Amino acids activate mTORC1 to release roe deer embryos from decelerated proliferation during diapause.

Embryonic diapause in mammals leads to a reversible developmental arrest. While completely halted in many species, European roe deer (Capreolus capreolus) embryos display a continuous deceleration of proliferation. During a 4-mo period, the cell doubling time is 2 to 3 wk. During this period, the preimplantation blastocyst reaches a diameter of 4 mm, after which it resumes a fast developmental pace to subsequently implant. The mechanisms regulating this notable deceleration and reacceleration upon developmental resumption are unclear. We propose that amino acids of maternal origin drive the embryonic developmental pace. A pronounced change in the abundance of uterine fluid mTORC1-activating amino acids coincided with an increase in embryonic mTORC1 activity prior to the resumption of development. Concurrently, genes related to the glycolytic and phosphate pentose pathway, the TCA cycle, and one carbon metabolism were up-regulated. Furthermore, the uterine luminal epithelial transcriptome indicated increased estradiol-17ß signaling, which likely regulates the endometrial secretions adapting to the embryonic needs. While mTORC1 was predicted to be inactive during diapause, the residual embryonic mTORC2 activity may indicate its involvement in maintaining the low yet continuous proliferation rate during diapause. Collectively, we emphasize the role of nutrient signaling in preimplantation embryo development. We propose selective mTORC1 inhibition via uterine catecholestrogens and let-7 as a mechanism regulating slow stem cell cycle progression.

One-week storage of refrigerated bovine milk does not affect the size, concentration, or molecular properties of extracellular vesicles.

Milk extracellular vesicles (EV) have gained extensive attention as promising diagnostic and therapeutic tools. Pre-analytical raw milk storage at low temperatures is an ordinary and usually necessary step after sample collection. It is known that direct freezing of unprocessed whole milk contaminates the native pool of milk EV with other cell structures. However, less evidence is available regarding prolonged cooling at 4°C. The current study assessed whether pre-analytical storage of bovine raw milk for several days affected EV isolation and further analysis. To confirm the independence from the health status of the mammary gland, we analyzed milk samples stored at 4°C for 1, 2, 3, and 7 d past collection, respectively, from 2 quarters of the same cow with different somatic cell counts (SCC). Seven days of refrigeration did not change the milk EV size, concentration, or morphology. We did not detect any changes in the EV cargo regarding the amount of protein and RNA, nor in the specific EV markers TSG101, CD9, and CD81 in milk from quarters with high and low SCC. Overall, we observed fewer CD81 and CD9 markers in quarters with high SCC. Moreover, we found no reduction in the mastitis-related miRNA bta-miR-223-3p, suggesting that refrigeration for several days up to 1 wk is a possible storage option compatible with further EV analyses. The findings of this study enhance the confidence that milk EV are highly stable in the raw milk matrix.

Novel role for conceptus signals in mRNA expression regulation by DNA methylation in porcine endometrium during early pregnancy†.

During early pregnancy, porcine conceptuses (the embryos with associated membranes) secrete estradiol-17ß (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 E2 affects gene expression by epigenetic mechanisms related to DNA methylation. Herein, we determined the effects of E2 and PGE2 alone, and a combined E2 + PGE2 treatment 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 E2 and PGE2 significantly 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 E2 treatment and in gilts on day 12 of pregnancy. Treatment with E2 alone and/or simultaneously with PGE2 altered endometrial DNA methylation of CpG sites of ADAMTS20, ADH1C, BGN, PSAT1, and WNT5A. Different CpG methylation patterns of ADAMTS20, BGN, DMBT1, RASSF1, and WNT5A were 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.

Reconsidering "low-dose"-Impacts of oral estrogen exposure during preimplantation embryo development.

Perturbations of estrogen signaling during developmental stages of high plasticity may lead to adverse effects later in life. Endocrine-disrupting chemicals (EDC) are compounds that interfere with the endocrine system by particularly mimicking the action of endogenous estrogens as functional agonists or antagonists. EDCs compose synthetic and naturally occurring compounds discharged into the environment, which may be taken up via skin contact, inhalation, orally due to contaminated food or water, or via the placenta during in utero development. Although estrogens are efficiently metabolized by the liver, the role of circulating glucuro- and/or sulpho-conjugated estrogen metabolites in the body has not been fully addressed to date. Particularly, the role of intracellular cleavage to free functional estrogens could explain the hitherto unknown mode of action of adverse effects of EDC at very low concentrations currently considered safe. We summarize and discuss findings on estrogenic EDC with a focus on early embryonic development to highlight the need for reconsidering low dose effects of EDC.

Endometrial extracellular matrix components do not change over the course of embryonic diapause and reactivation in the roe deer (Capreolus capreolus).

The modification of the endometrial extracellular matrix (ECM) is a crucial step for embryo implantation in many mammalian species. The embryo of the European roe deer (Capreolus capreolus) displays a 4-5 months long temporary reduction of developmental pace termed embryonic diapause. A reduction of epithelial cell height during diapause has previously been described. Co-occurring ECM modifications may contribute to the changes of the intra-uterine milieu during reactivation at which the embryo regains developmental velocity. We assessed the localization of five ECM proteins (collagen I and IV, fibronectin, laminin, and extracellular matrix protein 1) using immunohistochemistry in animals with early, late, and post-diapause (elongating) embryos. While our results confirmed the reduction of epithelial height during diapause, we only detected marginal differences in localization and staining intensities of the selected ECM proteins. Major ECM remodelling events in the roe deer endometrium are thus likely to occur only at implantation.

Circadian Rhythm Does Not Affect the miRNA Cargo of Bovine Raw Milk Extracellular Vesicles.

Extracellular vesicles (EVs) and their microRNA (miRNA) cargo have been proposed as possible mammary gland health biomarkers in cattle. However, throughout the day, the biologically active milk components, such as miRNAs, may change due to the dynamic nature of milk. The current study aimed to evaluate the circadian fluctuation of milk EVs miRNA cargo to assess the feasibility of milk EVs as future biomarkers for mammary gland health management. Milk from four healthy dairy cows was collected for four consecutive days in the two daily milking sessions in the morning and the evening. The isolated EVs were heterogeneous, intact, and carried the EV protein markers CD9, CD81, and TSG101, as shown by transmission electron microscopy and western blot. The miRNA sequencing results demonstrate that the abundance of miRNA cargo in milk EVs remained stable, unlike other milk components, such as somatic cells, that changed during milking sessions. These findings indicated that the miRNA cargo within milk EVs remains stable irrespective of the time of day, suggesting their potential utility as diagnostic markers for mammary gland health.

Determining extracellular vesicles properties and miRNA cargo variability in bovine milk from healthy cows and cows undergoing subclinical mastitis.

BACKGROUND: Subclinical mastitis, the inflammation of the mammary gland lacking clinical symptoms, is one of the most prevalent and costly diseases in dairy farming worldwide. Milk microRNAs (miRNAs) encapsulated in extracellular vesicles (EVs) have been proposed as potential biomarkers of different mammary gland conditions, including subclinical mastitis. However, little is known about the robustness of EVs analysis regarding sampling time-point and natural infections. To estimate the reliability of EVs measurements in raw bovine milk, we first evaluated changes in EVs size and concentration using Tunable Resistive Pulse Sensing (TRPS) during three consecutive days of sampling. Then, we analysed daily differences in miRNA cargo using small RNA-seq. Finally, we compared milk EVs differences from naturally infected udder quarters with their healthy adjacent quarters and quarters from uninfected udders, respectively. RESULTS: We found that the milk EV miRNA cargo was very stable over the course of three days regardless of the health status of the quarter, and that infected quarters did not induce relevant changes in milk EVs of adjacent healthy quarters. Chronic subclinical mastitis induced changes in milk EV miRNA cargo, but neither in EVs size nor concentration. We observed that the changes in immunoregulatory miRNAs in quarters with chronic subclinical mastitis were cow-individual, however, the most upregulated miRNA was bta-miR-223-3p across all individuals. CONCLUSIONS: Our results showed that the miRNA profile and particle size characteristics remained constant throughout consecutive days, suggesting that miRNAs packed in EVs are physiological state-specific. In addition, infected quarters were solely affected while adjacent healthy quarters remained unaffected. Finally, the cow-individual miRNA changes pointed towards infection-specific alterations.

Inhibition of lipopolysaccharide-induced suppression of luteal function in isolated perfused bovine ovaries.

Recently, we observed that lipopolysaccharide (LPS) suppresses corpus luteum (CL) function in isolated perfused ovaries. It remained unclear if this suppression was due to increased luteal PGF2α secretion or LPS-induced apoptosis. Therefore, possible impacts of PGF2α and LPS were inhibited by a non-steroidal anti-inflammatory drug (flunixin) and an endotoxin-binding agent (polymyxin B), respectively. Bovine ovaries with a mid-cycle CL were collected immediately after slaughter and perfused for 240 min. After 50 min of equilibration, either flunixin or polymyxin B (5 µg/ml of each) were added to the perfusion medium of six ovaries, respectively. All ovaries (n = 12) were treated with E. coli LPS (0.5 µg/ml) 60 min after the onset of perfusion, and received 500 I.U. of hCG after 210 min of perfusion. Progesterone and PGF2α were measured in the effluent perfusate every 10 and 30 min, respectively. Biopsies of the CL were collected every 60 min to determine the mRNA expression of the cytokine TNFA and factors of apoptosis (CASP3, -8). Flunixin-treatment inhibited the increase of PGF2α after LPS-challenge that was observed in the polymyxin B-treated (PX-LPS) ovaries. After hCG-stimulation, progesterone secretion increased (P < 0.05) in group PX-LPS but not in the flunixin-treated (F-LPS) ovaries. Compared to initial values before LPS-challenge, luteal mRNA expression of TNFA and CASP3 was increased (P < 0.05) in group F-LPS at 120 and 180 min, respectively, and those of CASP8 was decreased (P < 0.05) in PX-LPS at 60 and 120 min after LPS-treatment. In conclusion, although flunixin managed to inhibit PGF2α, it did not suffice to successfully prevent LPS-induced apoptosis. However, endotoxin-binding polymyxin B resulted in luteal responsiveness to hCG after LPS-challenge.

Transcriptome dynamics in early in vivo developing and in vitro produced porcine embryos.

BACKGROUND: The transcriptional changes around the time of embryonic genome activation in pre-implantation embryos indicate that this process is highly dynamic. In vitro produced porcine blastocysts are known to be less competent than in vivo developed blastocysts. To understand the conditions that compromise developmental competence of in vitro embryos, it is crucial to evaluate the transcriptional profile of porcine embryos during pre-implantation stages. In this study, we investigated the transcriptome dynamics in in vivo developed and in vitro produced 4-cell embryos, morulae and hatched blastocysts. RESULTS: In vivo developed and in vitro produced embryos displayed largely similar transcriptome profiles during development. Enriched canonical pathways from the 4-cell to the morula transition that were shared between in vivo developed and in vitro produced embryos included oxidative phosphorylation and EIF2 signaling. The shared canonical pathways from the morula to the hatched blastocyst transition were 14-3-3-mediated signaling, xenobiotic metabolism general signaling pathway, and NRF2-mediated oxidative stress response. The in vivo developed and in vitro produced hatched blastocysts further were compared to identify molecular signaling pathways indicative of lower developmental competence of in vitro produced hatched blastocysts. A higher metabolic rate and expression of the arginine transporter SLC7A1 were found in in vitro produced hatched blastocysts. CONCLUSIONS: Our findings suggest that embryos with compromised developmental potential are arrested at an early stage of development, while embryos developing to the hatched blastocyst stage display largely similar transcriptome profiles, irrespective of the embryo source. The hatched blastocysts derived from the in vitro fertilization-pipeline showed an enrichment in molecular signaling pathways associated with lower developmental competence, compared to the in vivo developed embryos.

Comparison of six breast cancer classifiers using qPCR.

MOTIVATION: Several gene expression-based risk scores and subtype classifiers for breast cancer were developed to distinguish high- and low-risk patients. Evaluating the performance of these classifiers helps to decide which classifiers should be used in clinical practice for personal therapeutic recommendations. So far, studies that compared multiple classifiers in large independent patient cohorts mostly used microarray measurements. qPCR-based classifiers were not included in the comparison or had to be adapted to the different experimental platforms. RESULTS: We used a prospective study of 726 early breast cancer patients from seven certified German breast cancer centers. Patients were treated according to national guidelines and the expressions of 94 selected genes were measured by the mid-throughput qPCR platform Fluidigm. Clinical and pathological data including outcome over five years is available. Using these data, we could compare the performance of six classifiers (scmgene and research versions of PAM50, ROR-S, recurrence score, EndoPredict and GGI). Similar to other studies, we found a similar or even higher concordance between most of the classifiers and most were also able to differentiate high- and low-risk patients. The classifiers that were originally developed for microarray data still performed similarly using the Fluidigm data. Therefore, Fluidigm can be used to measure the gene expressions needed by several classifiers for a large cohort with little effort. In addition, we provide an interactive report of the results, which enables a transparent, in-depth comparison of classifiers and their prediction of individual patients. AVAILABILITY AND IMPLEMENTATION: https://services.bio.ifi.lmu.de/pia/. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

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.

Spatial organization of endometrial gene expression at the onset of embryo attachment in pigs.

BACKGROUND: During the preimplantation phase in the pig, the conceptus trophoblast elongates into a filamentous form and secretes estrogens, interleukin 1 beta 2, interferons, and other signaling molecules before attaching to the uterine epithelium. The processes in the uterine endometrium in response to conceptus signaling are complex. Thus, the objective of this study was to characterize transcriptome changes in porcine endometrium during the time of conceptus attachment considering the specific localization in different endometrial cell types. RESULTS: Low-input RNA-sequencing was conducted for the main endometrial compartments, luminal epithelium (LE), glandular epithelium (GE), blood vessels (BV), and stroma. Samples were isolated from endometria collected on Day 14 of pregnancy and the estrous cycle (each group n = 4) by laser capture microdissection. The expression of 12,000, 11,903, 11,094, and 11,933 genes was detectable in LE, GE, BV, and stroma, respectively. Differential expression analysis was performed between the pregnant and cyclic group for each cell type as well as for a corresponding dataset for complete endometrium tissue samples. The highest number of differentially expressed genes (DEGs) was found for LE (1410) compared to GE, BV, and stroma (800, 1216, and 384). For the complete tissue, 3262 DEGs were obtained. The DEGs were assigned to Gene Ontology (GO) terms to find overrepresented functional categories and pathways specific for the individual endometrial compartments. GO classification revealed that DEGs in LE were involved in 'biosynthetic processes', 'related to ion transport', and 'apoptotic processes', whereas 'cell migration', 'cell growth', 'signaling', and 'metabolic/biosynthetic processes' categories were enriched for GE. For blood vessels, categories such as 'focal adhesion', 'actin cytoskeleton', 'cell junction', 'cell differentiation and development' were found as overrepresented, while for stromal samples, most DEGs were assigned to 'extracellular matrix', 'gap junction', and 'ER to Golgi vesicles'. CONCLUSIONS: The localization of differential gene expression to different endometrial cell types provided a significantly improved view on the regulation of biological processes involved in conceptus implantation, such as the control of uterine fluid secretion, trophoblast attachment, growth regulation by Wnt signaling and other signaling pathways, as well as the modulation of the maternal immune system.

Differential transcriptome dynamics during the onset of conceptus elongation and between female and male porcine embryos.

BACKGROUND: Porcine embryos undergo rapid differentiation and expansion between Days 8 and 12 before attaching to the maternal uterine epithelial surface after Day 13. It is known that maternal recognition of pregnancy and successful implantation are driven by mutual interactions between the elongated conceptus and the maternal endometrium. While most of the genes involved in regulation of embryo development are located on autosomal chromosomes, gene expression on sex chromosomes is modulating development through sex-specific transcription. To gain more insights into the dynamic transcriptome of preimplantation embryos at the onset of elongation and into X-linked gene expression, RNA-seq analyses were performed for single female and male porcine embryos collected on Days 8, 10, and 12 of pregnancy. RESULTS: A high number of genes were differentially expressed across the developmental stages (2174 and 3275 for Days 8 vs 10, and 10 vs 12, respectively). The majority of differentially expressed genes (DEGs) were involved in embryo elongation, development, and embryo-maternal interaction. Interestingly, a number of DEGs was found with respect to embryo sex (137, 37, and 56 on Days 8, 10 and 12, respectively). At Day 8, most of these DEGs were X-linked (96). Strikingly, the number of DEGs encoded on the X chromosome dramatically decreased from Day 10 to Day 12. CONCLUSIONS: The obtained results deepen the understanding about temporary transcriptomic changes in porcine embryos during the phase of conceptus elongation, meanwhile reveal dynamic compensation of X chromosome in the female and distinct transcriptional differences between female and male embryos.

Exposure of pregnant sows to low doses of estradiol-17ß impacts on the transcriptome of the endometrium and the female preimplantation embryos†.

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-17ß (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.

Endometrial luminal epithelial cells sense embryo elongation in the roe deer independent of interferon-tau†.

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.

Blastocysts exhibit sex-specific signalling of IFNT transcription, translation and activity.

Preimplantation bovine blastocyst supernatants exhibit sex-dependent antiviral activity, due to the ruminant pregnancy recognition signal interferon tau (IFNT). Differing potencies of IFNT variants have been supposed as cause, although evidence remains scarce. Here, we aimed at quantifying the sex-dependent IFNT production on transcriptional, translational and biological activity level in bovine blastocysts, to elucidate the origin of differences in antiviral activity between male and female blastocysts. Day 8 bovine blastocysts were co-cultured with endometrial stroma cells for 48 h. The embryonic IFNT mRNA expression was determined by quantitative reverse transcription followed by polymerase chain reaction (RT-qPCR). Additionally, the IFNT protein concentration was determined using a sensitive in-house developed IFNT-specific enzyme-linked immunosorbent assay (ELISA). The biological activity was assessed by quantifying the response of interferon-stimulated gene (ISG) expression in endometrial stroma cells. While the IFNT-specific ELISA displayed a limit of detection of 7.3 pg/mL, the stroma cell culture system showed to react to as little as 0.1 pg/mL IFNT in RT-qPCR analysis. The female blastocysts had a significant, 5.6-fold, 3.6-fold and 5.2-fold higher IFNT production than male blastocysts as determined by transcript abundance, protein concentration and, protein activity, respectively. Additionally, all parameters correlated positively, and therefore, we conclude that female blastocysts most likely have an increased IFNT gene and protein expression rather than expressing more potent IFNT variants.

Cell type-specific endometrial transcriptome changes during initial recognition of pregnancy in the mare.

Previous endometrial gene expression studies during the time of conceptus migration did not provide final conclusions on the mechanisms of maternal recognition of pregnancy (MRP) in the mare. This called for a cell type-specific endometrial gene expression analysis in response to embryo signals to improve the understanding of gene expression regulation in the context of MRP. Laser capture microdissection was used to collect luminal epithelium (LE), glandular epithelium and stroma from endometrial biopsies from Day 12 of pregnancy and Day 12 of the oestrous cycle. RNA sequencing (RNA-Seq) showed greater expression differences between cell types than between pregnant and cyclic states; differences between the pregnant and cyclic states were mainly found in LE. Comparison with a previous RNA-Seq dataset for whole biopsy samples revealed the specific origin of gene expression differences. Furthermore, genes specifically differentially expressed (DE) in one cell type were found that were not detectable as DE in biopsies. Overall, this study revealed spatial information about endometrial gene expression during the phase of initial MRP. The conceptus induced changes in the expression of genes involved in blood vessel development, specific spatial regulation of the immune system, growth factors, regulation of prostaglandin synthesis, transport prostaglandin receptors, specifically prostaglandin F receptor (PTGFR) in the context of prevention of luteolysis.

Cell type-specific analysis of transcriptome changes in the porcine endometrium on Day 12 of pregnancy.

BACKGROUND: Along with trophoblast elongation (Days 10 to 12), estradiol is secreted in increasing amounts for recognition of pregnancy. Endometrial secretions driven by ovarian progesterone and conceptus signals are essential for conceptus growth and development. Results of transcriptome analyses of whole endometrial tissue samples in the pig indicated the need for cell type-specific endometrial gene expression analysis for a better understanding of transcriptome changes associated with establishment of pregnancy. RESULTS: The most distinct transcriptome profile and the majority of differentially expressed genes (DEGs) were identified in luminal epithelium (LE). Many DEGs were found only in the cell type-specific analysis. The functional classification of DEGs identified in specific endometrial cell types revealed various distinct functions and pathways. Genes related to immune activation, estrogen signaling pathway, embryo development, and cell proliferation were upregulated in LE of pregnant gilts. Genes involved in sterol biosynthetic and metabolic processes and extracellular matrix were upregulated in stroma. Genes associated with cell communication such as via exosomes and vesicles were found as differential in LE, glandular epithelium (GE), and stroma (S). CONCLUSIONS: This study revealed that conceptus signals induce different transcriptomic regulations in the endometrial compartments/cell types related to their specific function during recognition and establishment of pregnancy.

Small RNA-seq analysis of single porcine blastocysts revealed that maternal estradiol-17beta exposure does not affect miRNA isoform (isomiR) expression.

BACKGROUND: The expression of microRNAs (miRNAs) is essential for the proper development of the mammalian embryo. A maternal exposure to endocrine disrupting chemicals during preimplantation bears the potential for transgenerational inheritance of disease through the epigenetic perturbation of the developing embryo. A comprehensive assembly of embryo-specific miRNAs and respective isoforms (isomiR) is lacking to date. We aimed at revealing the sex-specific miRNA expression profile of single porcine blastocysts developing in gilts orally exposed to exogenous estradiol-17 (E2). Therefore we analyzed the miRNA profile specifically focusing on isomiRs and potentially embryo-specific miRNAs. RESULTS: Deep sequencing of small RNA (small RNA-seq) result in the detection of miRNA sequences mapping to known and predicted porcine miRNAs as well as novel miRNAs highly conserved in human and cattle. A set of highly abundant miRNAs and a large number of rarely expressed miRNAs were identified by using a small RNA analysis pipeline, which was integrated into a novel Galaxy workflow specifically benefits incompletely annotated species. In particular, orthologue species information increased the total number of annotated miRNAs, while mapping to other non-coding RNAs avoided falsely annotated miRNAs. Neither the low nor the high dose of E2 treatment (10 and 1000 µ E2/kg body weight daily, respectively) affected the miRNA profile in blastocysts despite the distinct differential mRNA expression and DNA methylation found in previous studies. The high number of generated sequence reads enabled a comprehensive analysis of the isomiR repertoire showing various templated and non-templated modifications. Furthermore, potentially blastocyst-specific miRNAs were identified. CONCLUSIONS: In pre-implantation embryos, numerous distinct isomiRs were discovered indicating a high complexity of miRNA expression. Neither the sex of the embryo nor a maternal E2 exposure affected the miRNA expression profile of developing porcine blastocysts. The adaptation to the continuous duration of the E2 treatment might explain the lack of an effect.