Analysis of the transcriptome of bovine endometrial cells isolated by laser micro-dissection (2): impacts of post-partum negative energy balance on stromal, glandular and luminal epithelial cells.

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.

Analysis of the transcriptome of bovine endometrial cells isolated by laser micro-dissection (1): specific signatures of stromal, glandular and luminal epithelial cells.

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.

Analysis of blood parameters and molecular endometrial markers during early reperfusion in two ovine models of uterus transplantation.

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.

Progress and challenges in developing organoids in farm animal species for the study of reproduction and their applications to reproductive biotechnologies.

Within the past decades, major progress has been accomplished in isolating germ/stem/pluripotent cells, in refining culture medium and conditions and in establishing 3-dimensional culture systems, towards developing organoids for organs involved in reproduction in mice and to some extent in humans. Haploid male germ cells were generated in vitro from primordial germ cells. So were oocytes, with additional support from ovarian cells and subsequent follicle culture. Going on with the female reproductive tract, spherical oviduct organoids were obtained from adult stem/progenitor cells. Multicellular endometrial structures mimicking functional uterine glands were derived from endometrial cells. Trophoblastic stem cells were induced to form 3-dimensional syncytial-like structures and exhibited invasive properties, a crucial point for placentation. Finally, considering the embryo itself, pluripotent embryonic cells together with additional extra-embryonic cells, could self-organize into a blastoid, and eventually into a post-implantation-like embryo. Most of these accomplishments have yet to be reached in farm animals, but much effort is devoted towards this goal. Here, we review the progress and discuss the specific challenges of developing organoids for the study of reproductive biology in these species. We consider the use of such organoids in basic research to delineate the physiological mechanisms involved at each step of the reproductive process, or to understand how they are altered by environmental factors relevant to animal breeding. We evaluate their potential in reproduction of animals with a high genetic value, from a breeding point of view or in the context of preserving local breeds with limited headcounts.

Subclinical endometritis in dairy cattle is associated with distinct mRNA expression patterns in blood and endometrium.

Cattle with subclinical endometritis (SCE) are sub-fertile and diagnosing subclinical uterine disease remains a challenge. The hypothesis for this study was that endometrial inflammation is reflected in mRNA expression patterns of peripheral blood leucocytes. Transcriptome profiles were evaluated in healthy cows and in cows with SCE using circulating white blood cells (WBC) and endometrial biopsy samples collected from the same animals at 45-55 days postpartum. Bioinformatic analyses of microarray-based transcriptional data identified gene profiles associated with distinct biological functions in circulating WBC and endometrium. In circulating WBC, SCE promotes a pro-inflammatory environment, whereas functions related to tissue remodeling are also affected in the endometrium. Nineteen differentially expressed genes associated with SCE were common to both circulating WBC and the endometrium. Among these genes, transcript abundance of immune factors C3, C2, LTF, PF4 and TRAPPC13 were up-regulated in SCE cows at 45-55 days postpartum. Moreover, mRNA expression of C3, CXCL8, LTF, TLR2 and TRAPPC13 was temporally regulated during the postpartum period in circulating WBC of healthy cows compared with SCE cows. This observation might indicate an advantageous modulation of the immune system in healthy animals. The transcript abundance of these genes represents a potential source of indicators for postpartum uterine health.

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.

Peripheral thyroid hormone levels and hepatic thyroid hormone deiodinase gene expression in dairy heifers on the day of ovulation and during the early peri-implantation period.

BACKGROUND: Before the onset of fetal thyroid hormone production, the transplacental delivery of maternal thyroid hormones is necessary for embryonic and fetal development. Therefore, the adaptation of maternal thyroid hormone metabolism may be important for pregnancy success and embryo survival. The aims of this study were to determine the thyroid hormone levels during the early peri-implantation period until day 18 and on the day of ovulation, to determine whether pregnancy success is dependent on a "normothyroid status" and to determine whether physiological adaptations in maternal thyroid hormone metabolism occur, which may be necessary to provide sufficient amounts of biologically active T3 to support early pregnancy. Therefore, blood samples obtained on the day of ovulation (day 0) and days 14 and 18 of the Holstein-Friesian heifers (n = 10) during the respective pregnant, non-pregnant and negative control cycles were analyzed for thyroid-stimulating-hormone (TSH), thyroxine (T4) and triiodothyronine (T3). Liver biopsies (day 18) from pregnant and respective non-pregnant heifers were analyzed for mRNA expression of the most abundant hepatic thyroid hormone deiodinase (DIO1) by real time qPCR. RESULTS: Although liver DIO1 mRNA expression did not differ between the pregnant and non-pregnant heifers on day 18, the serum concentrations of TSH and T3 on day 18 were higher in non-pregnant heifers compared to pregnant heifers (P < 0.05). Moreover, T3 decreased between day 0 and 18 in pregnant heifers (P < 0.001). CONCLUSIONS: In conclusion, no associations between thyroid hormone patterns on day 18 and pregnancy success were detected. During the early peri-implantation period, TSH and T3 may be affected by the pregnancy status because both TSH and T3 were lower on day 18 in pregnant heifers compared to non-pregnant dairy heifers. In further studies, the thyroid hormone axis should be evaluated throughout the entire gestation to confirm these data and identify other possible effects of pregnancy on the thyroid hormone axis in cattle.

Serum IGFBP4 concentration decreased in dairy heifers towards day 18 of pregnancy.

This study was conducted to determine if the main components of the somatotropic axis change during the early phase of pregnancy in the maternal blood system and whether differences exist on day 18 after pregnancy recognition by the maternal organism. Blood samples of pregnant heifers (Holstein Friesian; n = 10 after embryo transfer) were obtained on the day of ovulation (day 0), as well as on days 7, 14, 16 and 18 and during pregnant, non-pregnant and negative control cycles. The concentrations of progesterone (P4), oestrogen, growth hormone (GH), insulin-like growth factor-1 and -2 (IGF1, -2) and IGF-binding protein-2, -3 and -4 (IGFBP2, -3, -4) were measured. The mRNA expressions of growth hormone receptor 1A, IGF1, IGF2, IGFBP2, IGFBP3 and IGFBP4 were detected using RT-qPCR in liver biopsy specimens (day 18). In all groups, total serum IGF1 decreased from day 0 to 16. Notably, IGFBP4 maternal blood concentrations were lower during pregnancy than during non-pregnant cycles and synchronized control cycles. It can be speculated that the lower IGFBP4 in maternal blood may result in an increase of free IGF1 for local action. Further studies regarding IGFBP4 concentration and healthy early pregnancy are warranted.

A novel subnucleocapsid nanoplatform for mucosal vaccination against influenza virus that targets the ectodomain of matrix protein 2.

In this study, subnucleocapsid nanorings formed by the recombinant nucleoprotein (N) of the respiratory syncytial virus were evaluated as a platform to anchor heterologous antigens. The ectodomain of the influenza virus A matrix protein 2 (M2e) is highly conserved and elicits protective antibodies when it is linked to an immunogenic carrier, making it a promising target to develop universal influenza vaccines. In this context, one or three M2e copies were genetically linked to the C terminus of N to produce N-M2e and N-3M2e chimeric recombinant nanorings. Mice were immunized intranasally with N-M2e or N-3M2e or with M2e or 3M2e control peptides. N-3M2e-vaccinated mice showed the strongest mucosal and systemic antibody responses. These mice presented a reduced viral load and minor weight loss, and all survived upon challenge with influenza virus A/PR8/34 (H1N1) (PR8). We compared the intranasal route to the subcutaneous route of N-3M2e immunization. Only the intranasal route induced a strong local IgA response and led to the protection of mice upon challenge. Finally, we demonstrated that the induction of anti-M2e antibodies by N-3M2e is not impaired by preexisting anti-N immunity. Overall, these results show that the N nanoring is a potent carrier for mucosal delivery of vaccinal antigens.

Human genetic polymorphisms in T1R1 and T1R3 taste receptor subunits affect their function.

Umami is the typical taste induced by monosodium glutamate (MSG), which is thought to be detected by the heterodimeric G protein-coupled receptor, T1R1 and T1R3. Previously, we showed that MSG detection thresholds differ substantially between individuals and we further showed that nontaster and hypotaster subjects are associated with nonsynonymous single polymorphisms occurring in the T1R1 and T1R3 genes. Here, we show using functional expression that both amino acid substitutions (A110V and R507Q) in the N-terminal ligand-binding domain of T1R1 and the 2 other ones (F749S and R757C), located in the transmembrane domain of T1R3, severely impair in vitro T1R1/T1R3 response to MSG. A molecular model of the ligand-binding region of T1R1/T1R3 provides a mechanistic explanation supporting functional expression data. The data presented here support causal relations between the genotype and previous in vivo psychophysical studies in human evaluating sensitivity to MSG.

Nonsynonymous single nucleotide polymorphisms in human tas1r1, tas1r3, and mGluR1 and individual taste sensitivity to glutamate.

Several studies indicate an essential role of the heterodimer Tas1R1-Tas1R3 for monosodium l-glutamate (MSG) detection, although others suggest alternative receptors. Human subjects show different taste sensitivities to MSG, and some are unable to detect the presence of glutamate. Our objective was to study possible relations between phenotype (sensitivity to glutamate) and genotype (polymorphisms in candidate glutamate taste receptors tas1r1, tas1r3, mGluR4, and mGluR1) at the individual level. The sensitivity was measured with a battery of tests to distinguish the effect of sodium ions from the effect of glutamate ions in MSG. A total of 142 genetically unrelated white French subjects were categorized into 27 nontasters (specific ageusia), 21 hypotasters, and 94 tasters. Reverse transcriptase polymerase chain reaction and immunohistochemistry showed expression of tas1r1, tas1r3, and alpha-gustducin in fungiform papillae in all 12 subjects tested, including subjects who presented specific ageusia for glutamate. Amplification and sequencing of cDNA and genomic DNA allowed the identification of 10 nonsynonymous single nucleotide polymorphisms (nsSNPs) in tas1r1 (n = 3), tas1r3 (n = 3), and mGluR1 (n = 4). In our sample of subjects, the frequencies of 2 nsSNPs, C329T in tas1r1 and C2269T in tas1r3, were significantly higher in nontasters than expected, whereas G1114A in tas1r1 was more frequent in tasters. These nsSNPs along with minor variants and other nsSNPs in mGluR1, including T2977C, account for only part of the interindividual variance, which indicates that other factors, possibly including additional receptors, contribute to glutamate sensitivity.

Tas1R1-Tas1R3 taste receptor variants in human fungiform papillae.

Monosodium glutamate as well as metabotropic and ionotropic glutamate receptor agonists have been reported to be perceived as umami by humans. In spite of the fact that Tas1R1-Tas1R3 has been shown to mediate most of the glutamate taste sensation in mice other candidate receptors have been put forward for which a clear role in detection is still lacking. This work was aimed at investigating the molecular determinants underlying umami taste detection in humans. First, we show evidence supporting expression of Tas1R1 and Tas1R3 but not mGluRs in the fungiform papillae of several individuals. Next, we report a number of naturally occurring L-glutamate taste receptor variants and their frequency in a population of Caucasian subjects. Detailed analysis of 9 non-synonymous single nucleotide polymorphisms from three L-glutamate taste GPCR candidates uncovers receptor specific clusters such that all substitutions in Tas1R1 are located in the extracellular N-terminal ligand-binding domain while in Tas1R3 they mostly affect residues in the seven transmembrane-spanning core domain responsible for the interaction with antagonists and allosteric modulators. In mGluR1, nsSNPs identified are clustered in the intracellular C-terminal tail, which is thought to play a role in signaling. Taken together, these results suggest that Tas1R1-Tas1R3 receptor variants found in human fungiform papillae might contribute to inter-individual differences of sensitivity to L-glutamate.