The Dynamics of microRNA Transcriptome in Bovine Corpus Luteum during Its Formation, Function, and Regression

The formation, function, and subsequent regression of the ovarian corpus luteum (CL) are dynamic processes that enable ovary cyclical activity. Studies in whole ovary tissue have found microRNAs (miRNAs) to by critical for ovary function. However, relatively little is known about the role of miRNAs in the bovine CL. Utilizing small RNA next-generation sequencing we profiled miRNA transcriptome in bovine CL during the entire physiological estrous cycle, by sampling the CL on days: d 1-2, d 3-4, and d 5-7 (early CL, eCL), d 8-12 (mid CL, mCL), d 13-16 (late CL, lCL), and d > 18 (regressed CL, rCL). We characterized patterns of miRNAs abundance and identified 42 miRNAs that were consistent significantly different expressed (DE) in the eCL relative to their expression at each of the analyzed stages (mCL, lCL, and rCL). Out of these, bta-miR-210-3p, -2898, -96, -7-5p, -183-5p, -182, and -202 showed drastic up-regulation with a fold-change of >= 2.0 and adjusted P < 0.01 in the eCL, while bta-miR-146a was downregulated at lCL and rCL vs. the eCL. Another 24, 11, and 21 miRNAs were significantly DE only between individual comparisons, eCL vs. the mCL, lCL, and rCL, respectively. Irrespective of cycle stage two miRNAs, bta-miR-21-5p and bta-miR-143 were identified as the most abundant miRNAs species and show opposing expression abundance. Whilst bta-miR-21-5p peaked in number of reads in the eCL and was significantly downregulated in the mCL and lCL, bta-miR-143 reached its peak in the rCL and is significantly downregulated in the eCL. MiRNAs with significant DE in at least one cycle stage (CL class) were further grouped into eight distinct clusters by the self-organizing tree algorithm (SOTA). Half of the clusters contain miRNAs with low-expression, whilst the other half contain miRNAs with high-expression levels during eCL. Prediction analysis for significantly DE miRNAs resulted in target genes involved with CL formation, functionalization and CL regression. This study is the most comprehensive profiling of miRNA transcriptome in bovine CL covering the entire estrous cycle and provides a compact database for further functional validation and biomarker identification relevant for CL viability and fertility

Developement of Diagnostic Tools to Decipher Para- and Endocrine Effects of IFN Tau

Reproductive success is indispensable for efficient animal husbandry of modern dairy farms. Nevertheless, embryonic losses occur in every second cow during the first three weeks of gestation. The comprehension of key factors involved in the reproduction events is thus a prerequisite to reveal dysregulations. The protein Interferon tau (IFN tau) is the major pregnancy recognition signal in ruminants. It is not only responsible to extend the luteal life span but also involved in establishment of uterine receptivity, embryo elongation, nutrient supply and antiviral protection. While IFN tau mainly acts in a paracrine manner via interferon receptor signalling on endometrial cells, endocrine actions in peripheral blood cells, corpus luteum and liver have also been described. As IFN tau is solely produced in trophoblast tissue during the critical pregnancy recognition phase and as its production is influenced by embryo characteristics and culture conditions, it may be an interesting molecular marker for pregnancy and embryo monitoring. Deeper understanding of paracrine and endocrine actions of IFN tau may therefore not only help to understand its influence in embryo-maternal crosstalk but also its potential as a biomarker. Therefore, the objective of this thesis was to provide the prerequisite for an in-depth investigation of IFN tau and its effects. For this purpose, a highly sensitive enzyme-linked Immunosorbent Assay (ELISA) for IFN tau quantification was developed and validated as well as a system to sense minute IFN activity. To monitor endocrine IFN tau or pregnancy induced effects, miRNA expression in milk was analysed and evaluated regarding its use for a bovine pregnancy detection system. The ELISA was established with polyclonal and monoclonal antibodies. They were generated in rabbits and rodents, respectively. Recombinantly produced IFN tau and IFN tau specific peptides were used as antigens. For IFN tau activity determination endometrial stroma cells (SC) were isolated, and cellular response to IFN tau exposure was determined via mRNA expression changes of ISG15, MX2 and OAS1. Both assays were biologically validated in a SC-blastocyst co-culture by determination of sex-dependent IFN tau production. The miRNA expression profiles of milk cells, skim milk and whole milk of pregnant and non-pregnant cows were determined using high-throughput sequencing and validated by RT-qPCR. The limit of detection of the IFN tau-ELISA was as low as 7 pg/ml in uterine flushings and embryo culture supernatants, which is lower than in any reported assay. The SC reacted to actions of only 0.4 pg/ml IFN tau. We thereby evidenced five times higher IFN tau production in female than in male blastocysts. Furthermore, the IFN tau activity, mRNA and protein expression were significantly correlated. The indirect analysis of endocrine IFN tau action, using miRNA expression changes in milk, on the other hand only displayed minor miRNA regulations with high variances that could not be validated in biological replicates. In conclusion, the miRNA analysis in milk lagged behind expectations, while the ELISA and SC culture form an excellent fundament for in-depth investigation of IFN tau and embryo development

The Dynamics of microRNA Transcriptome in Bovine Corpus Luteum during Its Formation, Function, and Regression

The formation, function, and subsequent regression of the ovarian corpus luteum (CL) are dynamic processes that enable ovary cyclical activity. Studies in whole ovary tissue have found microRNAs (miRNAs) to by critical for ovary function. However, relatively little is known about the role of miRNAs in the bovine CL. Utilizing small RNA next-generation sequencing we profiled miRNA transcriptome in bovine CL during the entire physiological estrous cycle, by sampling the CL on days: d 1–2, d 3–4, and d 5–7 (early CL, eCL), d 8–12 (mid CL, mCL), d 13–16 (late CL, lCL), and d &gt; 18 (regressed CL, rCL). We characterized patterns of miRNAs abundance and identified 42 miRNAs that were consistent significantly different expressed (DE) in the eCL relative to their expression at each of the analyzed stages (mCL, lCL, and rCL). Out of these, bta-miR-210-3p, −2898, −96, −7-5p, −183-5p, −182, and −202 showed drastic up-regulation with a fold-change of ≥2.0 and adjusted P &lt; 0.01 in the eCL, while bta-miR-146a was downregulated at lCL and rCL vs. the eCL. Another 24, 11, and 21 miRNAs were significantly DE only between individual comparisons, eCL vs. the mCL, lCL, and rCL, respectively. Irrespective of cycle stage two miRNAs, bta-miR-21-5p and bta-miR-143 were identified as the most abundant miRNAs species and show opposing expression abundance. Whilst bta-miR-21-5p peaked in number of reads in the eCL and was significantly downregulated in the mCL and lCL, bta-miR-143 reached its peak in the rCL and is significantly downregulated in the eCL. MiRNAs with significant DE in at least one cycle stage (CL class) were further grouped into eight distinct clusters by the self-organizing tree algorithm (SOTA). Half of the clusters contain miRNAs with low-expression, whilst the other half contain miRNAs with high-expression levels during eCL. Prediction analysis for significantly DE miRNAs resulted in target genes involved with CL formation, functionalization and CL regression. This study is the most comprehensive profiling of miRNA transcriptome in bovine CL covering the entire estrous cycle and provides a compact database for further functional validation and biomarker identification relevant for CL viability and fertility

Can milk cell or skim milk miRNAs be used as biomarkers for early pregnancy detection in cattle?

<div><p>The most critical phase of pregnancy is the first three weeks following insemination. During this period about 50% of high yielding lactating cows suffer embryonic loss prior to implantation, which poses a high economic burden on dairy farmers. Early diagnosis of pregnancy in cattle is therefore essential for monitoring breeding outcome and efficient production intervals. Regulated microRNAs (miRNAs) that reach easily accessible body fluids via a ‘liquid biopsy’ could be a new class of pregnancy predicting biomarkers. As milk is obtained regularly twice daily and non-invasively from the animal, it represents an ideal sample material. Our aim was to establish a pregnancy test system based on the discovery of small RNA biomarkers derived from the bovine milk cellular fraction and skim milk of cows. Milk samples were taken on days 4, 12 and 18 of cyclic cows and after artificial insemination, respectively, of the same animals (n = 6). miRNAs were analysed using small RNA sequencing (small RNA Seq). The miRNA profiles of milk cells and skim milk displayed similar profiles despite the presence of immune cell related miRNAs in milk cells. Trends in regulation of miRNAs between the oestrous cycle and pregnancy were found in miR-cluster 25~106b and its paralog cluster 17~92, miR-125 family, miR-200 family, miR-29 family, miR-15a, miR-21, miR-26b, miR-100, miR-140, 193a-5p, miR-221, miR-223, miR-320a, miR-652, miR-2898 and let-7i. A separation of cyclic and pregnant animals was achieved in a principal component analysis. Bta-miRs-29b, -221, -125b and -200b were successfully technically validated using quantitative real-time PCR, however biological validation failed. Therefore we cannot recommend the diagnostic use of these miRNAs in milk as biomarkers for detection of bovine pregnancy for now.</p></div

Foldchanges/ratios of regulated miRNA during oestrous cycle (cy) and pregnancy (p) measured in RT-qPCR.

<p>Foldchanges/ratios of regulated miRNA during oestrous cycle (cy) and pregnancy (p) measured in RT-qPCR.</p

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

Statistically significant differences in miRNA levels between milk cells and skim milk.

<p>Statistically significant differences in miRNA levels between milk cells and skim milk.</p

Correlation of miRNA expression between milk cells and skim milk.

<p>Correlation of miRNA expression between milk cells and skim milk.</p

log2 fold-change of selected miRNAs in the milk cellular and skim milk fraction measured with high throughput sequencing.

<p>log2 fold-change of selected miRNAs in the milk cellular and skim milk fraction (n = 6, except SM pregnant day 18 n = 5) measured with high throughput sequencing. All fold-changes and p-values for pregnant/cyclic data were calculated using Deseq2. p-values between cyclic (18c/4c) and pregnant Day 18/Day 4 ratios (18p/4p) were calculated using paired t-test on log2 fold-changes *: p<0.05, **: p< 0.01. Error bars are shown as log fold standard error. A) log2 fold-change between pregnancy and oestrous cycle on days 4, 12 and 18 B) log2 fold-change between Day 18 and Day 4 during oestrous cycle and pregnancy. C) PCA of log2 fold-change of the miRNAs: bta-miR-25, bta-miR-93, bta-miR-106b, bta-miR-125b, bta-miR-193a-5p, bta-miR-200b, bta-miR-200c, bta-miR-221, bta-miR-2898, bta-let-7i between days 4 and 18 (Day 18/Day 4) showing the separation of cyclic and pregnant animals.</p

Comparison of MC and SM miRNA profile.

<p>A) Correlation between miRNA mean values of all MC and SM NGS reads. Normalized NGS reads were used for calculation of Pearson Correlation Coefficient B) Heatmap of the top 100 miRNAs. Data was normalized, rld transformed and clustered using DESeq2 C) PCA showing MC and SM specific clustering. Clustering was performed using DESeq2.</p