Paul A. McGettigan

Conceptus-Induced Changes in the Endometrial Transcriptome: How Soon Does the Cow Know She Is Pregnant?

This study sought to determine the earliest response of the bovine uterine endometrium to the presence of the conceptus at key developmental stages of early pregnancy. There were no detectable differences in gene expression in endometria from pregnant and cyclic heifers on Days 5, 7, and 13 postestrus, but the expression of 764 genes was altered due to the presence of the conceptus at maternal recognition of pregnancy (Day 16). Of these 514 genes, MX2, BST2, RSAD2, ISG15, OAS1, USP18, IFI44, ISG20, SAMD9, EIF4E, and IFIT2 increased to the greatest extent in pregnant endometria (>8-fold log2 fold change increase). The expression of OXTR, Bt.643 (unofficial symbol), and KCNMA1 was reduced the most, but short-term treatment with recombinant ovine interferon tau (IFNT) in vitro or in vivo did not alter their expression. In vivo intrauterine infusion of IFNT induced the expression of EIF4E, IFIT2, IFI44, ISG20, MX2, RSAD2, SAMD9, and USP18. These results revealed for the first time that changes that occur in the endometrial transcriptome are independent of the presence of a conceptus until pregnancy recognition. The differentially expressed genes (including MX2, BST2, RSAD2, ISG15, OAS1, USP18, IFI44, ISG20, SAMD, and EIF4E) are a consequence of IFNT production by the conceptus. The identified genes represent known and novel early markers of conceptus development and/or return to cyclicity and may be useful to identify the earliest stage at which the endometrial response to the conceptus is detectable.

Transcriptomics in the RNA-seq era

The transcriptomics field has developed rapidly with the advent of next-generation sequencing technologies. RNA-seq has now displaced microarrays as the preferred method for gene expression profiling. The comprehensive nature of the data generated has been a boon in terms of transcript identification but analysis challenges remain. Key among these problems is the development of suitable expression metrics for expression level comparisons and methods for identification of differentially expressed genes (and exons). Several approaches have been developed but as yet no consensus exists on the best pipeline to use. De novo transcriptome approaches are increasingly viable for organisms lacking a sequenced genome. The reduction in starting RNA required has enabled the development of new applications such as single cell transcriptomics. The emerging picture of mammalian transcription is complex with further refinement expected with the integration of epigenomic data generated by projects such as ENCODE.

Genome sequencing of the extinct Eurasian wild aurochs, Bos primigenius, illuminates the phylogeography and evolution of cattle

BackgroundDomestication of the now-extinct wild aurochs, Bos primigenius, gave rise to the two major domestic extant cattle taxa, B. taurus and B. indicus. While previous genetic studies have shed some light on the evolutionary relationships between European aurochs and modern cattle, important questions remain unanswered, including the phylogenetic status of aurochs, whether gene flow from aurochs into early domestic populations occurred, and which genomic regions were subject to selection processes during and after domestication. Here, we address these questions using whole-genome sequencing data generated from an approximately 6,750-year-old British aurochs bone and genome sequence data from 81 additional cattle plus genome-wide single nucleotide polymorphism data from a diverse panel of 1,225 modern animals.ResultsPhylogenomic analyses place the aurochs as a distinct outgroup to the domestic B. taurus lineage, supporting the predominant Near Eastern origin of European cattle. Conversely, traditional British and Irish breeds share more genetic variants with this aurochs specimen than other European populations, supporting localized gene flow from aurochs into the ancestors of modern British and Irish cattle, perhaps through purposeful restocking by early herders in Britain. Finally, the functions of genes showing evidence for positive selection in B. taurus are enriched for neurobiology, growth, metabolism and immunobiology, suggesting that these biological processes have been important in the domestication of cattle.ConclusionsThis work provides important new information regarding the origins and functional evolution of modern cattle, revealing that the interface between early European domestic populations and wild aurochs was significantly more complex than previously thought.

Characterization of the equine skeletal muscle transcriptome identifies novel functional responses to exercise training

BackgroundDigital gene expression profiling was used to characterize the assembly of genes expressed in equine skeletal muscle and to identify the subset of genes that were differentially expressed following a ten-month period of exercise training. The study cohort comprised seven Thoroughbred racehorses from a single training yard. Skeletal muscle biopsies were collected at rest from the gluteus medius at two time points: T1 - untrained, (9 ± 0.5 months old) and T2 - trained (20 ± 0.7 months old).ResultsThe most abundant mRNA transcripts in the muscle transcriptome were those involved in muscle contraction, aerobic respiration and mitochondrial function. A previously unreported over-representation of genes related to RNA processing, the stress response and proteolysis was observed. Following training 92 tags were differentially expressed of which 74 were annotated. Sixteen genes showed increased expression, including the mitochondrial genes ACADVL, MRPS21 and SLC25A29 encoded by the nuclear genome. Among the 58 genes with decreased expression, MSTN, a negative regulator of muscle growth, had the greatest decrease.Functional analysis of all expressed genes using FatiScan revealed an asymmetric distribution of 482 Gene Ontology (GO) groups and 18 KEGG pathways. Functional groups displaying highly significant (P < 0.0001) increased expression included mitochondrion, oxidative phosphorylation and fatty acid metabolism while functional groups with decreased expression were mainly associated with structural genes and included the sarcoplasm, laminin complex and cytoskeleton.ConclusionExercise training in Thoroughbred racehorses results in coordinate changes in the gene expression of functional groups of genes related to metabolism, oxidative phosphorylation and muscle structure.

Sequential Analysis of Global Gene Expression Profiles in Immature and In vitro Matured Bovine Oocytes: Potential Molecular Markers of Oocyte Maturation

BackgroundWithout intensive selection, the majority of bovine oocytes submitted to in vitro embryo production (IVP) fail to develop to the blastocyst stage. This is attributed partly to their maturation status and competences. Using the Affymetrix GeneChip Bovine Genome Array, global mRNA expression analysis of immature (GV) and in vitro matured (IVM) bovine oocytes was carried out to characterize the transcriptome of bovine oocytes and then use a variety of approaches to determine whether the observed transcriptional changes during IVM was real or an artifact of the techniques used during analysis.Results8489 transcripts were detected across the two oocyte groups, of which ~25.0% (2117 transcripts) were differentially expressed (p < 0.001); corresponding to 589 over-expressed and 1528 under-expressed transcripts in the IVM oocytes compared to their immature counterparts. Over expression of transcripts by IVM oocytes is particularly interesting, therefore, a variety of approaches were employed to determine whether the observed transcriptional changes during IVM were real or an artifact of the techniques used during analysis, including the analysis of transcript abundance in oocytes in vitro matured in the presence of α-amanitin. Subsets of the differentially expressed genes were also validated by quantitative real-time PCR (qPCR) and the gene expression data was classified according to gene ontology and pathway enrichment. Numerous cell cycle linked (CDC2, CDK5, CDK8, HSPA2, MAPK14, TXNL4B), molecular transport (STX5, STX17, SEC22A, SEC22B), and differentiation (NACA) related genes were found to be among the several over-expressed transcripts in GV oocytes compared to the matured counterparts, while ANXA1, PLAU, STC1and LUM were among the over-expressed genes after oocyte maturation.ConclusionUsing sequential experiments, we have shown and confirmed transcriptional changes during oocyte maturation. This dataset provides a unique reference resource for studies concerned with the molecular mechanisms controlling oocyte meiotic maturation in cattle, addresses the existing conflicting issue of transcription during meiotic maturation and contributes to the global goal of improving assisted reproductive technology.

RNA Sequencing Reveals Novel Gene Clusters in Bovine Conceptuses Associated with Maternal Recognition of Pregnancy and Implantation

ABSTRACT Successful establishment and maintenance of pregnancy can be attained only through optimum conceptus-maternal cross talk. Despite significant progress in our understanding of the temporal changes in the transcriptome of the uterine endometrium, we have only a rudimentary knowledge of the genes and pathways governing growth and development of the bovine conceptus. In particular, very little information exists for the posthatching embryo and elongating conceptus. This period of development is arguably the most important, as approximately 40% of all embryonic loss occurs between Days 8 and 17 of pregnancy in cattle. Here, we describe the global transcriptome profile of the bovine conceptus at five key stages of its pre- and peri-implantation growth (Days 7, 10, 13, 16, and 19) using state-of-the-art RNA sequencing techniques. More than 287 million reads were generated at the five stages, and more than 22 700 unique transcripts were detected. Analysis of variance followed by self-organizing maps identified differentially regulated (P < 0.05) genes organized in nine gene clusters forming a sequential transcript dynamics across these developmental stages. Of particular interest, genes in clusters 3 (n = 236) and 6 (n = 1409) were significantly up-regulated on Days 16 and 19, suggesting a role in maternal recognition and initiation of implantation. This transcriptome analysis of the bovine conceptus will provide a blueprint of the dynamic changes in gene expression occurring during maternal recognition and implantation and will complement existing knowledge of the temporal changes in the endometrial transcriptome, thus facilitating a better understanding of conceptus-maternal cross talk during the peri-implantation period of pregnancy.

Evidence for an early endometrial response to pregnancy in cattle: both dependent upon and independent of interferon tau

The aims of this study were to 1) identify the earliest transcriptional response of the bovine endometrium to the presence of the conceptus (using RNAseq), 2) investigate if these genes are regulated by interferon tau (IFNT) in vivo, and 3) determine if they are predictive of the pregnancy status of postpartum dairy cows. RNAseq identified 459 differentially expressed genes (DEGs) between pregnant and cyclic endometria on day 16. Quantitative real-time PCR analysis of selected genes revealed PARP12, ZNFX1, HERC6, IFI16, RNF213, and DDX58 expression increased in pregnant compared with cyclic endometria on day 16 and were directly upregulated by intrauterine infusion of IFNT in vivo for 2 h (P < 0.05). On day 13 following estrous endometrial expression of nine genes increased [ARHGAP1, MGC127874, LIMS2, TBC1D1, FBXL7, C25H16orf71, LOC507810, ZSWIM4, and one novel gene (ENSBTAT00000050193)] and seven genes decreased (SERBP1, SRGAP2, AL7A1, TBK1, F2RL2, MGC128929, and WBSCR17; P < 0.05) in pregnant compared with cyclic heifers. Of these DEGs, significant differences in expression between pregnant and cyclic endometria were maintained on day 16 for F2RL2, LIMS2, LOC507810, MGC127874, TBC1D1, WBSCR17, and ZSWIM4 (P < 0.05) both their expression was not directly regulated by IFNT in vivo. Analysis of the expression of selected interferon-stimulated genes in blood samples from postpartum dairy cows revealed a significant increase (P < 0.05) in expression of ZXFX1, PARP12, SAMD9, and HERC6 on day 18 following artificial insemination in cows subsequently confirmed pregnant compared with cyclic controls. In conclusion, RNAseq identified a number of novel pregnancy-associated genes in the endometrium of cattle during early pregnancy that are not regulated by IFNT in vivo. In addition, a number of genes that are directly regulated by short term exposure to IFNT in vivo are differentially expressed on day 18 following estrus detection in the blood of postpartum dairy cows depending on their pregnancy status.

A Complete Mitochondrial Genome Sequence from a Mesolithic Wild Aurochs (Bos primigenius)

Background The derivation of domestic cattle from the extinct wild aurochs (Bos primigenius) has been well-documented by archaeological and genetic studies. Genetic studies point towards the Neolithic Near East as the centre of origin for Bos taurus, with some lines of evidence suggesting possible, albeit rare, genetic contributions from locally domesticated wild aurochsen across Eurasia. Inferences from these investigations have been based largely on the analysis of partial mitochondrial DNA sequences generated from modern animals, with limited sequence data from ancient aurochsen samples. Recent developments in DNA sequencing technologies, however, are affording new opportunities for the examination of genetic material retrieved from extinct species, providing new insight into their evolutionary history. Here we present DNA sequence analysis of the first complete mitochondrial genome (16,338 base pairs) from an archaeologically-verified and exceptionally-well preserved aurochs bone sample. Methodology DNA extracts were generated from an aurochs humerus bone sample recovered from a cave site located in Derbyshire, England and radiocarbon-dated to 6,738±68 calibrated years before present. These extracts were prepared for both Sanger and next generation DNA sequencing technologies (Illumina Genome Analyzer). In total, 289.9 megabases (22.48%) of the post-filtered DNA sequences generated using the Illumina Genome Analyzer from this sample mapped with confidence to the bovine genome. A consensus B. primigenius mitochondrial genome sequence was constructed and was analysed alongside all available complete bovine mitochondrial genome sequences. Conclusions For all nucleotide positions where both Sanger and Illumina Genome Analyzer sequencing methods gave high-confidence calls, no discrepancies were observed. Sequence analysis reveals evidence of heteroplasmy in this sample and places this mitochondrial genome sequence securely within a previously identified aurochsen haplogroup (haplogroup P), thus providing novel insights into pre-domestic patterns of variation. The high proportion of authentic, endogenous aurochs DNA preserved in this sample bodes well for future efforts to determine the complete genome sequence of a wild ancestor of domestic cattle.

Proteomic analysis of uterine fluid during the pre-implantation period of pregnancy in cattle

The aims of this study were (i) to characterize the global changes in the composition of the uterine luminal fluid (ULF) from pregnant heifers during pregnancy recognition (day 16) using nano-LC MS/MS; (ii) to describe quantitative changes in selected proteins in the ULF from days 10, 13, 16 and 19 by Isobaric tags for Relative and Absolute Quantification (iTRAQ) analysis; and (iii) to determine whether these proteins are of endometrial or conceptus origin, by examining the expression profiles of the associated transcripts by RNA sequencing. On day 16, 1652 peptides were identified in the ULF by nano-LC MS/MS. Of the most abundant proteins present, iTRAQ analysis revealed that RPB4, TIMP2 and GC had the same expression pattern as IFNT, while the abundance of IDH1, CST6 and GDI2 decreased on either day 16 or 19. ALDOA, CO3, GSN, HSP90A1, SERPINA31 and VCN proteins decreased on day 13 compared with day 10 but subsequently increased on day 16 (P<0.05). Purine nucleoside phosphorylase (PNP) and HSPA8 decreased on day 13, increased on day 16 and decreased and increased on day 19 (P<0.05). The abundance of CATD, CO3, CST6, GDA, GELS, IDHC, PNPH and TIMP2 mRNAs was greater (P<0.001) in the endometrium than in the conceptus. By contrast, the abundance of ACTB, ALDOA, ALDR, CAP1, CATB, CATG, GD1B, HSP7C, HSP90A, RET4 and TERA was greater (P<0.05) in the conceptus than in the endometrium. In conclusion, significant changes in the protein content of the ULF occur during the pre-implantation period of pregnancy reflecting the morphological changes that occur in the conceptus.

Effect of the metabolic environment at key stages of follicle development in cattle: focus on steroid biosynthesis

Cellular mechanisms that contribute to low estradiol concentrations produced by the preovulatory ovarian follicle in cattle with a compromised metabolic status are largely unknown. To gain insight into the main metabolic mechanisms affecting preovulatory follicle function, two different animal models were used. Experiment 1 compared Holstein-Friesian nonlactating heifers (n = 17) and lactating cows (n = 16) at three stages of preovulatory follicle development: 1) newly selected dominant follicle in the luteal phase (Selection), 2) follicular phase before the LH surge (Differentiation), and 3) preovulatory phase after the LH surge (Luteinization). Experiment 2 compared newly selected dominant follicles in the luteal phase in beef heifers fed a diet of 1.2 times maintenance (M, n = 8) or 0.4 M (n = 11). Lactating cows and 0.4 M beef heifers had higher concentrations of β-hydroxybutyrate, and lower concentrations of glucose, insulin, and IGF-I compared with dairy heifers and 1.2 M beef heifers, respectively. In lactating cows this altered metabolic environment was associated with reduced dominant follicle estradiol and progesterone synthesis during Differentiation and Luteinization, respectively, and in 0.4 M beef heifers with reduced dominant follicle estradiol synthesis. Using a combination of RNA sequencing, Ingenuity Pathway Analysis, and qRT-PCR validation, we identified several important molecular markers involved in steroid biosynthesis, such as the expression of steroidogenic acute regulatory protein (STAR) within developing dominant follicles, to be downregulated by the catabolic state. Based on this, we propose that the adverse metabolic environment caused by lactation or nutritional restriction decreases preovulatory follicle function mainly by affecting cholesterol transport into the mitochondria to initiate steroidogenesis.