Maternal Recognition Of Pregnancy Research Articles

115 Gene editing: Where are we on campus and where can it go?

Abstract Editing the genome of livestock provides solutions to otherwise intractable problems and can provide a tool to better understand the role of specific proteins during development. The potential benefit for production agriculture is so great that the USDA Agricultural Research Service has invested in 4 positions now located in a research unit at the University of Missouri with a focus currently on swine and poultry. Genome editing can result in introducing alleles from divergent populations or novel alleles to produce animals that have new proteins, modified proteins or animals that no longer produce a protein. Applications of genome editing include improving disease resistance (viral and bacterial), improving digestion, disseminating valuable genetics, improved thermoregulation, changing the body/milk composition, and creating hypoallergenic meat. Changes in these animals will improve animal welfare, animal productivity and the sustainability of animal agriculture. In addition to improving animal agriculture, gene editing has enabled a new understanding of maternal recognition of pregnancy in the pig and a better understanding of molecules involved in growth and development of pigs. With new developments in editing of just somatic cells [somatic cell gene editing (SCGE), rather than germline editing], it will be possible to edit some cells in existing animals and thus change their phenotype. SCGE technologies such as this are being developed via the biomedical community as models to lead to human clinical applications. One such application is to correct cells in the lungs that produce the CFTR protein. Correction of just 10% of the cells in the lungs is estimated to correct the lung symptoms associated with cystic fibrosis. Similar applications in production agriculture might produce animals that have novel phenotypes of an altered carcass composition or better use of nutrients. Application of genetic engineering technologies such as genome editing is limited by biology and the imagination of the investigator.

HH5 Double-Carrier Embryos Fail to Progress through Early Conceptus Elongation

Massive genotyping in cattle has uncovered several deleterious haplotypes that cause pre-term mortality. Holstein Haplotype 5 (HH5) is a deleterious haplotype present in the Holstein Friesian population that involves the ablation of the Transcription Factor B1 mitochondrial (TFB1M) gene. The developmental stage at which HH5 double-carrier (DC, homozygous) embryos or fetuses die remains unknown and this is a relevant information to estimate the economic losses associated to the inadvertent cross between carriers. To determine if HH5 DC survive to maternal recognition of pregnancy, embryonic day (E)14 embryos were flushed from superovulated carrier cows inseminated with a carrier bull. DC E14 conceptuses were recovered at Mendelian rates but they failed to achieve early elongation, as evidenced by a drastic (>26-fold) reduction in the proliferation of extraembryonic membranes compared with carrier or non-carrier embryos. To assess development at earlier stages, TFB1M knockout (KO) embryos -functionally equivalent to DC embryos- were generated by CRISPR technology and cultured to the blastocyst stage -Day (D)8- and to the early embryonic disc stage -D12-. No significant effect of TFB1M ablation was observed on the differentiation and proliferation of embryonic lineages and relative mtDNA content up to D12. In conclusion, HH5 DC embryos are able to develop to early embryonic disc stage but fail to undergo early conceptus elongation, required for pregnancy recognition.

11 Transcriptomic changes in response to form of selenium on the interferon-tau signaling mechanism in the caruncular tissue of beef heifers at maternal recognition of pregnancy

Abstract It is necessary to supplement selenium (Se) to the diet of beef cattle in regions where the soils and forages are deficient in this trace mineral. Supplementation with Se conventionally occurs using an inorganic form (ISe) although the organic forms (OSe) are available when cattle consume forage. Previously we have investigated the effects of supplemental Se as ISe, or a 1:1 mixture of ISe to OSe (MIX) on grazing beef cows and heifers and have observed significant effects on the corpus luteum (CL) and endometrium. The MIX form of Se increased systemic progesterone (P4) during the early luteal phase of the estrous cycle at a time that can significantly improve early embryonic development. We subsequently investigated the effects of MIX form Se versus ISe on the mRNA transcripts encoding IFNτ- and P4-induced proteins in the caruncular (CAR) tissue of the uterine endometrium at maternal recognition of pregnancy (MRP). MIX-supplemented heifers had a decrease in the relative abundance of mRNAs encoding DGAT2, FGF2, IFIT3, ISG15, MX1, OAS2, and RSAD2 in CAR which was coupled with significantly longer conceptuses at MRP. Presently, we examined transcriptomic changes in the CAR tissue of the endometrium at MRP. Angus-cross heifers (n = 20) were exposed to a 45-d period of Se depletion (no supplemental Se) followed by a 45-day repletion period with ISe alone. Heifers were then randomly assigned to 90 days of treatment with a vitamin-mineral mix containing 35-ppm Se as ISe (n = 10), or 1:1 mixture of ISe and OSe (n = 10, MIX). Following insemination, heifers were killed at MRP (d 17) and their reproductive tracts collected. Only heifers with a fully intact recovered conceptus were utilized for further analyses (n = 6 per treatment). Transcriptomic analysis using RNA-sequencing was conducted using total mRNA from CAR biopsies. Differential gene expression was determined using Integrated Differential Expression and Pathway Analysis (iDEP.96), and DEG were subjected to canonical, functional, and network analyses using QIAGEN’s Ingenuity Pathway Analysis (IPA). RNA sequencing results were validated using qPCR. At P < 0.05, there was a total of 2,029 DEGs with 1,038 transcripts upregulated, and 991 transcripts downregulated in MIX versus ISe heifers. Saliently, the interferon JAK/STAT pathway signaling through the STAT1/2 heterodimer was significantly down regulated in CAR samples from MIX heifers, as were the following interferon-responsive genes: IFIT1, IFIT2, IFIT3, IRF1, IRF9, ISG15, ISG20, OAS2, RSAD2, and STAT2. Additionally, there were significant changes in mRNA contribution to the immunotolerant environment (IDO1, ISG20, SCARA1, and TGFB1) to allow the fetal allograft to closely interact with the maternal interface. In combination, these findings suggest more advanced preparation of the CAR and developing conceptus for placentation and to evade immune rejection by the maternal endometrium during implantation.

129 Selenium-form affects de novo cholesterol synthesis and catabolism of cholesterol and progesterone in the liver of beef heifers

Abstract A deficiency of selenium (Se) in soils and hence forages poses a challenge to producers in the southeast United States, necessitating supplementation of this trace mineral to the diet of pasture fed cattle. Se occurs in either inorganic (ISe) or organic (OSe) forms; ISe is conventionally available in commercial supplements, while OSe is available in grazed forages. We have investigated supplementation with either ISe or a 1:1 mixture of ISe:OSe (MIX) and demonstrated that MIX increases the relative abundance of mRNA encoding the low-density lipoprotein receptor (LDLR) and enzymes involved in de novo cholesterol synthesis in the corpus luteum, concurrent with a MIX-induced increase in systemic progesterone (P4) during the early luteal phase. We also observed a MIX-induced decrease in circulating cholesterol and low-density/very low-density lipoproteins during early gestation, and a MIX-induced increase in conceptus length at maternal recognition of pregnancy (MRP). The objective herein was to investigate the effect of form of Se (ISe versus MIX) on de novo cholesterol synthesis and cholesterol/steroid catabolism in the liver at MRP. We hypothesized form of Se would affect the abundance of transcripts that regulate synthesis, catabolism, and local concentrations of cholesterol and P4. Angus-cross heifers were supplemented with ISe or MIX for at least 90 d before insemination at observed estrus. On d 17 of pregnancy, heifers (n = 6/TRT) were killed and their reproductive tracts and conceptuses recovered. Hepatic samples were collected for qPCR analysis of mRNA transcripts encoding proteins involved in de novo cholesterol synthesis, regulation of cholesterol availability and steroid catabolism. Additionally, on d 7 and d 17 of pregnancy, we quantified systemic concentrations of high-density lipoproteins (HDL), indicative of cholesterol recycling to the liver. We quantified the relative abundance of 22 mRNAs and observed that heifers supplemented with MIX-form Se had an increase in the relative abundance of mRNA encoding the very low-density lipoprotein receptor (VLDLR, P = 0.01). Unexpectedly, we failed to detect an effect of TRT on the abundance of mRNA encoding the high-density lipoprotein receptor (SCARB1, P > 0.05) which was consistent with our inability to detect an effect on the serum concentration of total or free HDL (P > 0.05) during early pregnancy and at MRP. Of the 11 mRNA transcripts encoding enzymes involved in catabolism of cholesterol and progesterone, including steroid alpha reductases (SRD5A1 and SRD5A3) and cytochrome P450 enzymes (CYP4A2, CYP4A11, CYP11A1, and CYP17A1), we were unable to detect a difference in the relative abundance of any transcript. Overall, it appears that the form of Se is altering circulating concentrations of cholesterol and further affecting whole animal physiology and fertility; however, the mechanism involved remains to be fully elucidated and warrants further investigation.

121 Unraveling the biology of bovine conceptus elongation

Abstract The establishment of pregnancy is a well-regulated process that involves the development of a viable blastocyst, conceptus elongation, maternal recognition of pregnancy, implantation, and the onset of placentation. Pregnancy loss is a major factor limiting reproductive efficiency in livestock species. In cattle, most embryonic losses occur during the first month of gestation. The genetic and epigenetic regulation of embryonic and placental development in ruminants is still poorly understood. To investigate the transcriptome profile of the bovine conceptus during elongation, Angus heifers (n = 8) were superovulated and bred using semen from a proven high-fertility Bos indicus sire. Heifers were flushed on d 14 of gestation using a standard nonsurgical flushing technique. Recovered conceptuses were individually photographed and snap-frozen for total RNA extraction and bulk mRNA sequencing. Recovered conceptuses were classified based on size into ovoid (0.2 – 5mm; n = 6), tubular (<15 mm; n = 7), or filamentous (>16 mm; n = 7). Differentially expressed genes (DEG: FDR < 0.05) were determined using DESeq2 analysis and were functionally annotated using DAVID. The most pronounced transcriptomic changes were identified between the ovoid and tubular stages, with 5,937 DEGs (2,913 upregulated and 3,024 downregulated) and affected pathways were primarily involved in ribosome assembly and rRNA processing. There were 3,966 DEGs (2,069 upregulated and 1,897 downregulated) between the ovoid and filamentous stages, and the most significant pathways were involved in protein translation, embryonic development, and cell cycle. The transition between tubular to filamentous presented 1,435 DEGs (775 upregulated and 660 downregulated) and significant pathways were involved in protein degradation, cell division, cell cycle, protein stabilization, and mRNA splicing. Our findings indicate that the greatest alterations in the transcriptome occur precisely at the initiation of elongation, suggesting that the bovine conceptus undergoes major structural changes at the outset of this process. This major shift in gene expression is presumed to be necessary to support the subsequent exponential growth phase of elongation, required for maternal recognition of pregnancy.

99 Diversity of vaginal bacteria in lactating dairy cattle on pregnancy establishment

Abstract Establishment and maintenance of pregnancy has a substantial economic impact within the dairy industry each year. Recent research has indicated a potential role of the reproductive microbiome on impacting pregnancy status as a result of hormone concentrations and breeding strategies. However, the association between alpha (within sample metric) and beta (between samples metric) bacterial diversity with pregnancy establishment has not been extensively evaluated in dairy cattle. Thereby, the objective of this study was to analyze vaginal bacterial diversity in lactating dairy cows 1) between day of artificial insemination (AI; day 0) and maternal recognition of pregnancy (MRP; d 18), and 2) between resulting Open and Pregnant females. Lactating Holstein dairy cows (n = 53) were sampled of day of AI and day of MRP. Pregnancy determination occurred on day 32 via transrectal ultrasonography. Sterile swabs were inserted into the vagina, rotated eight times against the vaginal wall, and stored in sterile microcentrifuge tubes at -80°C. Swab samples were utilized for subsequent bacterial diversity analyses targeting the V4 hypervariable region of the 16S rRNA gene. Cows underwent transrectal ultrasonography on gestational day 32 for pregnancy determination. Sample quality was determined by FastQC before being processed with the qiime2 pipeline for taxonomic classification. Alpha diversity metrics were analyzed with Kruskal-Wallis and beta diversity was measured utilizing PERMANOVA in the qiime2 pipeline. At day 32 of gestation, the pregnancy rate was 47.2%. According to Faith’s Phylogenetic Diversity metric for alpha diversity, there was no significant difference in diversity richness (i.e., number of different bacterial species within a sample) between day of AI and MRP or between cows diagnosed as Open or Pregnant (P >0.05). Pielou’s Evenness metric (i.e., distribution or balance of bacterial species within a sample) for alpha diversity indicated greater diversity within samples at day of AI compared with day of MRP (P = 0.02). Shannon’s diversity index metric (i.e., richness and evenness within a sample) for alpha diversity also indicated greater diversity at day of AI compared with day of MRP (P = 0.04). However, there was no difference between resulting Open and Pregnant females for Pielou’s or Shannon’s metrics (P > 0.05). Distinctions between samples (beta diversity) were compared utilizing pairwise comparisons of the unweighted and weighted Unifrac distance metrics. Specifically, day of AI indicated lower diversity compared with day of MRP for both metrics (P < 0.01). However, upon comparisons of Open and Pregnant status, there was no difference between samples for unweighted or weighted Unifrac distances (P > 0.05). Based on these results, bacterial diversity within and between vaginal samples appear to shift between day of AI and day of MRP. Additional studies are needed to elucidate the mechanisms altering bacterial diversity during early gestation in lactating dairy cows.

MUC1 regulation in the left and right uterine horns and conceptus trophectoderm during the peri-implantation period of dromedary camel

Pregnancy maintenance in dromedary camels poses significant challenges, including early embryonic loss in the left uterine horn (LH) and unsuccessful pregnancy in the right uterine horn (RH), suggesting a potential asynchrony between conceptus signaling and uterine receptivity. The transition of the uterine epithelium from a pre-receptive to a receptive state requires a delicate balance of adhesion-promoting and anti-adhesion molecules. Mucin-1 (MUC1) acts as an anti-adhesive molecule on the uterine luminal (LE) and glandular (GE) epithelium. Downregulation of MUC1 is believed to be crucial for successful embryo attachment in various mammals. This study aimed to investigate the temporospatial expression of MUC1 in the LH and RH on Days 8, 10, and 12 pregnant dromedaries and their conceptuses. Quantitative real-time polymerase chain reaction (qrt-PCR), Western blot analysis, immunohistochemistry, and immunofluorescence techniques were employed to assess MUC1 expression at the mRNA and protein levels. The results demonstrated a reduction in MUC1 mRNA expression on Day 8, then increased on Day 10, followed by a decrease on Day 12 in LH. While the RH exhibited progressive increases, peaking on Day 12. However, MUC1 expression constantly exhibited higher levels in RH than in LH in all days. Two bands were detected at 150-kDa and 180-kDa, with the highest intensity observed on Day 10. Spatially, MUC1 was localized in the apical, cytoplasmic, and lumen of uterine glands only. MUC1 was barely detectable on Day 8 but gradually increased on Days 10 and 12 in both horns. Likewise, the RH exhibited higher MUC1 signals than the LH on Days 10 and 12. In the conceptuses, MUC1 mRNA increased on Day 8, peaked on Day 10, and declined on Day 12. Notably, MUC1 protein was detected in both the trophectoderm and endoderm, with high expression observed on Day 10 and reduced by Day 12. In conclusion, the decrease in MUC1 expression on Day 8 in the LH may be associated with maternal recognition of pregnancy (MRP), and the increase on Day 10 may related to embryo protection and movement, while the subsequent decrease on Day 12 could be linked to the embryo attachment and preparation for the implantation. Conversely, the increase of MUC1 in the RH implies a role in the anti-adhesion mechanism. These findings contribute to understanding MUC1's involvement in reproductive processes and provide insights into the complex mechanisms underlying successful pregnancy establishment and maintenance in dromedary camels.

Transcriptomic analysis of bovine endometrial epithelial cells in response to interferon tau and hormone stimulation.

The embryonic loss during early stage of gestation is one of the major causes of infertility for domestic ruminants, causing huge economic losses to pasture. Maternal recognition of pregnancy and implantation are the crucial process for determining the successful establishment and development of pregnancy in cattle. The research on molecular mechanisms of pregnancy recognition will facilitate illustrating the complex process of pregnancy establishment and help to improve pregnancy outcomes. In this study, we performed transcriptomic analysis of primary bovine endometrial epithelial cells (BEND) with or without IFNT and hormones intervention through RNA sequencing. We eventually identified 608 differentially expressed genes (DEGs) including 409 up-regulated genes and 199 down-regulated genes in IFNT and hormones-treated group compared with control group. Gene Ontology (GO) enrichment analysis demonstrated that the majority of DEGs were implicated in immune system process, response to external stimulus, response to cytokine, regulation of response to stress. Results from KEGG analysis showed a significant enrichment of NOD-like receptor signaling pathway, antigen processing and presentation, necroptosis, oxidative phosphorylation, RIG-I-like receptor signaling pathway. Additionally, a set of promising candidate genes, including (USP18, STAT1, PSMB8, IFIH1, MX2, IFI44, DHX58, CASP8, DRAM1, CXCR4), were characterized by constructing an integrated interaction network. Specifically, the mRNA expression of HOXA11, PTGS1 and PTGS2 were remarkably suppressed by silencing DRAM1 under IFNT and hormone administration, thus speculating that DRAM1 might play a crucial role in early pregnancy by regulating endometrial function. The results of this study depicted a relatively comprehensive transcriptional profiles of BEND in response to IFNT and hormones, which contributes to a better understanding of gene interaction network and underlying regulatory mechanisms in endometrium of ruminants during early pregnancy.

Early transcriptomic changes in peripheral blood 7 days after embryo transfer in dairy cattle

A common goal of the dairy industry is to shorten the calving interval to reap several benefits associated with improved fertility. Early pregnancy detection is crucial to shorten this interval, allowing for prompt re-insemination of cows that failed to conceive after the first service. Currently, the industry lacks a method to accurately predict pregnancy within the first 3 wk. The polypeptide cytokine IFN-tau (IFNT) is the primary signal for maternal recognition of pregnancy in ruminants. As IFNT is released from the early conceptus, it initiates a cascade of effects, including upregulation of IFN-stimulated genes (ISG). Expression of ISG can be detected in the peripheral blood. The present study aimed to characterize peripheral transcriptomic changes, including the ISG, as early as d 7 after embryo transfer. A total of 170 Holstein heifers received in vitro-produced embryos. Whole blood was collected from these heifers within 24 h of the embryo transfer (d 0), d 7, and d 14 after embryo transfer. The heifers were divided into 2 groups, pregnant and nonpregnant, based on pregnancy diagnosis on d 28 via ultrasound. Total RNA was extracted from the peripheral blood of pregnant and nonpregnant heifers, pooled and sequenced. Expression analysis on d 7 heifers resulted in 13 significantly differentially expressed genes mostly related to innate immunity. Differential expression analysis comparing pregnant heifers on d 0 to the same heifers on d 14 showed 51 significantly differentially expressed genes. Eight genes were further quantified through reverse-transcription quantitative real-time PCR for biological validation. On d 7 after embryo transfer, mRNA transcriptions of EDN1, CXCL3, CCL4, and IL1A were significantly upregulated in pregnant heifers (n = 14) compared with nonpregnant heifers (n = 14), with respective fold changes of 8.10, 18.12, 29.60, and 29.97. Although on d 14 after embryo transfer, mRNA transcriptions of ISG15, MX2, OASY1, and IFI6 were significantly upregulated in the blood of pregnant heifers (n = 14) compared with the same heifers on d 0, with respective fold changes of 5.09, 2.59, 3.89, and 3.08. These findings demonstrate that several immune-related genes and ISG are activated during the first 2 wk after embryo transfer, which may explain how the maternal immune system accommodates the allogenic conceptus. To further investigate the diagnostic potentials of these genes, future studies are warranted to analyze the specificity and sensitivity of these biomarkers to predict early pregnancy.

Transcriptomic Changes in Response to Form of Selenium on the Interferon-Tau Signaling Mechanism in the Caruncular Tissue of Beef Heifers at Maternal Recognition of Pregnancy.

We have reported that selenium (Se) provided to grazing beef cattle in an inorganic (ISe) form versus a 1:1 mixture (MIX) of inorganic and organic (OSe) forms affects cholesterol biosynthesis in the corpus luteum (CL), the abundance of interferon tau (IFNτ) and progesterone (P4)-induced mRNAs in the caruncular (CAR) tissue of the endometrium, and conceptus length at maternal recognition of pregnancy (MRP). In this study, beef heifers were supplemented with a vitamin-mineral mix containing 35 ppm Se as ISe or MIX to achieve a Se-adequate status. Inseminated heifers were killed at MRP (d 17, n = 6 per treatment) for tissue collection. In CAR samples from MIX versus ISe heifers, qPCR revealed that mRNA encoding the thyroid regulating DIO2 and DIO3 was decreased (p < 0.05) and a complete transcriptomic analysis revealed effects on the interferon JAK-STAT1/2 pathway, including decreased expression of mRNAs encoding the classical interferon stimulated genes IFIT1, IFIT2, IFIT3, IRF1, IRF9, ISG15, OAS2, and RSAD2 (p < 0.05). Treatment also affected the abundance of mRNAs contributing to the immunotolerant environment (p < 0.05). In combination, these findings suggest more advanced preparation of the CAR and developing conceptus for implantation and to evade immune rejection by the maternal system in MIX- vs. ISe-treated heifers.

Uterine secretome: What do the proteins say about maternal-fetal communication in buffaloes?

The aim was to compare the UF proteomics of pregnant and non-pregnant buffalo during early pregnancy. Forty-four females were submitted to hormonal estrus synchronization and randomly divided into two groups: pregnant (n = 30) and non-pregnant (n = 14). The pregnant group was artificially inseminated and divided into a further two groups: P12 (n = 15) and P18 (n = 15). Conceptus and uterine fluid samples were collected during slaughter at, respectively, 12 and 18 days after insemination. Of all the inseminated females, only eight animals in each group were pregnant, which reduced the sample of the groups to P12 (n = 8) and P18 (n = 8). The non-pregnant group was also re-divided into two groups at the end of synchronization: NP12 (n = 7) and NP18 (n = 7). The UF samples were processed for proteomic analysis. The results were submitted to multivariate and univariate analysis. A total of 1068 proteins were found in the uterine fluid in both groups. Our results describe proteins involved in the conceptus elongation and maternal recognition of pregnancy, and their action was associated with cell growth, endometrial remodeling, and modulation of immune and antioxidant protection, mechanisms necessary for embryonic maintenance in the uterine environment. SignificanceUterine fluid is a substance synthesized and secreted by the endometrium that plays essential roles during pregnancy in ruminants, contributing significantly to embryonic development. Understanding the functions that the proteins present in the UF perform during early pregnancy, a period marked by embryonic implantation, and maternal recognition of pregnancy is of fundamental importance to understanding the mechanisms necessary for the maintenance of pregnancy. The present study characterized and compared the UF proteome at the beginning of pregnancy in pregnant and non-pregnant buffaloes to correlate the functions of the proteins and the stage of development of the conceptus and unravel their processes in maternal recognition of pregnancy. The proteins found were involved in cell growth and endometrial remodeling, in addition to acting in the immunological protection of the conceptus and performing antioxidant actions necessary for establishing a pregnancy.

Induced endometrial inflammation compromises conceptus development in dairy cattle†.

Endometrial inflammation is associated with reduced pregnancy per artificial insemination (AI) and increased pregnancy loss in cows. It was hypothesized that induced endometritis alters histotroph composition and induces inflammatory signatures on conceptus that compromise development. In Experiment 1, lactating cows were assigned to control (CON; n = 23) or to an intrauterine infusion of Escherichia coli and Trueperella pyogenes (ENDO; n = 34) to induce endometritis. Cows received AI 26days after treatment, and the uterine fluid and conceptuses were collected on day 16 after AI. In Experiment 2, Holstein heifers were assigned to CON (n = 14) or ENDO (n = 14). An embryo was transferred on day 7 of the estrous cycle, and uterine fluid and conceptuses were recovered on day 16. Composition of histotroph and trophoblast and embryonic disc gene expression were assessed. Bacterial-induced endometritis in lactating cows altered histotroph composition and pathways linked to phospholipid synthesis, cellular energy production, and the Warburg effect. Also, ENDO reduced conceptus length in cows and altered expression of genes involved in pathogen recognition, nutrient uptake, cell growth, choline metabolism, and conceptus signaling needed for maternal recognition of pregnancy. The impact of ENDO was lesser on conceptuses from heifers receiving embryo transfer; however, the affected genes and associated pathways involved restricted growth and increased immune response similar to the observed responses to ENDO in conceptuses from lactating cows. Bacterial-induced endometrial inflammation altered histotroph composition, reduced conceptus growth, and caused embryonic cells to activate survival rather than anabolic pathways that could compromise development.

Dose- and time-dependent effects of interferon tau on bovine endometrial gene expression

Failure by the developing conceptus to secrete sufficient interferon tau (IFNT), required for maternal recognition of pregnancy (MRP), at the appropriate time is related to early pregnancy loss in cattle. We aimed to test the hypothesis that there is a dose- and time-dependent relationship between IFNT and the endometrial expression of key interferon-stimulated genes (ISGs) involved in the signalling cascade leading to MRP in cattle. Candidate genes were identified first through a bioinformatic approach, where integrated transcriptomic data from two previous studies were analyzed to identify endometrial genes induced by IFNT. Next, expression of selected candidate genes was investigated in vitro in endometrial explants. Endometrial explants collected from cows (n = 8) in the late luteal phase of the estrous cycle were cultured in medium without (control) or with recombinant ovine IFNT (1, 10, 100 ng/mL) for 6 h. Simultaneously, endometrial explants were cultured in medium containing 100 ng/mL IFNT for different time periods (15 min, 30 min, 1 h, 3 h, 6 h). Gene expression was analyzed by RT-qPCR. We identified 54 endometrial genes responding to IFNT and to some degree to the conceptus, from which five ISGs (CMPK2, BPNT1, IFI35, TNFSF10 and TRIM38) were further selected for the dose- and time-dependent experiments. Classical ISGs (ISG15, OAS1, MX1 and MX2) were up-regulated (P < 0.05) in endometrium by 1 ng/mL IFNT. However, other selected ISGs (CMPK2, BPNT1, IFI35, TNFSF10 and TRIM38) were induced only by higher concentrations (10 and 100 ng/mL) of IFNT (P < 0.05). In terms of duration of exposure, IFNT at 100 ng/mL induced a significant (P < 0.05) increase in ISG15 and CMPK2 expression after 1 h incubation, while all other studied ISGs in the endometrium were upregulated when cultured for 3 or 6 h, but did not affect expression when the duration of culture was for 1 h or less. These results suggest that IFNT acts on the uterus in both a dose- and time-dependent manner in cattle and that timely exposure of the endometrium to sufficient IFNT is essential for appropriate signalling to ensure successful pregnancy establishment.

Equine endometrial Th1 and Th2 evaluation during early pregnancy

As in all mammals, continuous feto-maternal dialogue is essential for successful pregnancy in mares. Nevertheless, several features of pregnancy in the mare are unusual and differ markedly from equivalent events in other species. The nature of the signal by which the equine embryo biochemically indicates its presence in the mare´s uterus for pregnancy maintenance remains a mystery to the present day, but the equine conceptus must begin to release this factor resulting in maternal recognition of pregnancy around day 10 to day 13 after ovulation. In humans and some animal species, the Th1/Th2 balance is essential for immunological tolerance in pregnancy. This immunohistochemical study aimed to assess the T-bet (Th1) and GATA3 (Th2) lymphocyte populations in the endometrium on days 7, 10, and 13 after ovulation in cyclic and pregnant mares. Mares were randomly assigned to the day of sample collection in both groups. In the first cycle, endometrial biopsies from 30 cyclic mares were collected on days 7 (n = 10), 10 (n = 10), and 13 (n = 10) post-ovulation (Cyclic group). In the second cycle, the same mares were bred to a fertile stallion, and endometrial samples were collected on days 7, 10, and 13 post ovulation. Immediately after biopsies, uteri were flushed, and those with an embryo recovered were allotted to the Pregnant group. Of the 30 mares flushed, embryos were recovered from6 mares on day 7, 6 on day 10, and 6 on day 13. Samples from mares without embryo recovery were excluded from both groups. Immunohistochemistry was used to detect Th1 (T-bet PA5-28881, Invitrogen) or Th2 (GATA3 L50-823, Thermo Fisher) proteins in the endometrium. Only stained lymphocytes from the stratum compactum (SC) were counted. Five fields were analyzed at 400x magnification. A two-way ANOVA was performed to evaluate the absolute number of T-bet/GATA3 cells and their ratio as a dependent factor and experimental groups, day, and their interactions as independent variables. T-bet-stained cells decreased from days 7 to 13 in both the Cyclic (day 7=73 vs 13=48) and Pregnant groups (day 7=83 vs 13=39) (P=0.0179); no difference was observed between groups. GATA3 stained cells remained constant over the days, with more stained cells in the Pregnant (day 7=37; 10=35; 13=34) than in the Cyclic group (day 7=23; 10=12; 13=12) (P=0.0001). The Th1/Th2 ratio did not show a difference (P>0.2222). Th2 cells are recruited into the SC of the endometrium of the pregnant mare. These cells could be secreting interleukins and establishing an anti-inflammatory microenvironment in early pregnancy. In conclusion, the presence of Th2 cells in pregnant mares could be important for embryonic development and maternal recognition of pregnancy regarding immunological tolerance.

Insights into uterine fluid proteins and putative roles on maternal recognition of pregnancy

The embryo is dependent on uterine secretions in early pregnancy and notably around the time of maternal recognition of pregnancy (MRP). This study aimed to identify key proteins in the uterine fluid and embryo before and around the time of MRP. We hypothesized that uterine fluid has a unique signature of proteins around the time of MRP. Mares were bred to a fertile stallion until ten embryos were obtained for each of days 7, 10 and12 post-ovulation. A gynecological tampon was inserted in utero for collection of the uterine fluid. Embryo flushing was carried out with Lactated Ringers Solution. Recovered embryos were flushed in DPBS, and then stored in DPBS enriched with proteinase inhibitors at -80°C. Uterine fluid was centrifuged, and the supernatant was stored at -80°C until analysis. The embryo was sonicated, centrifuged as the uterine fluid, and then stored at -80°C until analysis. Total protein was determined by the acid bicinchoninic method, in-gel tryptic digestion was carried out and samples were analyzed by mass-spectrometry (ESI-Q-TOF-MS/MS). Gene-ontology was carried out on a Panther, vs17.0. Multivariate-analyses based on principle component analysis (PCA), and partial least squares discriminant analysis-variables importance (PLS-DA/VIP) score were performed with Metaboanalyst, v5.0 via R-studio. The total protein numbers found at Days 7, 10, and 12 were 75, 81 and 84 in the uterine fluid and 151, 139, and 178 inthe embryos, respectively. The most abundant proteins (VIP-score, α>1.5) present in the embryos at Days 7, 10, and 12 were 0, 6 and 2; whereas uterine fluid had 0, 3 and 2 for the same respective days. Molecular functions found on gene-ontology were catalytic activity and binding in both samples from embryos and uterine fluids. There was a remarkable change in PCA from Day 7 to Day 10/12 in the embryo and uterine fluid proteins. Proteins found in great abundance in the embryos PLS-DA/VIP-score analysis were related to protection against thermal-stress, protein folding, stabilization, translation, lipid-metabolism and cell-structure. Cathepsin-B is involved with endometrial remodeling and implantation, but its role in the horse remains to be determined. Uterine fluid had proteins associated with the transport of molecules with low-molecular-weight; in the upregulation of the ERK1- and ERK2-cascades, which are essential for cell-proliferation, differentiation, adhesion, migration, survival; and calcium/phosphorus metabolism. Herein is the first description of Stanniocalcin-1 in equine embryos. In conclusion, the proteins found in utero may play a role in the MRP and the embryo proteins were related to mobility and protection functions. Acknowledgements: The Mass Spectrometry Facility at Brazilian Biosciences National Laboratory (LNBio), CNPEM, Campinas, Brazil is thanked for their support with the use of equipment.

Differential gene expression in the equine endometrium and conceptus from Day 11 to 16 post-ovulation

Mobility of the equine conceptus is essential for maternal recognition of pregnancy (MRP) and is regulated by the conceptus's own derived prostaglandin secretion. Our objective was to compare prostaglandin-related genes and other genes of interest during the expected MRP time. Light breed mares were examined daily by transrectal ultrasound when in estrus until the day (D) of ovulation. Mares in the pregnant (P) group were bred by artificial insemination with >500 million progressively motile normal sperm from one fertile stallion until D0. Conceptuses were detected and measured via transrectal ultrasound and collected via uterine lavage using 0.9% NaCl through a transcervical bivona catheter. Endometrium was collected via a transcervical biopsy punch following conceptus retrieval. Endometrium of non-pregnant (NP) mares was collected at: D11 (NP11; n=6) and D13 (NP13; n=7), while endometrium from the pregnant horn and conceptuses was collected at D11 (P11; n=7), D13 (P13; n=8), and D16 (P16; n=6). Total RNA from trophoblast and endometrial samples was isolated and evaluated for expression of PTGS2, PTGES, mPGES2, PTGER4, PTGER2, PTGIS, PTGIR, HPGD, SLCO2A1, CRTC2, NR3C1, CBR1, CREB, NF-Kβ, SPP1, IGFBP1, ESR1 and DNTTIP2 using Real-time PCR. Mean threshold cycle was determined and normalized to the reference gene (GAPDH and ACTB) (ΔCT). Statistical analyses were performed using JMP Pro 16, and data was ranked, transformed if necessary, followed by a restricted maximum likelihood model with mare as arandom factor to test differences between Groups and Day. Regarding endometrial mRNA, NP11 had higher expression for NF-Kβ, PTGIS, PTGIR and CBR1 compared to NP13 (p<0.007). ESR1, mPGES2, PTGIS, and CBR1 mRNA were higher in P11 compared to P16 (p<0.05), while CRTC2 was higher in P16 compared to P11 (p<0.06). Lastly, NF-Kβ, PTGIR and CBR1 were higher in P11 compared to P13 (p<0.01), but NF-Kβ P13 was lower compared to P16 (p<0.04). Regarding the conceptuses, CREB and PTGFR expressions were higher in D16 compared to D11 (p<0.003), while CRTC2, NR3C1, NF-Kβ and SPP1 were higher in D16 compared to D11 and 13 (p<0.02). D13 conceptuses had lower expression of HPGD compared to D11 and 16 (p<0.02). Lastly, D11 conceptuses had higher expression of mPGES2, PTGIS and PTGER4 compared to D13 and D16 (p<0.03). Higher expression of D11 PTGIS from both tissues may increase nutrient and blood flow to the endometrium in support of the early conceptus. The upregulation of endometrial NF-Kβ at D11 may be a result of endometrial receptivity through the regulation of inflammation, and the up-regulation of HPGD may function to regulate PGE2 during early pregnancy. Understanding embryonic and endometrial prostaglandin regulation during MRP may help to develop future therapies to ameliorate pregnancy loss in the horse.

Global gene expression in equine endometrium around the time of maternal recognition of pregnancy

The conceptus-maternal communication during the preimplantation period, also known as maternal recognition of pregnancy (MRP), is crucial for the establishment and maintenance of pregnancy. This communication affects the endometrial transcriptome and proteome with subsequent reduction of endometrial pulsatile prostaglandin F2α secretion and the consequent extension of the corpus luteum's lifespan during early pregnancy in the mare. Also, this communication results in the preparation of the uterine environment for implantation. Any alteration of these processes will result in early embryonic death. Therefore, a better understanding of the normal endometrial transcriptomic changes around the time of MRP might be the key to understanding and preventing early embryonic loss. This study aimed to evaluate the endometrial transcriptome from Day 14 (Day 0 = ovulation) of pregnant and non-pregnant (i.e. diestrus) mares. RNA was isolated from pregnant (n=5) and non-pregnant (n=4) endometrial tissue samples. RNA-Seq was performed using Illumina NovaSeq6000 and differentially expressed genes (DEGs) were evaluated using DESeq.2 based upon an FDR<0.05. Our study identified 1193 DEGs (570 upregulated DEGs and 623 downregulated DEGs) in the pregnant endometrium as compared to the non-pregnant endometrium. In the current study, the pregnant endometrium was associated with downregulation (9.6-fold change) of mRNA forprostaglandin-endoperoxide synthase 2 (PTGS2, also known as cyclooxygenase-2 enzyme; COX2), which encodes for the rate-limiting enzyme in prostaglandin production. Gene ontology analysis of DEGs revealed that these genes were associated with relevant biological processes such as immune response (TNFAIP1, TNF, CXCR2, TNFSF15, IL15, TGFBR3, IL1B, TNFSF9, TLR8), regulation of trophoblast cell proliferation and migration (BOK, LIF, RBM46, TIMP1, CALR, GJA1), embryo growth and development (PTCH1, PTCH2R, DH10, GLI3, SHOX2, ZDBF2, EVX1, GREM2, KLF2, TWIST1), cell adhesion (COL28A1, EGFL6, ITGA3, FN1, NEDD9, LAMC2, RGMB, THBS4, RHOB, SELP, CHL1, CDHR1, MXRA8, FOLR3, NECTIN3), embryo implantation (MMP9 and LIF), placenta development (ADAM19, MAPK14, BIRC3), amino acids transportation (SLC1A1, SLC25A29, SLC36A2, SLC38A2, SLC7A5), steroid biosynthesis and metabolism (SRD5A1, HSD11B2, HSD17B7, CYP20A1), and progesterone receptors (PAQR5, and PGRMC1). Several of the DEGs identified in the current study are known to play a role in controlling endometrial receptivity to the conceptus in species other than the equine. These findings improve our understanding of the molecular changes taking place in equine endometrium around the time of MRP which might help us to understand and forestall early embryonic loss in the future.

Characterization of interferons in the equine conceptus and endometrium

Interferons (IFN) are involved in the maintenance and establishment of pregnancy in a variety of species. There are three types of IFNs, which include type I (IFN-α, β, ε, τ, and δ), type II (IFN-γ), and type III (IFN-λ1, λ2 and λ3). In the horse, IFNA, IFND and IFNO are expressed in the equine trophoblast, and IFNE is expressed in higher abundance in equine endometrium during the luteal phase. However, investigation of IFNs and their receptors during early equine pregnancy is still lacking. Our objective was to investigate the expression of IFN types I and II and their receptors, and other IFN related genes in equine conceptuses and endometrium during early pregnancy. Mares were examined daily by transrectal ultrasound when in estrus until the day (D) of ovulation (OV). Pregnant (P) group mares were bred using artificial insemination by a single fertile stallion. Conceptuses were detected and measured via transrectal ultrasound and collected via uterine lavage using 0.9% NaCl through a transcervical bivona catheter. Following conceptus retrieval, an endometrial biopsy was collected. Endometrial samples from nonpregnant (NP) mares were collected at D11 (NP11; n=6) and D13 (NP13; n=7), while endometrium and conceptuses were collected from pregnant mares at D11 (P11; n=7), D13 (P13; n=8), and D16 (P16; n=6). As the majority of nonpregnantmares would have undergone luteolysis, a D16 sample was not included in this study. Total RNA was isolated and evaluated for expression of interferon-related genes using Real-time PCR. The mean threshold cycle was determined and then normalized to the mean of the reference genes (GAPDH and ACTB) (ΔCT). A restricted maximum likelihood model was performed in JMP Pro 16 (Cary, NC) with mare as a random factor with significance set at P<0.05. There was no amplification for IFNB and IFND in the endometrium, and no amplification of IFNG, IFNB and IFNAR2 in the conceptus. In the endometrium, IFNAR1 and IFNGR1 had higher expression in P11 compared to P13 (P<0.02). Expression of endometrial IFNG mRNA was higher in P11 and P16 compared to P13 (P<0.004). There was no difference in the mRNA relative abundance of endometrial ISG15 and IFITM1 between groups or days. In the conceptus, IFNA, IFNO, and IFNGR2 expression were higher in D16 compared to other ages (P<0.05). D11 and D16 conceptuses had higher expression of IFNAR1 compared to D13 (P<0.0002), while IFNGR1 was higher in D16 compared to D11 (P<0.004). The role of interferons during maternal recognition of pregnancy in the horse is unknown. Unlike the pig, the horse embryo does not seem to express IFNG. Further investigation is underway.

Embryo-maternal interactions revealed by phosphoproteomic analysis of endometrial organoids exposed to embryo secretome

The equine embryo remains mobile and without physical attachment to the endometrium for a prolonged period in early pregnancy. During this time, the embryo only communicates with the maternal system via secreted compounds in its immediate environment. These interactions are poorly understood, and a definitive signal for maternal recognition of pregnancy remains elusive. In this study, we sought to develop a model to examine embryo-maternal interactions at close range in vitro, demonstrating that incubation of endometrial organoids with embryo-conditioned medium results in detectable changes in the endometrial phosphoproteome. Mare endometrial organoids were derived from isolated endometrial glands of uteri obtained immediately post-mortem. Embryos were recovered by uterine lavage from Standardbred mares at day 8-9 post-ovulation, washed and pre-cultured in BSA-containing medium under oil, and cultured in protein-free medium under oil. Embryo-conditioned and control media were collected at 12 h, centrifuged to remove debris and stored at −80°C until further analysis. For co-incubation experiments, organoids were primed with 10nM estradiol (48 h), then 10nM estradiol + 1 µM progesterone (48 h). Embryo-conditioned (n=3) or control (n=3) media were added at a dilution of 1:100 in organoid medium and co-incubated for 30 minutes. Organoids were then recovered and processed to remove media and secretome components (on ice and in the presence of phosphatase inhibitors). Cell pellets were frozen at -80˚C until protein extraction. Proteins were extracted with urea-based extraction buffer, reduced,alkylated, and digested with trypsin. Phosphopeptides were isolated using titanium dioxide before LC tandem mass spectrometry (MS/MS) using a Q-Exactive Plus hybrid quadrupole-Orbitrap MS system (Thermo Fisher Scientific, Bremen, Germany). Endometrial organoids were successfully cultured in vitro and their functional mimicry of in vivo endometrium was confirmed via multiple routes including histological analysis of gland-like structures, epithelial and endometrial markers, and RNA sequencing alongside isolated endometrial glands. Phosphoproteomic mass spectrometry of endometrial organoids revealed a profile of 181 phosphopeptides mapping to 90 protein IDs. Of these, 10 phosphopeptides were decreased (log2[fold-change] < -0.8) in co-incubated organoids, while 3 were uniquely detected in secretome-exposed organoids only. Most notably, we observed a decrease in the phosphorylated peptides of PGRMC1 (progesterone receptor membrane component 1). This protein forms part of a non-classical system of progesterone action, located in the cell membrane. A change in PGRMC1 phosphorylation suggests that an alternative system of progesterone receptor activation participates in equine embryo-maternal signaling. There were also changes in phosphorylation status of chaperone proteins essential to progesterone receptor assembly (e.g., HSP90AB1). In summary, this study describes the immediate response of mare endometrial organoids to compounds released by embryos, identifying novel mechanisms possibly implicated in signaling between the embryo and maternal system.

The Timing of the Maternal Recognition of Pregnancy Is Specific to Individual Mares.

The present experiment aimed at determining whether the timing of the maternal recognition of pregnancy (MRP) was specific to individual mares by determining when luteostasis, a failure to return to oestrus, reliably occurred in individuals following embryo reduction. Singleton (n = 150) and synchronous twin pregnancies (n = 9) were reduced in 10 individuals (5-29 reductions/mare) at pre-determined time points within days 10 (n = 20), 11 (n = 65), 12 (n = 47), 13 (n = 12) or 14 (n = 15) of pregnancy. Prior to embryo reduction, the vesicle diameter was measured in 71% (106/150) of the singleton pregnancies. The interovulatory interval (IOI) was recorded on 78 occasions in seven of the mares in either non-pregnant cycles (n = 37) or those in which luteolysis followed embryo reduction (n = 41). The earliest time post-ovulation at which the embryo reduction resulted in luteostasis in an individual was 252 h (mid-Day 10). Consistency in luteostasis following embryo reduction showed individual variation between mares (272-344 h). Binary logistic regression analysis showed an individual mare effect (p < 0.001) and an effect of the interval post-ovulation at which embryo reduction was undertaken (p < 0.001). However, there was no significant effect of vesicle diameter at the time of embryo reduction (p = 0.099), nor a singleton or twin pregnancy (p = 0.993), on the dependent of luteolysis or luteostasis. The median IOI between individual mares varied significantly (p < 0.05) but was not correlated to the timing of MRP. The timing of MRP varied between the mares but was repeatable in each individual. The factors and mechanisms underlying the individuality in the timing of MRP were not determined and warrant further study.