrial explants in media for 6 h. Explants were also cultured with media alone as a control or media containing 100 ng/mL IFNT. Total explant RNA was analyzed by RNA-Seq. Incubation of endometrium with IFNT or IVF- or AI-derived conceptuses changed (P ≤ 0.001) expression of 491, 498, and 576 transcripts, respectively, compared to the control. Further, 369 differentially expressed genes (DEGs) were common between explants exposed to IFNT or a conceptus. A total of 240 DEGs were uniquely altered by conceptuses (IVF- and AI-derived) but not IFNT. Of these transcripts, 46 were shared between the IVF and AI groups, while 61 and 133 were specific to IVF and AI conceptuses, respectively. Five genes [melanophilin (MLPH), prominin-2 (PROM2), myeloid associated differentiation marker (MYADM), vomeronasal 1 receptor 4 like (VN1R4L) and 5-hydroxytryptamine receptor 1A (HTR1A)] were more abundant in endometrium exposed to female compared to male conceptuses (P < 0.001). A single gene [ADP-ribosylation factor like GTPase 4C (ARL4C)] was more abundant in response to male conceptuses (P < 0.001) than female conceptuses. These data support the hypothesis that conceptus regulation of gene expression in the endometrium is complex and involves factors other than IFNT that may have a biological role in pregnancy establishment.Summary SentenceElongating bovine conceptuses modify the adjacent endometrial transcriptome in a manner dependent and independent of interferon tau and related to conceptus origin and sex.

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Highlights presented at the congress of the European Society for Reproductive Immunology in Budapest (Hungary), March 29 - April 01, 2014 The authors are grateful to the technical staff and former students for their valuable contributions to this topic. The authors thank Dr. Michel Guillomot (INRA, Jouy-en-Josas) for the gift of the microphotography; International audience; In terms of contribution to pregnancy, the mother not only produces gametes, but also hosts gestation, whose progression in the uterus is conditioned by early events during implantation. In ruminants, this period is associated with elongation of the extra-embryonic tissues, gastrulation of the embryonic disk and cross-talk with the endometrium. Recent data have prompted the need for accurate staging of the bovine conceptus and shown that asynchrony between elongation and gastrulation processes may account for pregnancy failure. Data mining of endometrial gene signatures has allowed the identification of molecular pathways and new factors regulated by the conceptus (e.g. FOXL2, SOCS6). Interferon-tau has been recognised to be the major signal of pregnancy recognition, but prostaglandins and lysophospholipids have also been demonstrated to be critical players at the conceptus-endometrium interface. Interestingly, up-regulation of interferon-regulated gene expression has been identified in circulating immune cells during implantation, making these factors a potential source of non-invasive biomarkers for early pregnancy. Distinct endometrial responses have been shown to be elicited by embryos produced by artificial insemination, in vitro fertilisation or somatic cell nuclear transfer. These findings have led to the concept that endometrium is an early biosensor of embryo quality. This biological property first demonstrated in cattle has been recently extended and associated with embryo selection in humans. Hence, compromised or suboptimal endometrial quality can subtly or deeply affect embryo development, with visible and sometimes severe consequences for placentation, foetal development, pregnancy outcome and the long-term health of the offspring.

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In vivo and in vitro studies showed that E. coli LPS and BoHV4 often cooperate to induce endometritis. In this study, we used an in vitro model to characterize their effects on proliferation and death of bovine endometrial epithelial cells (EC). Bovine uteri from luteal phase cows (CL histology) were collected from slaughterhouse and EC primary culture established. EC from 9 cows, on passages 4 to 6 were challenged with 0(Control, C) 2, 4, 8, 12, or 16 µg/ml LPS. The numbers of attached cells were counted 72 h later. Variation in cell number (%) = (number of LPS treated-number C)/number C, was analyzed by ANOVA (SAS 9.1, PROCGLM, 3classes of cells number ( 10 x 105), 5 classes of LPS. EC from 2 cows were exposed for 5 days to dilutions (10-6 to 10-1) of one BoHV4 strain and the variation in cell number analyzed. LPS strongly stimulated proliferation of endometrial epithelial cells without increasing cell death. LPS effect was dose dependent but not in a linear manner; the maximum stimulation (+20% to 40%) being observed for the 8 µg/ml dosage (p < 0.0001). Exposure of EC to BoHV4 induced cell death, the LD50 being observed for the 10-4 to 10"3 dilutions and no or

Remerciements à l'Unité UCEA de Bressonvilliers, à V. Gelin et A. Neveu pour leur aide.; Apoptotic activity is a common physiological process which culminates at the blastocyst stage in the preimplantation embryo of many mammals. The degree of embryonic cell death can be influenced by the oocyte microenvironment. However, the prognostic significance of the incidence of apoptosis remains undefined. Prostaglandin E2 (PGE2) derived from prostaglandin G/H synthase-2 (PTGS2) activity is a well-known prosurvival factor that is mainly studied in oncology. PGE2 is the predominant PTGS2-derived prostaglandin present in the oocyte microenvironment during the periconceptional period. Using an in vitro model of bovine embryo production followed by transfer and collection procedures, we investigated the impact of periconceptional PGE2 on the occurrence of spontaneous apoptosis in embryos and on subsequent in vivo posthatching development. Different periconceptional PGE2 environments were obtained using NS-398, a specific inhibitor of PTGS2 activity, and exogenous PGE2. We assessed the level of embryonic cell death in blastocysts at day 8 postfertilization by counting total cell numbers, by the immunohistochemical staining of active caspase-3, and by quantifying terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling signals and apoptosis regulator (BCL-2/BAX) mRNA expression. Morphometric parameters were used to estimate the developmental stage of the embryonic disk and the extent of trophoblast elongation on day 15 conceptuses. Our findings indicate that periconceptional PGE2 signaling durably impacts oocytes, conferring increased resistance to spontaneous apoptosis in blastocysts and promoting embryonic disk development and the elongation process during preimplantation development.

E. coli is one of the most frequently involved bacteria in uterine diseases. Lipopolysaccharide (LPS) is a component of the outer membrane of Gram-negative bacteria involved in pathogenic processes leading to post-partum metritis and endometritis in cattle. It also causes inflammation of the endometrium. The increase of cell proliferation by LPS is part of the inflammatory process. The aim of this study was to investigate possible changes in protein expression in relation to the proliferative response of bEECs after challenge with E. coli-LPS. In vitro culture of bEECs was performed from cow genital tracts collected at a slaughterhouse. In passage 5, bEECs from each of 9 cows (3 series of 3 cows) were exposed to 0, 8, and 16 μg ml(-1) LPS for 72 h. At time 0 and 72 h later, attached cells/living cells were counted and for each time and LPS dosage, cells were frozen for proteomic analyses. All samples from the 3 series were analyzed by 2-D gel electrophoresis coupled to MALDI-TOF/TOF mass spectrometry. The samples from the first series were subjected to shotgun nLC-MS/MS analysis. From the whole differential proteomics analysis, 38 proteins were differentially expressed (p < 0.05 to p < 0.001) following exposure to LPS. Among them, twenty-eight were found to be up-regulated in the LPS groups in comparison to control groups and ten were down-regulated. Differentially expressed proteins were associated with cell proliferation and apoptosis, transcription, destabilization of cell structure, oxidative stress, regulation of histones, allergy and general cell metabolism pathways. The de-regulations induced by LPS were consistent with the proliferative phenotype and indicated strong alterations of several cell functions. In addition, some of the differentially expressed proteins relates to pathways activated at the time of implantation. The specific changes induced through those signals may have negative consequences for the establishment of pregnancy.

In the present study, we examined the lysophosphatidic acid (LPA) pathway in the ovine uterus during the estrous cycle and early pregnancy. With the use of quantitative reverse transcription PCR, expression of LPAR1 and LPAR3 was analyzed. Both receptors were present in the ovine uterus. Immunolocalization showed that LPAR1 was mainly present in the stroma of the ovine endometrium, whereas LPAR3 was mostly restricted to epithelial compartments. In luminal and glandular epithelia, LPAR1 and LPAR3 levels were affected by pregnancy status, day, or the day-by-status interaction, whereas in stroma the receptors were not modified. Analysis of the whole endometrium from ovariectomized ewes showed that the expression of LPAR3 but not LPAR1 was regulated by the administration of progesterone. However, the examination of receptors at cellular levels showed that progesterone increases LPAR1 and LPAR3 in glandular epithelium and, in a minor extent, in endometrial stroma. Emerging evidence suggests that LPA is an essential component in the estrous cycle and early pregnancy regulation. We demonstrated that LPA induced stress fiber formation in ovine uterine epithelial cells, suggesting that LPA may be involved in cytoskeleton reorganization occurring cyclically in ovine uterus.

Full papers will be published in Recent Advances in Animal Nutrition – 2008 Nottingham University Press

We investigated the lysophosphatidic acid (LPA) pathway during early pregnancy in sheep. LPA was detected in the uteri of early-stage pregnant ewes. Using quantitative RT-PCR, the expression of autotaxin, the LPA-generating enzyme, was found in the endometrium and conceptus. In the latter autotaxin, transcript levels were low on d 12–14 and increased on d 15–16, in parallel with the level of LPA. Autotaxin was localized in the luminal epithelium and superficial glands of the endometrium and in trophectoderm cells of the conceptus. The expression of G protein-coupled receptors for LPA was also examined in the ovine conceptus. LPA receptor LPAR1 and LPAR3 transcripts were expressed during early pregnancy and displayed a peak on d 14, whereas the highest level of protein for both receptors was observed at d 17. LPAR1 was localized in cellular membranes and nuclear compartments of the trophectoderm cells, whereas LPAR3 was revealed only in membranes. LPA activated phosphorylation of the MAPK ERK1/2 in ovine trophectoderm-derived cells. Moreover, the bioactive lipid increased the proliferation of trophectoderm cells in culture, as shown by thymidine and bromodeoxyuridine incorporation. Furthermore, LPA induced changes to the organization of β-actin and α-tubulin, suggesting a role for it in rearrangement of trophectoderm cells cytoskeleton. Because a link had previously been established between prostaglandin and LPA pathways, we analyzed the effect of LPA on prostaglandin synthesis. LPA induced an increase in the release of prostaglandin F2α and prostaglandin E2, with no significant modifications to cytosolic phospholipase A2α and prostaglandin synthase-2 expression. Taken together, our results suggest a new role for LPA-mediated signaling in the ovine conceptus at the time of implantation.Lysophosphatidic acid (LPA) receptor 1 (R1) and LPAR3 mediate signaling of lysophosphatidic acid produced by autotaxin and induce prostaglandin biosynthesis and cytoskeleton changes in ovine trophectoderm cells at implantation time.

Prostaglandin E2(PGE2) and progesterone appear to be critical mediators of cumulus expansion and the resumption of oocyte meiosis. The aim of this study was to identify the types of prostaglandin E synthase (PTGES) expressed in the bovine cumulus–oocyte complex (COC), to characterize their temporal expression during the periconceptional interval using anin vitromodel of maturation (IVM) and fertilization (IVF), and to compare their expression with the level of steroidogenic gene expression. Real-time RT-PCR analysis revealed that enzymes related to the PGE2biosynthesis pathway were mainly expressed during IVM. Transcripts encoding PTGES1–3 were detected in bovine COCs. Only the expression of PTGES1 significantly increased during IVM whereas that of PTGES2 and PTGES3 remained unchanged. The induction of PTGES1 expression paralleled the induction of prostaglandin G/H synthase-2 (PTGS2) expression and the amounts of PGE2secreted by maturing COCs. Concomitantly, cholesterol side chain cleavage cytochrome P450 expression was significantly upregulated in maturing COCs and the high level of expression persisted in fertilized COCs. The expression of the StAR protein remained constant during IVM and then decreased significantly during IVF. Expression of the progesterone catabolic-related enzyme, 20α-hydroxysteroid dehydrogenase significantly decreased throughout the periconceptional interval. This was associated with a rising level of progesterone released by COCs in the culture media. In conclusion, our results suggest that the periconceptional differentiation of the bovine COC includes the transient induction of PGE2biosynthetic activity via the PTGS2/PTGES1 pathway during the maturation period and the increasing ability to produce progesterone from the immature to the fertilized stages.

Following hatching, pre-elongated conceptuses undergo elongation by intense proliferation, until implantation. We investigated the changes in gene expression associated with these physiological events using human cDNA arrays containing 2370 known genes. Comparison of pre-elongated, elongated, and implanting trophoblasts allowed the determination of 313 expressed genes, 63 of which were differentially regulated. These were classified into four functional families. Pre-elongated trophoblasts were characterized by preferential expression of genes involved in protein trafficking, whereas only latter developmental stages expressed cell signaling genes and receptors. Among the 63 developmentally regulated genes, four exhibited the highest levels of expression (TMSB10, CTNNA1, NMP1, and CX3CL1). Each of these also represents a functional family and display a specific expression pattern. One of them, CX3CL1 (CX3C chemokine, also known as fractalkine), is a chemokine that seems to have potential importance in trophoblast development, and which deserves further clarification of its role in implantation.

Mitochondria have a broad range of functions that affect reproduction, and structural as well as quantitative variation in mtDNA has been associated with gamete quality and reproductive success. To investigate the mitochondria effect on in vitro embryo production, we collected oocytes by ultrasound-guided follicular aspiration from donor cows known to differ in the developmental capacity, measured by the blastocyst formation rate, of their oocytes. To evaluate the potential effects of mtDNA and mitochondrial function on oocyte quality, the donor cows' mtDNA control region was sequenced and, after pairwise comparisons of polymorphisms, animals were grouped into two major haplogroups. The number of mtDNA molecules per oocyte was quantified by real-time PCR, and the adenosine triphosphate (ATP) content was measured in each oocyte to identify variations between haplogroups. Overall, ATP stocks in oocytes of the two haplogroups differed significantly (P < 0.05; means +/- SEM) both at the germinal vesicle and metaphase II stages (2.8 +/- 0.06 pmol vs. 2.6 +/- 0.07 pmol and 2.9 +/- 0.1 pmol vs. 2.3 +/- 0.06 pmol, respectively). The proportion of development to blastocyst was significantly different between haplogroups (22.3 +/- 2.1 % vs. 36.7 +/- 2.9 %). The number of mtDNA molecules per oocyte was highly variable (377 327 +/- 14 104, ranging from 2.0 x 10(3) to 1.2 x 10(6)) but not significantly different between the two haplogroups; significant differences were observed between animals without any apparent relationship to blastocyst production. These data suggest that mitochondria and mtDNA haplogroup affect the developmental capacity of bovine oocytes in vitro.

IGF system expression has been largely explored in the bovine follicular wall whereas it remains poorly studied in the COC. Using semi-quantitative RT-PCR and Western blot analysis, we have investigated spatial and temporal expression of IGF-1, IGFR-1, IGFBP-2, IGFBP-4, as well as gonadotropin receptors in bovine COC during oocyte maturation. In addition, we have compared changes in the IGF system and FSHR expression during in vitro maturation in TCM199 alone or in the presence of 10 ng/ml of EGF. The transcripts for IGFR-1 and IGFBP-2 were detected in cumulus and germinal cells whereas IGF-1, IGFBP-4 and FSHR mRNA were restricted to cumulus cells. Topography of the IGF system and gonadotropin receptor expression within COC were unaffected by the maturation step. In contrast, levels of IGFBP-2 and FSHR expression decreased (P < 0.05) in matured COC. Under defined culture conditions, IGFBP-2 and FSHR mRNA expression remained at a high level in TCM199 alone and were significantly reduced (P < 0.05) in the presence of 10 ng/ml EGF after a 24 h period of in vitro maturation. In conclusion, our results demonstrate a cell-specific pattern of IGF system member gene expression within bovine COC suggesting interaction between the somatic and germinal compartments. In addition, synchronized changes in the pattern of COC IGFBP-2 and FSHR expression during oocyte maturation suggest possible synergistic actions between IGF-1 and FSH.

The mechanisms that lead to the onset of human parturition are still unknown, although selected critical factors have been identified. To investigate the changes in myometrial gene expression associated with parturition, we used two macroarrays each containing 1176 different complementary human cDNA clones. Methods involving hierarchical clustering and conventional statistical analysis allowed us to generate a profile of genes expression at three stages of late pregnancy: preterm (29 wk amenorrhea); full term, not in labor (38 wk amenorrhea); and full term in labor (39 wk amenorrhea). Only 4% of the genes investigated were differentially expressed between the preterm and term groups (P < 0.05). These genes could be clustered as groups of either down-regulated or up-regulated transcripts. The changes in transcript abundance were particularly marked between the preterm and term stages of gestation, whereas the differences between term not in labor and term in labor were less pronounced. The parturition was characterized by a massive down-regulation of a large panel of developmental, cell adhesion molecule and proliferation-related genes, along with the up-regulation of inflammatory, contraction and apoptosis associated genes. We propose that the mechanisms of parturition consist primarily in the arrest of the processes of myometrial development, a step that might be essential to allow the uterus to recover appropriate contractile function before delivery.

Although numerous studies have examined global mRNA expression in the whole endometrium, very few studies aimed at characterising the global mRNA profile at specific cellular types within this tissue. In this study, we hypothesised that metabolic status during the postpartum period can affect specifically gene expression of endometrial luminal, glandular epithelial and stromal cells. To achieve this, endometrial biopsies were performed in 22 postpartum cows receiving either a control diet (n = 12, aiming for 35 kg/d of energy-corrected milk, ECM) or a mild restricted diet (n = 10, aiming for 25 kg/d of ECM). The diet affected the metabolic status of the cows as documented in Ntallaris et al. (2017 Theriogenology 90, 276-283). Three endometrial biopsies were collected for each cow between 12 and 15 days post-oestrus (oestrus was synchronized with CIDR and all cows had progesterone concentrations >5 ng mL−1 at time of biopsy). Biopsies were frozen in Optimal Cutting Temperature compound and then 10-µm sections were obtained by using cryostat. Slides were fixed in ethanol and stained using cresol violet. Luminal and glandular epithelial cells as well as stromal cells were harvested by laser capture microdissection using the PixCell II LCM System. Following capture, mRNA was extracted from each cellular types using the Arcturus PicoPure RNA Isolation (Thermo Fisher Scientific, Waltham, MA, USA). A genome-wide transcriptomic profile of endometrial cells from biopsies was generated with RNAseq. Samples were sequenced in 50-length paired. The average number of reads across all samples was near 100 million. Reads were mapped on the UMD3.1 assembly of the Bos taurus genome using Tophat. Quantification of gene expression has been performed using HTSeq with annotations from Ensembl. The comparisons of interest have been performed using DESEqn 2 (R package) with the statistical method proposed by Love et al. (2014 Genome Biol. 15, 550). With an adjusted P-value of 0.05, 8, 571, and 2658 genes were found significantly differentially expressed between animals fed with low or control diets for glandular, stromal, and luminal cells respectively. These results provide novel insights into mechanisms regulating endometrium physiology in postpartum dairy cows. The number of differentially expressed genes according to diet was much higher in luminal epithelial and stromal cells than in glandular cells providing evidence that these cells express differential responses to the metabolic environment to which they are exposed. Therefore, transcriptomic analyses based only on full tissue, as possibly biased by differences in cell numbers of different types could be not fully relevant. We acknowledge the financial support of ‘Prolific’ (EU grant KBBE; 31176) and RMUTSV (Thailand)

Prostaglandin D2 synthase (PGDS) is a major epididymal secretory protein in several species. We quantified PGDS in ram and bull semen using a specific antiserum. Strong variations in PGDS concentration existed between animals. In the bull, the highest concentrations were found preferentially in animals with normal or high fertility, as was previously suggested. However, low concentrations were found in males with all ranges of fertility, suggesting that the function of PGDS either is not necessary for male fertility or can be assumed by other proteins when its concentration is low. In the ram and stallion, cDNA and deduced protein sequences of PGDS were obtained by reverse transcription-polymerase chain reaction and showed that PGDS possessed the sequences involved in the three-dimensional folding characteristic of the lipocalin family and a cysteine at position 65 that is involved in the enzymatic activity. The enzymatic activity of PGDS was estimated in the ram by in vitro incubation of epididymal-isolated tubules with radioactive arachidonic acid. Prostaglandin (PG) D2 represented approximately 10% of the PGs produced in the lumen, irrespective of the presence or absence of luminal PGDS, suggesting that this protein is not involved in PGD2 biosynthesis. These results were corroborated by the absence of conversion of PGH2 to PGD2 when epididymal fluids were incubated with PGH2. In the rat, inhibition of PG biosynthesis in vivo by nonsteroidal anti-inflammatory drugs for 60 days did not change spermatozoa mobility or male fertility. It is likely that PGDS, which has a structure similar to that of lipocalin, functions as a lipophilic carrier protein, because we have shown that epididymal PGDS binds retinoic acid and testosterone in vitro.

Ovine endometrium showed transient expression of high concentrations of the inducible isoform of cyclooxygenase, cyclooxygenase 2 (COX-2), whereas the constitutive isoform, cyclooxygenase 1 (COX-1), was expressed at much lower concentrations and did not change. In this study, the pattern of prostaglandin synthesis in endometrial luminal cells was investigated in relation to their COX-2 content. Endometrial cells from cyclic or pregnant ewes at days 9, 12, 14 and 16 were isolated and analysed for the presence of COX-1 and COX-2 proteins using western blot analysis. Freshly isolated cells were incubated with 0.5 microCi [3H]arachidonic acid ml-1. Radioactive cyclooxygenase metabolites were analysed by reverse-phase HPLC. Luminal cells produced mainly PGF2 alpha, PGE2, PGD2 and 13,14-dihydro-15-keto PGF2 alpha and to a lesser extent 6-keto PGF1 alpha, thromboxane B2 and 13,14-dihydro-15-keto PGE2. The production of PGE2 and PGD2 was proportional to the cellular concentration of COX-2. PGE2 and PGD2 release was low on day 9 when COX-2 was not expressed, whereas high concentrations of PGE2 and PGD2 were synthesized on days 12-14 when COX-2 was highly expressed, reaching 100 ng microgram-1 cellular protein. In contrast, the basal production of PGF2 alpha did not appear to be related to COX-2 concentration and was greatest on day 16. Moreover, the release of PGF2 alpha was maintained at steady state values between days 9 and 14 by the production of 13,14-dihydro-15-keto PGF2 alpha. Although PGF2 alpha output was lower at day 16 of pregnancy compared with the oestrous cycle, no difference was observed in the pattern of prostaglandin synthesis between pregnant and non-pregnant ewes.

In this study we investigated expression of the two isoforms of the prostaglandin-forming enzyme, cyclooxygenase-1 (Cox-1) and cyclooxygenase-2 (Cox-2), in sheep embryos. Using Western blot and immunohistochemical analyses, we demonstrated that Cox-2 was highly expressed in embryos from Day 8 to Day 17 of development whereas Cox-1 was undetectable during this time. The expression of Cox-2 was developmentally regulated. It was maximal between Days 14 and 16. There was a 30-fold increase in Cox-2 content per protein extract between Day 10 and Day 14, corresponding to a 50,000-fold increase in the whole embryo. The expression of Cox-2 declined after Day 16 to become undetectable by Day 25 of pregnancy. Cox-2 was localized in the trophoblastic cells and was not detected in the inner cell mass. The [3H]arachidonic acid metabolites synthesized by Cox-2-rich conceptuses were analyzed by HPLC after short-term embryo culture. Day 14 conceptuses released mainly cyclooxygenase metabolites and to a lesser extent lipoxygenase derivatives. Cyclooxygenase products were 6-keto-prostaglandin (PGF)1alpha 18.2% (+/- 4.2), thromboxane-B2 22.51% (+/- 15.9), PGF2alpha 21% (+/- 11), PGE2 14.5% (+/- 7.4), and PGD2 2.7% (+/- 2.6). Taken together, these results suggest an important role for the Cox-2-dependent cyclooxygenase metabolites during embryo development.

To establish parameters predicting the quality of bovine oviduct epithelial cell-conditioned media, we compared media conditioned by oviduct cells from cows at Day 2 (n = 3) and Day 15 (n = 3) of the estrous cycle. In addition, we tested the influence of time of conditioning. Media were evaluated for their embryotrophic activity using a cumulus cell co-culture system as a control. The same media were tested for their mitogenic activity on NIH 3T3 cells and for chemical parameters, including total protein, and de novo synthesized protein as well as for concentrations of glucose, lactate and ammonium. Analysis of variance did not reveal a significant effect by stage of the estrous cycle on the embryotrophic activity of conditioned media. However, there was a significant effect by time of conditioning on the proportion of 5- to 8-cell embryos (P < 0.01) and of blastocysts and hatched blastocysts (P < 0.05). None of the conditioned media (19 to 31% blastocysts) was superior to the cumulus cell co-culture system (32% blastocysts). In the conditioned media, the proportion of 5- to 8-cell embryos correlated positively with mitogenic activity on 3T3 cells (r = 0.64; P < 0.05), whereas the proportion of blastocysts was not significantly correlated with this parameter. In summary, our results provide evidence for an effect of time of conditioning on embryotrophic activity of oviduct epithelial cell-conditioned media. The fact that mitogens for NIH 3T3 cells affect the proportion of 5- to 8-cell embryos but not of blastocysts suggests different culture requirements for early and late preimplantation stage development of bovine embryos.

In this study we investigated the expression of the two cyclooxygenases, cox-1 and -2, in sheep uterine tissues during the estrous cycle and early pregnancy. We identified the cox-2 isoform in the ovine uterus by Western blot and demonstrated that the two cyclooxygenases exhibited different patterns of expression. Cox-1 was expressed at steady state levels in the endometrium during the estrous cycle and comparable stages of pregnancy. In contrast, cox-2 was highly and transiently expressed from days 12-15 of the estrous cycle and declined thereafter to undetectable levels. Endometrium from early pregnant ewes showed a similar pattern of cox-2 expression, although there was a slower decrease beyond day 15. Immunohistochemical studies demonstrated that cox-1 was localized in both epithelial and stromal cells, whereas cox-2 was localized solely in the luminal epithelium and to a lesser extent in the superficial glands. Treatment of ovariectomized ewes with steroids indicated that expression of cox-1 remained at constant levels whatever the treatment. In contrast, endometrial cox-2 was highly induced by a 10-day progesterone treatment. Estradiol slightly increased cox-2 expression but only after progesterone priming. Collectively these results suggest that the developing ability of the uterus to synthesize PGs is due to the induction of cox-2.

Lipopolysaccharide (LPS) is a component of the outer membrane of gram-negative bacteria and is involved in postpartum uterine infection in cattle. Lipopolysaccharide causes inflammation of the endometrium and the activation of immune and pro-inflammatory pathways in uterine cells has been well documented. This study was performed to investigate the effects of LPS on epithelial cells from whole-genome information, and this abstract focuses on genes and pathways involved in the regulation of implantation. Following in vitro culture of bovine endometrial epithelial cells (bEEC), passage 4 epithelial cell samples from 3 cows were exposed to 0, 2, and 8 µg mL−1 LPS (Sigma L2630, Escherichia coli O111:B4, Sigma Chemical Co., St. Louis, MO, USA) for 24 h. At time 0 and at 24 h for each LPS dosage, RNA was extracted by using the All prep DNA/RNA Universal kit (Qiagen, Valencia, CA, USA). Samples were analysed by RNA sequencing performed in the SciLife Laboratory in Uppsala. Differentially expressed genes (DEG) were identified by using Ensemble genes as a reference. No DEG were found between 2 and 8 µg mL−1 LPS-treated samples and at 24 h 2035 DEG were identified (Benjamini-Hochberg adjusted P-value

Pregnancy-associated glycoproteins (PAGs) constitute a multigenic family of aspartic proteinases expressed in the trophoblast of the ruminant placenta. InBos taurus, this family comprises 21 members segregated into ancient and modern phylogenetic groups. Ancient PAGs have been reported to be synthesized throughout the trophoblastic cell layer whereas modern PAGs are produced by binucleate cells of cotyledons. The aim of this study was to investigate modern and ancient PAGs during gestation in cotyledonary and intercotyledonary tissues. To obtain convincing and innovative results despite the high sequence identity shared between PAGs, we designed specific tools such as amplification primers and antibodies. Using real-time RT-PCR, we described the transcript expression of 16 bovine PAGs. Overall, PAGs are characterized by an increase in their expression during gestation. However, we demonstrated a segregation of modern PAGs in cotyledons and of ancient PAGs in the intercotyledonary chorion, except for the ancient PAG2 expressed in cotyledons. By raising specific antibodies against the modern PAG1 and ancient PAG11 and PAG2, we established the expression kinetics of the proteins using western blotting. Immunohistochemistry showed that PAGs were produced by specific cellular populations: PAG1 by binucleate cells in the whole trophoblastic layer, PAG11 was localized in binucleate cells of the intercotyledonary trophoblast and the chorionic plate of the cotyledon, while PAG2 was produced in mononucleate cells of the internal villi of the cotyledon. These results revealed a highly specific regulation of PAG expression and cell localization as a function of their phylogenetic status, suggesting distinct biological functions within placental tissues.

Secretion of interferon tau (IFN tau) by trophoblastic tissue has been shown to be the first embryonic signal for pregnancy recognition. Therefore we tried to derive biologically active trophoblastic tissue by in vitro techniques. Since conventional in vitro conditions for bovine embryo development were not sufficient for long-term culture, we tested more complex culture conditions, including Menezo B2 or Buffalo rat liver (BRL) cell-conditioned medium, for their ability to support proliferation and IFN tau secretion by in vitro-derived trophoblastic tissue. IFN tau activity was determined by using a biological assay based on the inhibition of the cytopathic effect of vesicular stomatitis virus on Madin-Darby bovine kidney cells. When cultures of individual hatched blastocysts were started in 60-microliters drops of BRL cell-conditioned medium, mean IFN tau secretion (antiviral units/ml/48 h) corresponded to 1200 on Day 11 and to 5000 on Day 13 (p 10(5) antiviral units/ml/48 h on Day 23m, stayed high for about 1 wk, and then slowly declined to levels below 10(3) antiviral U/ml/48 h. The specificity of the cytoprotective effect of IFN tau was tested by Western blot analysis and by immunoneutralization with use of a polyclonal antiserum specific to IFN tau. Our results demonstrate that viable trophoblastic tissue can be maintained entirely in vitro and secretes high amounts of IFN tau.

Chantier qualité GA; International audience; Undernutrition before and after calving has a detrimental effect on the fertility of dairy cows. The effect of nutritional stress was previously reported to influence gene expression in key tissues for metabolic health and reproduction such as the liver and the genital tract early after calving, but not at breeding, that is, between 70 and 90 days post-partum. This study investigated the effects of pre- and post-partum mild underfeeding on global gene expression in the oviduct, endometrium and corpus luteum of eight multiparous Holstein cows during the early and middle phases of an induced cycle 80 days post-partum. Four control cows received 100% of energy and protein requirements during the dry period and after calving, while four underfed received 80% of control diet. Oestrous synchronization treatment was used to induce ovulation on D80 post-partum. Oviducts, ovaries and the anterior part of each uterine horn were recovered surgically 4, 8, 12 and 15 days after ovulation. Corpora lutea were dissected from the ovaries, and the endometrium was separated from the stroma and myometrium in each uterine horn. The oviduct segments were comprised of ampulla and isthmus. RNAs from ipsi- and contralateral samples were pooled on an equal weight basis. In each tissue, gene expression was assessed on a custom bovine 10K array. No differentially expressed gene (DEG) in the corpus luteum was identified between underfed and control, conversely to 293 DEGs in the oviduct vs 1 in the endometrium under a false discovery rate (FDR)

FOXL2, a winged-helix/forkhead domain transcription factor, is a key gene involved in the differentiation and biological functions of the ovary. In a recent transcriptomic analysis, we found that FOXL2 expression in bovine caruncular endometrium was different from that in intercaruncular endometrium. In order to gain new insights into FOXL2 in this tissue, we determined the expression of this transcription factor during the estrous cycle and the establishment of pregnancy in cattle. The endometrial expression of FOXL2 did not vary during maternal recognition of pregnancy (Days 16‐20). Using an in vivo bovine model and primary cell cultures, we showed that FOXL2 was not an interferon-tau target gene. Both FOXL2 transcript and protein were expressed from Day 5 to Day 20 of the estrous cycle, and their levels showed a significant increase during the luteolytic phase. A 2-day progesterone supplementation in heifers led to a clear down-regulation of FOXL2 protein levels, suggesting the negative impact of progesterone on FOXL2 expression. Immunohistochemistry data revealed the localization of FOXL2 in endometrial stromal and glandular cells. FOXL2 subcellular distribution was shown to be nuclear in endometrial samples collected during the luteolytic period, while it was not detected in nuclei during the luteal phase and at implantation. Collectively, our findings provide the first evidence that FOXL2 is involved in the regulation of endometrial tissue physiology. bovine, endometrium, estrous cycle, FOXL2, pregnancy

Erratum in Biol Reprod. 2012 Oct;87(4):102. [corrected to Vitorino Carvalho, Anaïs]; FOXL2, a winged-helix/forkhead domain transcription factor, is a key gene involved in the differentiation and biological functions of the ovary. In a recent transcriptomic analysis, we found that FOXL2 expression in bovine caruncular endometrium was different from that in intercaruncular endometrium. In order to gain new insights into FOXL2 in this tissue, we determined the expression of this transcription factor during the estrous cycle and the establishment of pregnancy in cattle. The endometrial expression of FOXL2 did not vary during maternal recognition of pregnancy (Days 16-20). Using an in vivo bovine model and primary cell cultures, we showed that FOXL2 was not an interferon-tau target gene. Both FOXL2 transcript and protein were expressed from Day 5 to Day 20 of the estrous cycle, and their levels showed a significant increase during the luteolytic phase. A 2-day progesterone supplementation in heifers led to a clear down-regulation of FOXL2 protein levels, suggesting the negative impact of progesterone on FOXL2 expression. Immunohistochemistry data revealed the localization of FOXL2 in endometrial stromal and glandular cells. FOXL2 subcellular distribution was shown to be nuclear in endometrial samples collected during the luteolytic period, while it was not detected in nuclei during the luteal phase and at implantation. Collectively, our findings provide the first evidence that FOXL2 is involved in the regulation of endometrial tissue physiology.

Eicosanoids are metabolites of arachidonic acid produced by cyclooxygenases (COXs) or lipoxygenases (LOXs). They mediate inflammation and mucus secretion in chronic pulmonary inflammatory diseases. The gel-forming mucin MUC5AC is over-expressed in the airways of patients with these diseases. MUC5AC expression is mediated by an extracellular signal-regulated kinase (ERK)/Sp1 dependent mechanism. Our aim was to study the role of eicosanoids and their signalling pathways in MUC5AC expression. Inhibitors of 12-LOX, but not those of COX, 5-LOX or 15-LOX, reduce MUC5AC expression induced by phorbol myristate acetate (PMA) in the bronchial epithelial cell line NCI-H292. These inhibitors also abrogate the production of whole mucus by cell monolayers. Two forms of 12-LOX (R and S) exist in mammals. Using siRNAs we show that 12R-LOX but not 12S-LOX is involved in MUC5AC expression induced by PMA, lipopolysaccharide or transforming growth factor-α. 12R-LOX also participates in MUC2 and MUC5B expression, although to a lesser extent than for MUC5AC. Contrarily, 12R-LOX silencing does not modify interleukin-8 production. 12-LOX inhibitors reduce ERK activation and Sp1 translocation induced by PMA. Moreover, the 12R-LOX product 12(R)-hydroxyeicosatetraenoic acid, induces MUC5AC expression, ERK activation and Sp1 translocation. 12R-LOX is involved in MUC5AC expression. This occurs via ERK- and Sp1-signalling pathways.

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During the periovulatory period, the induction of prostaglandin G/H synthase-2 (PTGS2) expression in cumulus cells and associated prostaglandin E2 (PGE2) production are implicated in the terminal differentiation of the cumulus-oocyte complex. During the present study, the effects of the PTGS2/PGE2 pathway on the developmental competence of bovine oocytes were investigated using an in vitro model of maturation, fertilization, and early embryonic development. The specific inhibition of PTGS2 activity with NS-398 during in vitro maturation (IVM) significantly restricted mitogen-activated protein kinase (MAPK) activation in oocytes at the germinal vesicle breakdown stage and reduced both cumulus expansion and the maturation rate after 22 h of culture. In addition, significantly higher rates of abnormal meiotic spindle organization were observed after 26 h of culture. Periconceptional PTGS2 inhibition did not affect fertilization but significantly reduced the speed of embryo development. Embryo output rates were significantly decreased on Day 6 postfertilization but not on Day 7. However, total blastomere number was significantly lower in embryos obtained after PTGS2 inhibition. The addition of PGE2 to IVM and in vitro fertilization cultures containing NS-398 overrode oocyte maturation and early embryonic developmental defects. Protein and mRNA expression for the prostaglandin E receptor PTGER2 were found in oocytes, whereas the PTGER2, PTGER3, and PTGER4 subtypes were expressed in cumulus cells. This study is the first to report the involvement of PGE2 in oocyte MAPK activation during the maturation process. Taken together, these results indicate that PGE2-mediated interactions between somatic and germ cells during the periconceptional period promote both in vitro oocyte maturation and preimplantation embryonic development in cattle.