Bovine scavenger receptor class A (SR-A) exhibit specific patterns of regulation in the endometrium during the oestrous cycle and early pregnancy.

In mammals, tight regulation of maternal endometrial function is critical for pregnancy success. In bovine species, endometrial expression of members of the scavenger receptor class A (SR-A) has been listed in high-throughput analyses, but very little is known about the involvement of these immune factors during implantation in mammals. To provide first insights into the contribution of SR-A to endometrial physiology, we analysed the expression and regulation of all members of SR-A (SR-A1, SR-A3-SR-A6) during the oestrous cycle and early pregnancy in cattle. Levels of SR-A1 were increased on Day 20 of pregnancy, whereas SR-A3 levels were increased on Day 13 of the oestrous cycle and of the pregnancy. Although SR-A4 levels were reduced on Day 20 of the oestrous cycle, they remained high in pregnant animals. SR-A5 levels increased by Day 13 of the oestrous cycle and decreased on Day 20, but remained high in pregnant animals. Interferon-τ does not affect SR-A gene expression, whereas progesterone regulates the expression of the SR-A3 and SR-A5 transcripts. Endometrial SR-A3 appeared significantly higher in cows carrying invitro-produced embryos than in AI cows. Our data suggest that members of the SR-A family are involved in endometrial remodelling and regulation of endometrial gland physiology, both processes being critical for implantation in mammals.

Localisation of stem cell factor, stanniocalcin-1, connective tissue growth factor and heparin-binding epidermal growth factor in the bovine uterus at the time of blastocyst formation.

Early embryonic losses before implantation account for the highest rates of reproductive failure in mammals, in particular when in vitro-produced embryos are transferred. In the present study, we used molecular biology techniques (real-time quantitative polymerase chain reaction), classical immunohistochemical staining coupled with confocal microscopy and proteomic analysis (multiple reaction monitoring and western blot analysis) to investigate the role of four growth factors in embryo-uterine interactions during blastocyst development. Supported by a validated embryo transfer model, the study investigated: (1) the expression of stem cell factor (SCF), stanniocalcin-1 (STC1), connective tissue growth factor (CTGF) and heparin-binding epidermal growth factor-like growth factor (HB-EGF) in bovine uterine fluid; (2) the presence of SCF, STC1, CTGF and HB-EGF mRNA and protein in the bovine endometrium and embryos; and (3) the existence of reciprocal regulation between endometrial and embryonic expression of SCF, STC1, CTGF and HB-EGF. The results suggest that these growth factors most likely play an important role during preimplantation embryo development in cattle. The information obtained from the present study can contribute to improving the performance of in vitro culture technology in cattle and other species.

Energy and lipid metabolism gene expression of D18 embryos in dairy cows is related to dam physiological status.

We analyzed the change in gene expression related to dam physiological status in day (D)18 embryos from growing heifers (GH), early lactating cows (ELC), and late lactating cows (LLC). Dam energy metabolism was characterized by measurement of circulating concentrations of insulin, glucose, IGF-1, nonesterified fatty acids, ß-hydroxybutyrate, and urea before embryo flush. The metabolic parameters were related to differential gene expression in the extraembryonic tissues by correlation analysis. Embryo development estimated by measuring the length of the conceptuses and the proportion of expected D18 gastrulating stages was not different between the three groups of females. However, embryo metabolism was greatly affected by dam physiological status when we compared GH with ELC and GH with LLC but to a lesser extent when ELC was compared with LLC. Genes involved in glucose, pyruvate, and acetate utilization were upregulated in GH vs. ELC conceptuses (e.g., SLC2A1, PC, ACSS2, ACSS3). This was also true for the pentose pathway ( PGD, TKT), which is involved in synthesis of ribose precursors of RNA and DNA. The pathways involved in lipid synthesis were also upregulated in GH vs. ELC. Despite similar morphological development, the molecular characteristics of the heifers' embryos were consistently different from those of the cows. Most of these differences were strongly related to metabolic/hormone patterns before insemination and during conceptus free-life. Many biosynthetic pathways appeared to be more active in heifer embryos than in cow embryos, and consequently they seemed to be healthier, and this may be more conducive to continue development.

Hepatoma-derived growth factor: from the bovine uterus to the in vitro embryo culture.

Early in cow embryo development, hepatoma-derived growth factor (HDGF) is detectable in uterine fluid. The origin of HDGF in maternal tissues is unknown, as is the effect of the induction on developing embryos. Herein, we analyze HDGF expression in day 8 endometrium exposed to embryos, as well as the effects of recombinant HDGF (rHDGF) on embryo growth. Exposure to embryos did not alter endometrial levels of HDGF mRNA or protein. HDGF protein localized to cell nuclei in the luminal epithelium and superficial glands and to the apical cytoplasm in deep glands. After uterine passage, levels of embryonic HDGF mRNA decreased and HDGF protein was detected only in the trophectoderm. In fetal fibroblast cultures, addition of rHDGF promoted cell proliferation. In experiments with group cultures of morulae in protein-free medium containing polyvinyl alcohol, adding rHDGF inhibited blastocyst development and did not affect cell counts when the morulae were early (day 5), whereas it enhanced blastocyst development and increased cell counts when the morulae were compact (day 6). In cultures of individual day 6 morulae, adding rHDGF promoted blastocyst development and increased cell counts. Our experiments with rHDGF indicate that the growth factor stimulates embryonic development and cell proliferation. HDGF is synthesized similarly by the endometrium and embryo, and it may exert embryotropic effects by autocrine and/or paracrine mechanisms.

SOCS genes expression during physiological and perturbed implantation in bovine endometrium.

In mammals, suppressor of cytokine signalling (CISH, SOCS1 to SOCS7) factors control signalling pathways involved in the regulation of numerous physiological processes including pregnancy. In order to gain new insights into the biological functions of SOCS in the endometrium, a comprehensive analysis of SOCS gene expression was carried out in bovine caruncular (CAR) and intercaruncular (ICAR) tissues collected i) during the oestrous cycle, ii) at the time of maternal recognition of pregnancy and at implantation in inseminated females, iii) following uterine interferon-tau (IFNT) infusion at day 14 post-oestrus, iv) following a period of controlled intravaginal progesterone release and v) following transfer of embryos by somatic-cell nuclear transfer (SCNT). The regulatory effects of IFNT on in vitro cultured epithelial and stromal cells were also examined. Altogether, our data showed that CISH, SOCS4, SOCS5 and SOCS7 mRNA levels were poorly affected during luteolysis and pregnancy. In contrast, SOCS1, SOCS2, SOCS3 and SOCS6 mRNA levels were strongly up-regulated at implantation (day 20 of pregnancy). Experimental in vitro and in vivo models demonstrated that only CISH, SOCS1, SOCS2 and SOCS3 were IFNT-induced genes. Immunohistochemistry showed an intense SOCS3 and SOCS6 staining in the nucleus of luminal and glandular epithelium and of stromal cells of pregnant endometrium. Finally, SOCS3 expression was significantly increased in SCNT pregnancies in keeping with the altered immune function previously reported in this model of compromised implantation. Collectively, our data suggest that spatio-temporal changes in endometrial SOCS gene expression reflect the acquisition of receptivity, maternal recognition of pregnancy and implantation.

Expression and localization of ARTEMIN in the bovine uterus and embryos.

Artemin a member of the glial cell line-derived neurotrophic factor (GDNF) family is present in mice and human preimplantation embryos, and reproductive tract, during early pregnancy promoting embryo development in vitro. The presence of artemin in cattle embryos and reproductive tract, however, is unknown. In the present work we identified for first time artemin in bovine uterine fluid (UF) (Western blot), endometrium (RT-PCR, Western blot and immunohistochemistry) and embryos (RT-PCR and immunohistochemistry) during early preimplantation development. In addition, GFRalpha3, a component of the artemin receptor was localized in blastocysts produced in vitro. Individually developing embryos released ARTEMIN in culture medium and triggered ARTEMIN mRNA down-regulation in epithelial cells from endometrial cell cultures. Our results suggest that ARTEMIN derived from early embryos and maternal reproductive tract may exert important roles during early development in cattle.

Interferon stimulated genes as peripheral diagnostic markers of early pregnancy in sheep: a critical assessment.

We investigated the diagnostic reliability of pregnancy detection using changes in interferon stimulated gene (ISG) messenger RNA (mRNA) levels in circulating immune cells in ewes. Two different groups of ewes (an experimental group, experiment 1 and a farm group, experiment 2) were oestrus-synchronized and blood sampled on day 14 (D0=day of insemination in control animals, experiment 1) and day 15 (experiment 2). Real-time PCR were performed to evaluate the abundance of different ISG mRNAs. In the experimental group, peripheral blood mononuclear cells of 29 ewes born and bred in experimental facilities were isolated using a Percoll gradient method. Gene expression for Chemokine (C-X-C motif) ligand 10 (CXCL10), Myxovirus (influenza virus) resistance 1 (MX1) and Signal transducer and activator of transcription 1 (STAT1) mRNA were, respectively, 8.3-fold, 6.1-fold and 2.7-fold higher (P0.10) in CXCL10, STAT1, MX1, Myxovirus (influenza virus) resistance 2 (MX2) and ISG15 ubiquitin-like modifier (ISG15) mRNA expression were found between pregnant and non-pregnant ewes. The ROC curves and the hierarchical classification generated from the real-time PCR data failed to discriminate between pregnant and non-pregnant animals. In this group of animals, our results show a strong variability in ISG expression patterns: 17% of animals identified as non-pregnant by the five tests were in fact pregnant, only 52% of pregnant animals had at least two positive results (two genes above threshold), whereas up to five positive results (five genes above threshold) were needed to avoid misclassification. In conclusion, this study illustrates the high variability in ISG expression levels in immune circulating cells during early pregnancy and, therefore, highlights the limits of using ISG expression levels in blood samples, collected on PAXgene® tubes on farms, for early pregnancy detection in sheep.

Early embryonic and endometrial regulation of tumor necrosis factor and tumor necrosis factor receptor 2 in the cattle uterus.

Tumor necrosis factor (TNF) alpha likely mediates embryomaternal communication in mammals. In bovine, we have previously found that the uterine fluid of heifers that carried early embryos shows downregulation in the TNF and nuclear factor κB system. In this work, we assessed the expression of TNF and its receptor TNFR2 in the bovine endometrium and embryos during blastocyst development. Moreover, to explore the endometrial immune response to early embryos, we analyzed the number of CD45 leukocytes in the bovine endometrium. Day 8 endometrium and blastocyst recovered from animals after transfer of Day 5 embryos showed TNF and TNFR2 mRNA transcription and protein colocalization. The presence of embryos increased endometrial TNF and TNFR2 protein, whereas endometrial leukocytes decreased. Blastocysts exposed to the uterine tract had undetectable levels of TNF and lower levels of TNFR2 mRNA. These results suggest that the endometrium might lower the TNF concentration in the blastocyst by (1) regulating TNF secretion into the uterine fluid and (2) inducing decreased TNF and TNFR2 mRNA transcription in the embryo. Thus, TNF and TNFR2 might participate in early embryomaternal communication.

Comparative gene mapping: a fine-scale survey of chromosome rearrangements between ruminants and humans.

A total of 202 genes were cytogenetically mapped to goat chromosomes, multiplying by five the total number of regional gene localizations in domestic ruminants (255). This map encompasses 249 and 173 common anchor loci regularly spaced along human and murine chromosomes, respectively, which makes it possible to perform a genome-wide comparison between three mammalian orders. Twice as many rearrangements as revealed by ZOO-FISH were observed. The average size of conserved fragments could be estimated at 27 and 8 cM with humans and mice, respectively. The position of evolutionary breakpoints often correspond with human chromosome sites known to be vulnerable to rearrangement in neoplasia. Furthermore, 75 microsatellite markers, 30 of which were isolated from gene-containing bacterial artificial chromosomes (BACs), were added to the previous goat genetic map, achieving 88% genome coverage. Finally, 124 microsatellites were cytogenetically mapped, which made it possible to physically anchor and orient all the linkage groups. We believe that this comprehensive map will speed up positional cloning projects in domestic ruminants and clarify some aspects of mammalian chromosomal evolution.

Identification by PCR of Fusarium culmorum strains producing large and small amounts of deoxynivalenol.

Thirty deoxynivalenol-producing F. culmorum strains, isolated from wheat grains, were incubated in vitro and analyzed for trichothecene production. Seventeen strains produced more than 1 ppm of deoxynivalenol and acetyldeoxynivalenol and were considered high-deoxynivalenol-producing strains, whereas 13 F. culmorum strains produced less than 0.07 ppm of trichothecenes and were considered low-deoxynivalenol-producing strains. For all strains, a 550-base portion of the trichodiene synthase gene (tri5) was amplified and sequenced. According to the tri5 data, the F. culmorum strains tested clustered into two groups that correlated with in vitro deoxynivalenol production. For three high-producing and three low-producing F. culmorum strains, the tri5-tri6 intergenic region was then sequenced, which confirmed the two separate clusters within the F. culmorum strains. According to the tri5-tri6 sequence data, specific PCR primers were designed to allow differentiation of high-producing from low-producing F. culmorum strains.