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During early pregnancy, porcine conceptuses (the embryos with associated membranes) secrete estradiol-17β (E2)—their major signal for maternal recognition of pregnancy—and prostaglandin E2(PGE2). Both hormones induce prominent changes of the endometrial transcriptome in vivo. Studies on endometrial pathologies have shown that E2affects gene expression by epigenetic mechanisms related to DNA methylation. Herein, we determined the effects of E2and PGE2alone, and a combined E2+ PGE2treatment administered into the uterine lumen in vivo on the expression and activity of DNA-methyltransferases (DNMTs) and on CpG methylation patterns of selected genes in porcine endometrium. To compare the effect of treatment with the physiological effect of pregnancy, endometria from day 12 pregnant/cyclic gilts were included. Both E2and PGE2significantly reduced the expression of DNMTs. Likewise, the expressions of DNMT1 and DNMT3A were decreased on day 12 of pregnancy compared to the estrous cycle. DNMT activity increased in endometrial samples following E2treatment and in gilts on day 12 of pregnancy. Treatment with E2alone and/or simultaneously with PGE2altered endometrial DNA methylation of CpG sites of ADAMTS20, ADH1C, BGN, PSAT1, and WNT5A. Different CpG methylation patterns of ADAMTS20, BGN, DMBT1, RASSF1, and WNT5Awere found in the endometrium on day 12 of pregnancy compared to day 12 of the estrous cycle. Significant correlations were detected between CpG methylation and gene expression for ADAMTS20, ADH1C, BGN, DMBT1, PSAT1, and WNT5A. Our results indicate that CpG methylation induced by embryonic signals may contribute to regulating endometrial gene expression during pregnancy establishment.Estradiol-17β acting alone and/or simultaneously with PGE2in vivo alters DNA methylation and regulates the expression of genes in the porcine endometrium during early pregnancy.Graphical Abstract

Numerous intrauterine changes take place across species during embryo development. Following fertilization in July/August, the European roe deer (Capreolus capreolus) embryo undergoes diapause until embryonic elongation in December/January. Embryonic elongation prior to implantation is a common feature among ungulates. Unlike many other ruminants, the roe deer embryo does not secrete interferon-tau (IFNτ). This provides the unique opportunity to unravel IFNτ-independent signaling pathways associated with maternal recognition of pregnancy (MRP). This study aimed at identifying the cell-type-specific endometrial gene expression changes associated with the MRP at the time of embryo elongation that are independent of IFNτ in roe deer. The messenger RNA (mRNA) expression of genes known to be involved in embryo-maternal communication in cattle, pig, sheep, and mice was analyzed in laser capture microdissected (LMD) endometrial luminal, glandular epithelial, as well as stromal cells. The mRNA transcript abundances of the estrogen (ESR1), progesterone receptor (PGR), and IFNτ-stimulated genes were lower in the luminal epithelium in the presence of an elongated embryo compared to diapause. Retinol Binding Protein-4 (RBP4), a key factor involved in placentation, was more abundant in the luminal epithelium in the presence of an elongated embryo. The progesterone receptor localization was visualized by immunohistochemistry, showing an absence in the luminal epithelium and an overall lower abundance with time and thus prolonged progesterone exposure. Our data show a developmental stage-specific mRNA expression pattern in the luminal epithelium, indicating that these cells sense the presence of an elongated embryo in an IFNτ-independent manner.

Maternal exposure to estrogens can induce long-term adverse effects in the offspring. The epigenetic programming may start as early as the period of preimplantation development. We analyzed the effects of gestational estradiol-17β (E2) exposure with two distinct low doses, corresponding to the acceptable daily intake “ADI” and close to the no-observed-effect level “NOEL”, and a high dose (0.05, 10, and 1000 μg E2/kg body weight daily, respectively). The E2 doses were orally applied to sows from insemination until sampling at day 10 of pregnancy and compared to carrier-treated controls leading to a significant increase in E2 in plasma, bile and selected somatic tissues including the endometrium in the high-dose group. Conjugated and unconjugated E2 metabolites were as well elevated in the NOEL group. Although RNA-sequencing revealed a dose-dependent effect of 14, 17, and 27 differentially expressed genes (DEG) in the endometrium, single embryos were much more affected with 982 DEG in female blastocysts of the high-dose group, while none were present in the corresponding male embryos. Moreover, the NOEL treatment caused 62 and 3 DEG in female and male embryos, respectively. Thus, we detected a perturbed sex-specific gene expression profile leading to a leveling of the transcriptome profiles of female and male embryos. The preimplantation period therefore demonstrates a vulnerable time window for estrogen exposure, potentially constituting the cause for lasting consequences. The molecular fingerprint of low-dose estrogen exposure on developing embryos warrants a careful revisit of effect level thresholds.Maternal oral low-dose estrogen exposure during the preimplantation period specifically targeted female embryos by inducing a male-like gene expression profile.

The pre-implantation period is prone to embryonic losses in bovine. Embryo–maternal communication is crucial to support embryo development. Thereby, factors of the uterine fluid (UF) are of specific importance. The maternal diet can affect the UF composition. Since omega 3 fatty acids (omega 3 FA) are considered to be beneficial for reproduction, we investigated if dietary omega 3 FA affected factors in the UF related to embryo elongation. Angus heifers (n = 37) were supplemented with either 450 g of rumen-protected fish oil (omega 3 FA) or sunflower oil (omega 6 FA) for a period of 8 weeks. Following cycle synchronization and artificial insemination, the uteri were flushed post mortem to recover the embryos on day 15 of pregnancy. The UF and tissue samples of endometrium and corpus luteum (CL) were collected. Strikingly, the embryo elongation in the omega 3 group was enhanced compared to the omega 6 group. No differences were observed in uterine prostaglandins, even though the endometrial concentration of their precursor arachidonic acid was reduced in omega 3 compared to omega 6 heifers. The dietary FA neither led to differential expression of target genes in endometrium nor CL nor to a differential abundance of low-density lipoprotein cholesterol, cortisol or amino acids in the UF. Interestingly, the omega 3 group displayed a higher plasma progesterone concentration during luteal growth than the omega 6 group, possibly promoting embryo elongation. Further research should include an ovarian perspective to understand the functional link between dietary omega 3 FA and reproductive outcome.The pre-implantation embryo elongation in bovine is affected by dietary omega 3/6 fatty acids likely due to an effect on the ovary rather than a change of the uterine secretome.

Establishment and maintenance of pregnancy in equids is only partially understood. To provide new insights into early events of this process, we performed a systematic analysis of transcriptome changes in the endometrium at Days 8 and 12 of pregnancy. Endometrial biopsy samples from pregnant and nonpregnant stages were taken from the same mares. Composition of the collected biopsy samples was analyzed using quantitative stereological techniques to determine proportions of surface and glandular epithelium and blood vessels. Microarray analysis did not reveal detectable changes in gene expression at Day 8, whereas at Day 12 of pregnancy 374 differentially expressed genes were identified, 332 with higher and 42 with lower transcript levels in pregnant endometrium. Expression of selected genes was validated by quantitative real-time RT-PCR. Gene set enrichment analysis, functional annotation clustering, and cocitation analysis were performed to characterize the genes differentially expressed in Day 12 pregnant endometrium. Many known estrogen-induced genes and genes involved in regulation of estrogen signaling were found, but also genes known to be regulated by progesterone and prostaglandin E2. Additionally, differential expression of a number of genes related to angiogenesis and vascular remodeling suggests an important role of this process. Furthermore, genes that probably have conserved functions across species, such as CRYAB, ERRFI1, FGF9, IGFBP2, NR2F2, STC1, and TNFSF10, were identified. This study revealed the potential target genes and pathways of conceptus-derived estrogens, progesterone, and prostaglandin E2 in the equine endometrium probably involved in the early events of establishment and maintenance of pregnancy in the mare.

Uterine secretions have a dominant impact on the environment in which embryo development takes place. The uterine serpins (SERPINA14, previously known as UTMP) are found most abundantly during pregnancy in the uterus of ruminants. Although progesterone is currently assumed to be the major regulator of SERPINA14 expression, our recent study of transcriptome changes in bovine endometrium during the estrous cycle unexpectedly detected a marked upregulation of SERPINA14mRNA levels at estrus. The present study describes the full-length mRNA sequence, genomic organization, and putative promoter elements of the SERPINA14gene. The SERPINA14mRNA abundance was quantified by real-time RT-PCR in intercaruncular endometrium at several time points during the estrous cycle and early pregnancy. Highest levels were found at estrus, followed by a dramatic decrease and a moderate expression during the luteal phase. Transcript levels were higher in pregnant endometrium compared with controls at Day 18. At estrus, immunoreactive protein was localized in deep glandular epithelium, and Western blotting concomitantly showed the 52-kDa form in uterine flushings. SERPINA14mRNA was significantly upregulated in glandular endometrial cells in vitro after stimulation with estradiol-17beta and progesterone, but not after interferon-tau treatment. Our results clearly demonstrate that SERPINA14 appears distinctly in bovine endometrium during the estrus phase. A supporting role toward providing a well-prepared endometrial environment for passing gametes, especially sperm, is assumed.

Initiation and maintenance of pregnancy are critically dependent on an intact embryo-maternal communication in the preimplantation period. To get new insights into molecular mechanisms underlying this complex dialog, a holistic transcriptome study of endometrium samples from Day 18 pregnant vs. nonpregnant twin cows was performed. This genetically defined model system facilitated the identification of specific conceptus-induced changes of the endometrium transcriptome. Using a combination of subtracted cDNA libraries and cDNA array hybridization, 87 different genes were identified as upregulated in pregnant animals. Almost one half of these genes are known to be stimulated by type I interferons. For the ISG15ylation system, which is assumed to play an important role in interferon tau (IFNT) signaling, mRNAs of four potential components (IFITM1, IFITM3, HSXIAPAF1, and DTX3L) were found at increased levels in addition to ISG15and UBE1L. These results were further substantiated by colocalization of these mRNAs in the endometrium of pregnant animals shown by in situ hybridization. A functional classification of the identified genes revealed several different biological processes involved in the preparation of the endometrium for the attachment and implantation of the embryo. Specifically, elevated transcript levels were found for genes involved in modulation of the maternal immune system, genes relevant for cell adhesion, and for remodeling of the endometrium. This first systematic study of maternal transcriptome changes in response to the presence of an embryo on Day 18 of pregnancy in cattle is an important step toward deciphering the embryo-maternal dialog using a systems biology approach.

We previously identified a microdeletion (Del) in the maternally imprinted PEG3 domain in cattle that results in loss of paternal MIMT1expression and causes late-term abortion and stillbirth. The mutation, when inherited from the sire, is semilethal for his progeny, with 85% mortality. Here we precisely delineate the deletion and describe comparative analyses of fetuses carrying the deletion with wild-type (WT) siblings. Global DNA methylation analysis of cotyledon tissue revealed greater global CpG methylation in fetuses with the deletion (P= 0.003). Gene expression microarray analyses identified increased NPSR1A, IL1RN, NOS3, IL4R, ZDHHC22, and SMOC2expression in fetuses carrying the deletion and decreased GRID1, PLG, and IGF1expression. Involvement of the NPSR1A, IL1RN, NOS3, and IL4Rgenes suggests that some form of restriction related to blood supply, perhaps hypoxemia, may play a role in the pathological mechanism. Also, imprinted genes known to play a role in mammalian prenatal development, specifically IGF2, DLK1, MEST, AST1, PEG3, APEG3, and H19, showed differential expression. The most striking difference was abundant abnormal expression of the neuropeptide S receptor 1 (NPSR1)gene in placental cotyledon tissue of 7 of 11 MIMT1Del/WTfetuses. The similarity of this proportion to that of the semilethal mortality rate suggests that abnormal NPSR1expression may be linked to death or survival of MIMT1Del/WTfetuses. NPSR1is expressed as two isoforms (A and B), and isoform A was detected in MIMT1Del/WTcotyledons. NPSR1Ais normally not expressed in placenta. Its role in the stillbirth phenomenon has yet to be elucidated, but it may provide a useful prognostic indicator.

Interferon tau (IFNT), a type I IFN similar to alpha IFNs (IFNA), is the pregnancy recognition signal produced by the ruminant conceptus. To elucidate specific effects of bovine IFNT and of other conceptus-derived factors, endometrial gene expression changes during early pregnancy were compared to gene expression changes after intrauterine application of human IFNA2. In experiment 1, endometrial tissue samples were obtained on Day (D) 12, D15, and D18 postmating from nonpregnant or pregnant heifers. In experiment 2, heifers were treated from D14 to D16 of the estrous cycle with an intrauterine device releasing IFNA2 or, as controls, placebo lipid extrudates or PBS only. Endometrial biopsies were performed after flushing the uterus. All samples from both experiments were analyzed with an Affymetrix Bovine Genome Array. Experiment 1 revealed differential gene expression between pregnant and nonpregnant endometria on D15 and D18. In experiment 2, IFNA2 treatment resulted in differential gene expression in the bovine endometrium. Comparison of the data sets from both studies identified genes that were differentially expressed in response to IFNA2 but not in response to pregnancy on D15 or D18. In addition, genes were found that were differentially expressed during pregnancy but not after IFNA2 treatment. In experiment 3, spatiotemporal alterations in expression of selected genes were determined in uteri from nonpregnant and early pregnant heifers using in situ hybridization. The overall findings of this study suggest differential effects of bovine IFNT compared to human IFNA2 and that some pregnancy-specific changes in the endometrium are elicited by conceptus-derived factors other than IFNT.

RNA ultra deep sequencing was used to strive for the generation of a complete catalog of transcriptome changes in bovine endometrium during the pre-implantation period. Endometrial tissue samples were collected from Simmental heifers after slaughter at days 15 (D15) and 18 (D18) of pregnancy and at the corresponding days of the estrous cycle (n=4 per group). RNAseq libraries starting from polyA RNA were prepared and analyzed on an Illumina Genome Analyzer IIx. For the D18 libraries 36 bp paired-end reads (sequences) were generated sequencing one sample per flow-cell lane resulting in more than 40 million uniquely mapped paired-end reads with 4 to 12 million reads per sample. These reads corresponded to approx. 15,000 annotated genes. For the D15 libraries 100 million uniquely mapped reads (54 bp paired-end) were produced ranging from 7 to 26 million reads per sample that corresponded to approx. 16,000 genes. Based on the results of the mapping to known genes normalized expression values (reads per kilobase mRNA and million reads, RPKM) were calculated for every sample and used for statistical analysis (SAM Excel Add-in). At D18 664 differentially expressed genes (DEGs) were identified (FDR 1%, at least 2-fold change), from which 414 were up-regulated and 250 down-regulated in pregnant endometrium. Analysis of the same RNA samples with Affymetrix Bovine Genome Arrays resulted in 336 DEGs (279 up- and 57 down-regulated). At D15, both RNAseq and microarray analysis revealed only up-regulated genes, namely 167 and 108 DEGs (FDR 5%, at least 2-fold change), respectively. Of the RNAseq DEGs, which were represented and detectable on the microarray, 90% (Day18) and 84% (D15) also showed differential expression in the microarray analysis indicating high consistency between RNAseq and Affymetrix microarray data. Comparison of RNAseq DEGs of D18 and D15 revealed that almost 90% of the genes up-regulated at D15 were also up-regulated at D18 of pregnancy. Direct comparison of RNAseq datasets from D15 and D18 pregnant endometrium showed that nearly all D15 DEGs had similar or higher expression levels in D18 pregnant endometrium. For selected genes differential expression was confirmed using quantitative real-time RT-PCR. Both, RNAseq and Affymetrix results showed good correlation to the qPCR results. Finally, analysis of differential splicing and 3' UTR variants based on RNAseq data is in progress to get further insights into the complex gene regulation networks in the bovine endometrium during early pregnancy. In conclusion, based on the higher sensitivity and detection of transcripts not represented on commercially available bovine microarrays, transcriptome analysis using RNAseq provides significant additional information on differential mRNA expression in bovine endometrium during early pregnancy. Furthermore, the expression values obtained by RNAseq have a more quantitative character than microarray results allowing direct comparison of different RNAseq datasets. Research supported by the German BMBF (FUGATOplus Compendium).(poster)