Initiation of Conceptus Elongation Coincides with an Endometrium Basic Fibroblast Growth Factor (FGF2) Protein Increase in Heifers.

Fibroblast growth factors (FGF) play an important role during embryo development. To date, the role of FGF and the respective receptors (FGFR) during the preimplantation phase in cattle are not fully characterized. We examined FGF1, FGF2, FGFR1, FGFR2, and FGFR3 in cyclic and early pregnant heifers at Days 12, 15, and 18 after insemination (Day 0). Endometrial FGF1 mRNA transcript abundance in heifers varied significantly with respect to the day after insemination, the pregnancy status, and their interaction. The expression was higher in nonpregnant than in pregnant heifers at Day 18. The conceptus transcripts abundance of FGFR2 and FGFR3 were significantly lower at Day 15 than 18. In the endometrium, FGF1 protein abundance significantly decreased from Day 12 onwards and FGF2 protein abundance showed a minor, but a significant increase at Day 15 in comparison to Days 12 and 18. We concluded that the decrease in FGF1 mRNA expression in pregnant heifers at Day 18 points towards a potential contribution of FGF1 in the preimplantation process. Additionally, successful embryo elongation might require a spatiotemporal FGF2 protein increase in the endometrium.

Vascular Endothelial Growth Factor A and VEGFR-1 Change during Preimplantation in Heifers.

Vascular endothelial growth factor A (VEGFA) plays a critical angiogenic role in the endometrium of placentalia during preimplantation. The role of VEGFA and its receptors is not fully characterised in bovine reproduction. We analysed the mRNA expression of VEGFA isoforms 121, 165 and 189, and VEGF receptors 1 and 2 in three experimental settings (A, B and C). We compared intercaruncular endometrium of cyclic to pregnant heifers at Days 12, 15 and 18 post insemination (Day 0), and between Day 15 and Day 18 conceptuses (A). We further compared caruncular versus intercaruncular endometrium at Day 15 (B), and endometrium of heifers carrying embryos originating from somatic cell nuclear transfer (SCNT) versus in vitro fertilisation (IVF) at Day 18 (C). Endometrial VEGFA protein was localised and quantified. Pregnant heifers displayed lower intercaruncular endometrial mRNA expression of VEGFA-121 (p = 0.045) and VEGFA-189 (p = 0.009) as well as lower VEGFA protein abundance (p < 0.001) at Day 15. The VEGFA protein was localised in intercaruncular luminal, glandular epithelium and in tunica muscularis of blood vessels. At Day 15, caruncular endometrium displayed higher VEGFA mRNA expression than intercaruncular endometrium (p < 0.05). Intercaruncular endometrial VEGFA protein at Day 18 was higher in abundance in SCNT than in IVF (p = 0.038). Therefore, during preimplantation in cattle, there may be a need for timely physiological reduction in intercaruncular endometrial VEGFA expression in favour of the caruncular area to facilitate a gradient towards the implantation sites. A higher expression of VEGFA in SCNT may predispose for later placentation abnormalities frequently observed following SCNT.

Novel sampling procedure to characterize bovine subclinical endometritis by uterine secretions and tissue.

Subclinical endometritis (SE) in cattle is defined as clinically unapparent inflammation of the endometrium. It is reported to impair fertility in affected cows and causes economic loss within the dairy industry. A gold standard for diagnosis of SE has not been set. Uterine cytology and histopathology are both applied, but low agreement between these methods has been described. The objective of the present study was to assess the capability of uterine secretions (US) as a new medium for diagnosis of SE. A novel sampling tool was applied to retrieve US as well as cytological, histological and bacteriological samples of the endometrium after a singular passage through the cervix in 108 dairy cows (43-62 days post-partum [dpp]). To assess the quality of the US samples, a proteome analysis of samples from five healthy donors was performed, demonstrating that in vivo sampling of US was feasible and generated samples suitable for diagnostic purposes. Diagnosis of SE was realized by the combination of clinical, cytological, and histopathological findings. Quantitative analysis of pro- and anti-inflammatory cytokines (interleukin (IL)1B, IL6, IL8, IL17A, IL10) in US was conducted using AlphaLISA-technology. RNAlater-fixed endometrial biopsies were used for gene expression analysis of the cytokines IL1B, IL6, IL8, IL10 and tumor necrosis factor alpha (TNFα) as well as the prostaglandin-endoperoxide synthase 2 (PTGS2) and the antimicrobial peptide S100A9 by reverse transcription quantitative real-time polymerase chain reaction (RT-qPCR). Cows were assigned to groups according to their uterine health status. A large group of animals (n = 83) displayed no signs of endometritis (E.NEG). Cytological and histopathological examination revealed low agreement; hence, animals with SE were differentiated into SE(cyto) and SE(histo) groups (n = 7 and n = 13, respectively). One animal in group SE(cyto + histo) as well as four animals with signs of clinical endometritis (CE) were excluded from further analysis. SE(cyto) showed significantly higher median concentrations of IL1B, IL8 and IL17A in US as well as a significantly higher median expression of IL1B, IL8 and IL10 in endometrial biopsies compared to E.NEG. No significant differences were found for IL6 and IL10 in US and IL6, TNFα, PTGS2 and S100A9 in endometrial tissue between these groups. SE(histo) presented no differences concerning the analyzed parameters compared to E.NEG. In conclusion, a method to sample US was successfully established in dairy cows. The cytokines IL1B, IL8 and IL17A are promising candidates in diagnosing cytological endometritis by US. Further assessment of US might contribute to a better understanding of the pathological mechanisms leading to chronic endometrial inflammation and to impaired fertility in affected cows.

Transcriptome changes in the porcine endometrium during the preattachment phase.

The porcine conceptus undergoes rapid differentiation and expansion of its trophoblastic membranes between Days 11 and 12 of gestation. Concomitant with trophoblast elongation, production of conceptus estrogen, the porcine embryonic pregnancy recognition signal, increases. Conceptus attachment to the uterine surface epithelium starts after Day 13, initiating epitheliochorial placentation. To analyze the transcriptome changes in the endometrium in the course of maternal recognition of pregnancy, deep sequencing of endometrial RNA samples of Day 12 pregnant animals (n = 4) and corresponding nonpregnant controls (n = 4) was performed using RNA sequencing (RNA-Seq). Between 30 000 000 and 35 000 000 sequence reads per sample were produced and mapped to the porcine genome (Sscrofa10.2). Analysis of read counts revealed 2593 differentially expressed genes (DEGs). Expression of selected genes was validated by the use of quantitative real-time RT-PCR. Bioinformatics analysis identified several functional terms specifically overrepresented for up-regulated or down-regulated genes. Comparison of the RNA-Seq data from Days 12 and 14 of pregnancy was performed at the level of all expressed genes, the level of the DEG, and the level of functional categories. This revealed specific gene expression patterns reflecting the different functions of the endometrium during these stages (i.e., recognition of pregnancy and preparation for conceptus attachment). Genes related to mitosis, immune response, epithelial cell differentiation and development, proteolysis, and prostaglandin signaling and metabolism are discussed in detail. This study identified comprehensive transcriptome changes in porcine endometrium associated with establishment of pregnancy and could be a resource for targeted studies of genes and pathways potentially involved in regulation of this process.

Comparison of the effects of early pregnancy with human interferon, alpha 2 (IFNA2), on gene expression in bovine endometrium.

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.

Bovine endometrial metallopeptidases MMP14 and MMP2 and the metallopeptidase inhibitor TIMP2 participate in maternal preparation of pregnancy.

Early embryonic development is critically dependent on both maternal preparation and embryonic signalling of pregnancy. Matrix metallopeptidases (MMP) contribute to spatial and temporal matrix remodeling in the bovine endometrium. In this study we observed distinct changes in expression of MMP2, MMP14, and the metallopeptidase inhibitor TIMP2 between different phases of the estrous cycle indicating an endocrine regulation. An increase of TIMP2 protein abundance was ascertained in the uterine lumen during the time of embryo elongation. The expression pattern and cellular localization correlate well with the assumed effects of MMPs on release and activation of cytokines and growth factors directing cell migration, differentiation, and vascularization during this pivotal period of development. Specifically, active MMP2 in the endometrium may determine the allocation of growth factors supporting conceptus development. The presence of a day 18 conceptus in vivo and day 8 blastoysts in vitro induced endometrial TIMP2 mRNA expression. The results imply that TIMP2 is involved in very early local maternal recognition of pregnancy. Matrix metallopeptidases are likely to participate in remodeling processes preparing a receptive endometrium for a timely and precise regulation of embryo development.

Evidence for estrogen-dependent uterine serpin (SERPINA14) expression during estrus in the bovine endometrial glandular epithelium and lumen.

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 SERPINA14 mRNA levels at estrus. The present study describes the full-length mRNA sequence, genomic organization, and putative promoter elements of the SERPINA14 gene. The SERPINA14 mRNA 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. SERPINA14 mRNA 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.

The endometrium responds differently to cloned versus fertilized embryos.

Although somatic cell nuclear transfer (SCNT) cloning is more efficient in cattle than in any other species tested so far, there is a high rate of pregnancy failure that has been linked to structural and functional abnormalities of the placenta. We tested the hypothesis that these changes may originate from disturbed embryo-maternal interactions in the peri-implantation period. Therefore, we evaluated the response of the endometrium to SCNT embryos (produced from 7 different fetal fibroblast cell lines) as compared with embryos derived from in vitro fertilization (IVF). SCNT embryos and IVF embryos were cultured under identical conditions to the blastocyst stage (day 7) and were transferred to corresponding recipients, which were slaughtered at day 18 of pregnancy. The mRNA profiles of endometrium samples were obtained using a custom cDNA microarray enriched for transcripts differentially expressed in the endometrium and/or oviduct epithelium during the estrous cycle and/or early pregnancy. Overall, the variation in mRNA profiles was greater in the SCNT group than in the IVF group. Furthermore, 58 transcripts were differentially abundant in endometria from SCNT and IVF pregnancies. Prominent examples are orphan nuclear receptor COUP-TFII and connexin 43, both known to play important roles in uterine receptivity and conceptus placentation. These findings suggest that placental failure in bovine clone pregnancies may originate from abnormal embryo-maternal communication that develops during the peri-implantation period. Endometrium transcriptome profiles may serve as a tool to evaluate SCNT embryos for their ability to establish pregnancy and develop a functional placenta.

Quantitative monitoring of pluripotency gene activation after somatic cloning in cattle.

The development of somatic cell nuclear transfer (SCNT) embryos critically depends on appropriate reprogramming and expression of pluripotency genes, such as Pou5f1/POU5F1 (previously known as Oct4/OCT4). To study POU5F1 transcription activation in living bovine SCNT embryos without interference by maternal POU5F1 mRNA, we generated chromosomally normal fetal fibroblast donor cells stably carrying a mouse Pou5f1 promoter-driven enhanced green fluorescent protein (EGFP) reporter gene at a single integration site without detectable EGFP expression. Morphologic and quantitative analyses of whole-mount SCNT embryos by confocal microscopy revealed robust initial activation of the Pou5f1 reporter gene during the fourth cell cycle. In Day 6 SCNT embryos EGFP expression levels were markedly higher than in Day 4 embryos but varied substantially between individual embryos, even at comparable cell numbers. Embryos with low EGFP levels had far more morphologically abnormal cell nuclei than those with high EGFP levels. Our data strongly suggest that bovine SCNT embryos consistently start activation of the POU5F1 promoter during the fourth cell cycle, whereas later in development the expression level substantially differs between individual embryos, which may be associated with developmental potential. In fibroblasts from phenotypically normal SCNT fetuses recovered on Day 34, the Pou5f1 reporter promoter was silent but was activated by a second round of SCNT. The restoration of pluripotency can be directly observed in living cells or SCNT embryos from such Pou5f1-EGFP transgenic fetuses, providing an attractive model for systematic investigation of epigenetic reprogramming in large mammals.

The variability of ovum pick-up response and in vitro embryo production from monozygotic twin cows.

Oocytes were recovered by ovum pick up (OPU) from nine pairs of monozygotic twin German Simmental cows. The hypothesis was that there is less variability between identical twins versus among non-related individuals in the variation in the recovery of oocytes by OPU and in the efficiency of in vitro embryo production. Estrous cycles were synchronized with two doses of cloprostenol, 11 days apart. Beginning 3-4 days after synchronized estrus, OPU was done twice weekly (every 3 or 4 days; total of 11 sessions). The influence of repeated OPU on estrous cyclicity was established by estrus detection, plasma progesterone concentrations, and ovarian ultrasonography. There were no differences among days of collection for the number and quality of cumulus oocyte-complexes (COCs), and rates of cleavage and blastocyst formation. A total of 1,661 COCs, including 657 (39.6%) good-quality COCs, were recovered. From 1,457 (87.7%) cultured COCs, 827 zygotes cleaved and 314 blastocysts were produced on Day 7. The total number of COCs and the blastocyst rates varied among pairs of monozygotic twins; within pairs, only slight differences were observed. In conclusion, recovery of COCs and production of embryos had substantially less variation within pairs of monozygotic twins than among non-related cattle.

Monozygotic twin model reveals novel embryo-induced transcriptome changes of bovine endometrium in the preattachment period.

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 ISG15 and 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.

Holistic differential analysis of embryo-induced alterations in the proteome of bovine endometrium in the preattachment period.

During the peri-implantation period, molecular signaling between embryo and endometrium (layer of tissue lining the uterus lumen) is supposed to be crucial for the maintenance of pregnancy. To investigate embryo-induced alterations in the proteome of bovine endometrium in the preattachment period (day 18), we used monozygotic cattle twins (generated by embryo splitting) as a model eliminating genetic variability as a source for proteome differences. One of the twins was pregnant after the transfer of two in vitro produced blastocysts, while the corresponding twin received a sham-transfer and served as a nonpregnant control. The two-dimensional fluorescence difference gel electrophoresis (2-D DIGE) analysis of the endometrium samples of three twin pairs (pregnant/nonpregnant) revealed four proteins with significantly higher abundance (p < 10(-9)) in each sample derived from the pregnant animals: Rho GDP dissociation inhibitor beta; 20 alpha-hydroxysteroid dehydrogenase (20 alpha-HSD); soluble NADP(+)-dependent isocitrate dehydrogenase 1; and acyl-CoA-binding protein. To verify the accuracy of the 2-D DIGE quantification, the abundances of 20 alpha-HSD were quantified by a targeted cleavable isotope-coded affinity tag (ICAT) approach. The mass spectrometry-based ICAT quantification matched perfectly the results obtained by 2-D DIGE quantification, demonstrating the accuracy of our data. These results demonstrate that our model (monozygotic twins) in combination with the appropriate analytical tools is particularly suitable for the detection of the proteins involved in the embryo-maternal interactions.

Maternal-fetal transplacental leakage of mitochondrial DNA in bovine nuclear transfer pregnancies: potential implications for offspring and recipients.

The synepitheliochorial placenta of ruminants is constructed of multiple tissue layers that separate maternal and fetal blood. In nuclear transfer cloned ruminants, however, placental anomalies such as abnormal vascular development and hemorrhagic cotyledons have been reported. We have investigated the possible exchange of genetic material between somatic cell nuclear transfer cloned (SCNT) bovine fetuses and recipients at day 80 of gestation using mitochondrial DNA (mtDNA) as a marker. Twenty-three recovered SCNT-fetuses and their recipients were screened for divergent and thus informative mtDNA combinations. Twenty-one fetuses generated by in vitro fertilization (IVF) or multiple ovulation embryo transfer (MOET) and the corresponding recipients served as controls. A search for recipient mtDNA haplotype in DNA extracts from fetal blood by PCR-RFLP analysis revealed three cases of chimerism (two SCNT, one IVF) among a total of 19 informative fetus-recipient pairs (eight SCNT, seven IVF, four MOET). Placental anomalies have also been observed in some IVF fetuses and the present data therefore suggests transplacental leakage of cell components or cells from the recipient into some fetuses generated by in vitro techniques. Further studies are necessary to determine (i) the nature of leaked material, (ii) whether there is bi-directional leakage, and (iii) whether leaked material is present in recipients and calves after parturition, i.e. whether leakage takes place in vivo. If recipients were chimeric for DNA or cells derived from genetically modified SCNT (or IVF) embryos, their subsequent utilization might be affected.