Maternal metabolism affects endometrial expression of oxidative stress and FOXL2 genes in cattle.

Intensive selection for milk production has led to reduced reproductive efficiency in high-producing dairy cattle. The impact of intensive milk production on oocyte quality as well as early embryo development has been established but few analyses have addressed this question at the initiation of implantation, a critical milestone ensuring a successful pregnancy and normal post-natal development. Our study aimed to determine if contrasted maternal metabolism affects the previously described sensory properties of the endometrium to the conceptus in cattle. Following embryo transfer at Day 7 post-oestrus, endometrial caruncular (CAR) and intercaruncular (ICAR) areas were collected at Day 19 from primiparous postpartum Holstein-Friesian cows that were dried-off immediately after parturition (i.e., never milked; DRY) or milked twice daily (LACT). Gene quantification indicated no significant impact of lactation on endometrial expression of transcripts previously reported as conceptus-regulated (PLET1, PTGS2, SOCS6) and interferon-tau stimulated (RSAD2, SOCS1, SOCS3, STAT1) factors or known as female hormone-regulated genes (FOXL2, SCARA5, PTGS2). Compared with LACT cows, DRY cows exhibited mRNA levels with increased expression for FOXL2 transcription factor and decreased expression for oxidative stress-related genes (CAT, SOD1, SOD2). In vivo and in vitro experiments highlighted that neither interferon-tau nor FOXL2 were involved in transcriptional regulation of CAT, SOD1 and SOD2. In addition, our data showed that variations in maternal metabolism had a higher impact on gene expression in ICAR areas. Collectively, our findings prompt the need to fully understand the extent to which modifications in endometrial physiology drive the trajectory of conceptus development from implantation onwards when maternal metabolism is altered.

Specific expression patterns and cell distribution of ancient and modern PAG in bovine placenta during pregnancy.

Pregnancy-associated glycoproteins (PAGs) constitute a multigenic family of aspartic proteinases expressed in the trophoblast of the ruminant placenta. In Bos 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.

Pivotal role for monocytes/macrophages and dendritic cells in maternal immune response to the developing embryo in cattle.

In mammals, successful pregnancy is dependent in part on the adaptation or regulation of the maternal immune system to prevent the rejection of the embryonic semiallograft. A modification in Th cell function and secretion is a requirement for the establishment and maintenance of pregnancy. Although there is strong evidence from studies in humans and mice linking successful pregnancy with the predominance of Th2-type immunity, the situation in cattle remains unclear. This study describes the characterization of the immune response of the bovine maternal endometrium to the presence of a developing embryo, with specific emphasis on the macrophage and dendritic cell populations and associated factors, using quantitative real-time PCR, in situ hybridization, and immunohistochemistry. Furthermore, in vivo and in vitro models were developed to investigate the potential role of progesterone and interferon-tau (IFNT) in the regulation of these immune factors. There was a marked increase in the population of CD14(+) cells and CD172a-CD11c(+) cells in the endometrium in response to pregnancy, which was paralleled by increased mRNA expression of a number of non-Th-associated factors, including IL12B and IL15, and downregulation of IL18. In addition, we identified several novel IFNT- and progesterone-regulated factors, including IL12B, MCP1, MCP2, PTX3, RSAD2, and TNFA, whose regulation may be critical to pregnancy outcome. Our findings give center stage to non-Th cells, such as monocytes/macrophages and dendritic cells, in the bovine immune response to the semiallogenic embryo. In conclusion, we propose that in cattle, successful pregnancy establishment is associated with a dramatic regulation of the cytokine network, primarily by endometrial monocytes/macrophages and dendritic cells.

PTGS2-related PGE2 affects oocyte MAPK phosphorylation and meiosis progression in cattle: late effects on early embryonic development.

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.

Differential expression of the enzymatic system controlling synthesis, metabolism, and transport of PGF2 alpha in human fetal membranes.

The present study investigated the expression of genes and proteins associated with PGF2alpha biosynthesis, catabolism, and transport in matched amnion and choriodecidua of human term placenta. The concentration of PGF2alpha within fetal membranes depends on the balance between complex enzymatic systems responsible for, respectively, its synthesis-by prostaglandin-endoperoxide synthase 2 (PTGS2) and members of the aldo-keto reductase (AKR) family, AKR1C3 and AKR1B1-and its catabolic inactivation-through hydroxy-prostaglandin-dehydrogenase (HPGD). We observed that AKR1C3 shows equal basal expression (mRNA and protein) in choriodecidua and amnion but that AKR1B1 exhibits preferential expression in the choriodecidua. Expression of HPGD and solute carrier organic anion transporter family member 2A1 (SLCO2A1) was found primarily in the choriodecidua. We also evaluated whether an inflammatory environment induced by the gram-negative bacterial endotoxin lipopolysaccharide (LPS) affects expression of each candidate enzymes. The amnion responded to LPS with a small but significant decrease of AKR1B1 mRNA expression. In contrast, we found a significant increase in PTGS2 and AKR1C3 mRNA expression in choriodecidua after LPS challenge, but such regulation was confirmed only at protein levels for PTGS2 and not for AKR1C3. Our results suggest that the choriodecidua appears to be the main tissue, which expresses maximally all the components (synthesis, degradation, and transport) controlling PGF2alpha levels.

Gene expression profiles of bovine caruncular and intercaruncular endometrium at implantation.

At implantation the endometrium undergoes modifications necessary for its physical interactions with the trophoblast as well as the development of the conceptus. We aim to identify endometrial factors and pathways essential for a successful implantation in the caruncular (C) and the intercaruncular (IC) areas in cattle. Using a 13,257-element bovine oligonucleotide array, we established expression profiles at day 20 of the estrous cycle or pregnancy (implantation), revealing 446 and 1,295 differentially expressed genes (DEG) in C and IC areas, respectively (false discovery rate = 0.08). The impact of the conceptus was higher on the immune response function in C but more prominent on the regulation of metabolism function in IC. The C vs. IC direct comparison revealed 1,177 and 453 DEG in cyclic and pregnant animals respectively (false discovery rate = 0.05), with a major impact of the conceptus on metabolism and cell adhesion. We selected 15 genes including C11ORF34, CXCL12, CXCR4, PLAC8, SCARA5, and NPY and confirmed their differential expression by quantitative RT-PCR. The cellular localization was analyzed by in situ hybridization and, upon pregnancy, showed gene-specific patterns of cell distribution, including a high level of expression in the luminal epithelium for C11ORF34 and MX1. Using primary cultures of bovine endometrial cells, we identified PTN, PLAC8, and CXCL12 as interferon-tau (IFNT) target genes and MSX1 and CXCR7 as IFNT-regulated genes, whereas C11ORF34 was not an IFNT-regulated gene. Our transcriptomic data provide novel molecular insights accounting for the biological functions related to the C or IC endometrial areas and may contribute to the identification of potential biomarkers for normal and perturbed early pregnancy.

Lysophosphatidic acid signaling during embryo development in sheep: involvement in prostaglandin synthesis.

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 beta-actin and alpha-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 F2alpha and prostaglandin E2, with no significant modifications to cytosolic phospholipase A2alpha 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.

Expression of genes involved in prostaglandin E2 and progesterone production in bovine cumulus-oocyte complexes during in vitro maturation and fertilization.

Prostaglandin E(2) (PGE(2)) 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 an in vitro model 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 PGE(2) biosynthesis 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 PGE(2) secreted 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, 20alpha-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 PGE(2) biosynthetic activity via the PTGS2/PTGES1 pathway during the maturation period and the increasing ability to produce progesterone from the immature to the fertilized stages.

Molecular evidence for a critical period in mural trophoblast development in bovine blastocysts.

Embryonic and extra-embryonic lineages are separated at the blastocyst stage in the mouse at the onset of implantation but well ahead of implantation in most mammals. To provide information on the development of the trophoblast lineage in late-implanting bovine embryos, we combined the use of molecular markers defining embryonic and extra-embryonic lineages in the mouse with a transcriptomic approach dedicated to the early steps of the elongation process, a characteristic feature of blastocyst development in ruminants. In this study, we present molecular evidence for differences between the cow and the mouse in the programming of trophoblast differentiation. This different programming encompasses: (i) the expression of epiblast specifying genes (Oct-4, Nanog) in bovine trophoblast cells at the onset of elongation, (ii) the transcription of proliferation markers in early elongating blastocysts, (iii) the early detection of trophoblast-specific transcripts related to extra-embryonic tissue's differentiation (Hand1, Ets2, IFN-tau) and (iv) the identification of a new transcript (c12) which displays a reciprocal pattern to that of Oct-4 and Nanog genes in the embryonic cells and for which no equivalent has thus far been found in the mouse. Altogether, these results tended to show that early elongation is a critical transition in bovine trophoblast development.

Suppressor of cytokine signalling (SOCS) genes are expressed in the endometrium and regulated by conceptus signals during early pregnancy in the ewe.

It is established that the conceptus-endometrium dialogue involves cytokines, growth factors and hormones. Given the crucial functions of the suppressor of cytokine signaling (SOCS) family proteins in cytokine signalling, we analyzed the expression and the regulation of CIS and SOCSs 1-3 transcripts during early pregnancy in the ovine endometrium. An overall stimulation of the SOCS transcripts was described in the pregnant ewes with two specific patterns. Unilaterally pregnant ewes confirmed the conceptus-produced factors as regulators of the SOCSs 1-3 expression at day 16 of pregnancy. Intrauterine injection of recombinant ovine interferon tau (IFNtau) in cyclic ewes stimulated the expression of the SOCS mRNA with various potencies, therefore suggesting that the SOCS could take part in the negative regulation of the IFNtau signalling pathway.

Mammalian lipocalin-type prostaglandin D2 synthase in the fluids of the male genital tract: putative biochemical and physiological functions.

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.