Massive dysregulation of genes involved in cell signaling and placental development in cloned cattle conceptus and maternal endometrium.

A major unresolved issue in the cloning of mammals by somatic cell nuclear transfer (SCNT) is the mechanism by which the process fails after embryos are transferred to the uterus of recipients before or during the implantation window. We investigated this problem by using RNA sequencing (RNA-seq) to compare the transcriptomes in cattle conceptuses produced by SCNT and artificial insemination (AI) at day (d) 18 (preimplantation) and d 34 (postimplantation) of gestation. In addition, endometrium was profiled to identify the communication pathways that might be affected by the presence of a cloned conceptus, ultimately leading to mortality before or during the implantation window. At d 18, the effects on the transcriptome associated with SCNT were massive, involving more than 5,000 differentially expressed genes (DEGs). Among them are 121 genes that have embryonic lethal phenotypes in mice, cause defects in trophoblast and placental development, and/or affect conceptus survival in mice. In endometria at d 18, <0.4% of expressed genes were affected by the presence of a cloned conceptus, whereas at d 34, ∼36% and <0.7% of genes were differentially expressed in intercaruncular and caruncular tissues, respectively. Functional analysis of DEGs in placental and endometrial tissues suggests a major disruption of signaling between the cloned conceptus and the endometrium, particularly the intercaruncular tissue. Our results support a "bottleneck" model for cloned conceptus survival during the periimplantation period determined by gene expression levels in extraembryonic tissues and the endometrial response to altered signaling from clones.

A high-resolution whole-genome cattle-human comparative map reveals details of mammalian chromosome evolution.

Approximately 3,000 cattle bacterial artificial chromosome (BAC)-end sequences were added to the Illinois-Texas 5,000-rad RH (RH, radiation hybrid) map. The BAC-end sequences selected for mapping are approximately 1 Mbp apart on the human chromosomes as determined by blastn analysis. The map has 3,484 ordered markers, of which 3,204 are anchored in the human genome. Two hundred-and-one homologous synteny blocks (HSBs) were identified, of which 27 are previously undiscovered, 79 are extended, 26 were formed by previously unrecognized breakpoints in 18 previously defined HSBs, and 23 are the result of fusions. The comparative coverage relative to the human genome is approximately 91%, or 97% of the theoretical maximum. The positions of 64% of all cattle centromeres and telomeres were reassigned relative to their positions on the previous map, thus facilitating a more detailed comparative analysis of centromere and telomere evolution. As an example of the utility of the high-resolution map, 22 cattle BAC fingerprint contigs were directly anchored to cattle chromosome 19 [Bos taurus, (BTA) 19]. The order of markers on the cattle RH and fingerprint maps of BTA19 and the sequence-based map of human chromosome 17 [Homo sapiens, (HSA) 17] were found to be highly consistent, with only two minor ordering discrepancies between the RH map and fingerprint contigs. The high-resolution Illinois-Texas 5,000-rad RH and comparative maps will facilitate identification of candidate genes for economically important traits, the phylogenomic analysis of mammalian chromosomes, proofing of the BAC fingerprint map and, ultimately, aid the assembly of cattle whole-genome sequence.

Cytokine mRNA expression in B cells from bovine leukemia virus-infected cattle with persistent lymphocytosis.

We have characterized the expression of six cytokine mRNAs in highly purified B cells from bovine leukemia virus (BLV)-infected cows with persistent lymphocytosis. Selected cytokine mRNAs included those encoding tumor necrosis factor (TNF), lymphotoxin-alpha (LT-alpha), transforming growth factor-beta1 (TGF-beta1), interleukin-1beta (IL-1beta), interleukin-6 (IL-6) and interleukin-10 (IL-10). Fresh B cells from cows with persistent lymphocytosis constitutively transcribed TNF, LT-alpha and TGF-beta1 mRNAs. Although IL-1beta, IL-6 and IL-10 mRNAs were barely detectable in fresh B cells from cows with persistent lymphocytosis, transcripts encoding these cytokines were strongly and rapidly upregulated in B cells after cell culture. Results from this study provide the first evidence that B cells infected with BLV express specific cytokine mRNAs in vivo.

Reduced IL-2 and IL-4 mRNA expression in CD4+ T cells from bovine leukemia virus-infected cows with persistent lymphocytosis.

The role of T-helper (Th) responses in the subclinical progression of bovine leukemia virus (BLV) infection was explored by determining the contribution of CD4+ T cells to the expression of mRNAs encoding interferon-gamma (IFN-gamma), interleukin-2 (IL-2), interleukin-4 (IL-4), and interleukin-10 (IL-10) in BLV-infected cattle. Relative levels of mRNA encoding IFN-gamma, IL-2, IL-4, and IL-10 were measured in fresh and concanavalin A (Con A) activated peripheral blood mononuclear cells (PBMCs) and purified CD4+ T cells from cows seronegative to BLV (BLV-), seropositive without persistent lymphocytosis (BLV+PL-), and seropositive with PL (BLV+PL+) using a semiquantitative reverse transcription-polymerase chain reaction (RT-PCR) assay. The expressions of IFN-gamma, IL-2, and IL-4 mRNAs were significantly reduced in the PBMCs from BLV+PL+ cows as compared to BLV- cows. Reduced levels of IL-2 and IL-4 mRNAs were detected in fresh CD4+ T cells from BLV+PL+ cows. In contrast, Con A stimulated PBMCs and CD4+ T cells did not differ significantly in expression of IFN-gamma, IL-2, IL-10, or IL-4 mRNAs among the BLV infection groups. Using flow-sorted CD4+ T cells and semiquantitative RT-PCR the frequencies of CD4+ T cells transcribing IFN-gamma, IL-2, IL-4, and IL-10 mRNAs in the peripheral blood of BLV-, BLV+PL-, and BLV+PL+ cows were determined. There were no significant differences in the frequencies of CD4+ T cells expressing these cytokine mRNAs among animals in the different BLV infection categories. Thus, the observed differences in IL-2 and IL-4 mRNAs in CD4+ T cells were due to changes in steady-state mRNA levels expressed by individual cells and not to changes in the frequency of cells transcribing IL-2 and IL-4 mRNAs. These results demonstrate that the progression of BLV infection to PL is associated with reduced expression of classical Th1 and Th2 cytokines by CD4+ T cells, thus suggesting aberrant Th regulation in subclinically infected animals.