Eric Antoniou

Development and application of bovine and porcine oligonucleotide arrays with protein-based annotation.

The design of oligonucleotide sequences for the detection of gene expression in species with disparate volumes of genome and EST sequence information has been broadly studied. However, a congruous strategy has yet to emerge to allow the design of sensitive and specific gene expression detection probes. This study explores the use of a phylogenomic approach to align transcribed sequences to vertebrate protein sequences for the detection of gene families to design genomewide 70-mer oligonucleotide probe sequences for bovine and porcine. The bovine array contains 23,580 probes that target the transcripts of 16,341 genes, about 72% of the total number of bovine genes. The porcine array contains 19,980 probes targeting 15,204 genes, about 76% of the genes in the Ensembl annotation of the pig genome. An initial experiment using the bovine array demonstrates the specificity and sensitivity of the array.

A microarray analysis for genes regulated by interferon-τ in ovine luminal epithelial cells

Interferon-tau (IFNT) is released by preimplantation conceptuses of ruminant species and prepares the mother for pregnancy. Although one important function is to protect the corpus luteum from the luteolytic activity of prostaglandin-F 2alpha, IFNT most likely regulates a range of other physiological processes in endometrium. Here, an immortalized cell line from ovine uterine luminal epithelial cells was treated with IFNT for either 8 or 24 h. RNA was subjected to cDNA microarray analysis, with RNA from untreated cells as the reference standard. Of 15 634 genes, 1274 (8%) were IFNT responsive at P<0.01 and 585 at P<0.001 to at least one treatment. Of the latter, 356 were up-regulated and 229 down-regulated. Increasing IFNT concentrations from 10 ng/ml to 10 microg/ml had minor effects, and most genes up- or down-regulated at 8 h were regulated similarly at 24 h. Although IFNT influences many genes implicated in antiviral activity and apoptosis, its action also likely regulates prostaglandin metabolism, growth factors and their receptors, apoptosis and the nuclear factor (NF)-kappaB cascade, extracellular matrix accretion, angiogenesis, blood coagulation, and inflammation. In particular, it increased mRNA concentrations of genes related to the vascular endothelial growth factor R2 pathway of angiogenesis and down-regulated ones associated with hypoxia. Two genes implicated in the antiluteolytic actions of IFNT (encoding cyclooxygenase-2 and the oxytocin receptor respectively) were down-regulated in response to all treatments. IFNT targets a complex range of physiological processes during the establishment of pregnancy.

Testicular gene expression in male mice divergent for fertility after heat stress.

Fertility losses in male mice occur approximately 18-28 d after heat stress. The objective of this study was to identify gene expression differences in males highly versus lowly fertile after heat stress. Mature male mice were exposed to heat stress (35+/-1 degrees C; n=50) or thermoneutral (21+/-1 degrees C; n=10) conditions for 24 h (Day 0) and hemicastrated (Day 1) to collect tissue for gene expression analyses. Males were subjected to a mating test from Days 18 to 26 when variation in fertility was anticipated. A fertility index was used to rank heat-stressed males and identify those males resistant and susceptible to heat stress, respectively. Microarray analyses were conducted on testis tissues from control (n=5), heat stress resistant (n=5), and heat stress susceptible (n=5) males, and 225 genes were observed to be differentially expressed (P<0.05), including genes involved in chaperone (Canx, Hspcb1, and Tcp1) and catalytic (Fkpb6, Psma7, and Idh1) activity. Expression patterns of these genes were confirmed using real-time RT-PCR. Male progeny from selected sires were similarly divergent in fertility after heat stress. Testicular expression levels of Canx, Hspcb, and Tcp1 genes were determined in these progeny. Hspcb expression was moderately heritable (0.31+/-0.25); however, expression patterns of Canx and Tcp1 were not heritable.

Molecular Mechanisms Regulating Bovine Ovarian Follicular Selection

Transcription profiling of ovarian follicles. Understanding the mechanisms by which a single follicle is selected for further ovulation is important to control fertility in mammals. However, development of new treatments is limited by our poor understanding of molecular mechanisms regulating follicular selection. Our hypothesis is that genes involved in the control of cell proliferation and apoptosis are differentially regulated during follicular selection. Our objective was to identify these new genes. Bovine follicles were collected and gene expression levels were measured using microarrays. First, follicles were allocated to three groups, according to the time spent from the initiation of follicular wave to surgery (24 H, 36 H, and 48–60 H). Fifty‐seven genes are differentially expressed at a false discovery rate of 5%. These genes are involved in the control of lipid metabolism (P‐value = 0.0005), cell proliferation (0.007), cell death (0.003), cell morphology (0.003), and immune response (0.003). Follicles were also grouped into four categories, according to the expected time of deviation (early deviation; 8 mm, mid‐deviation; 8.5 mm, late deviation; 9 mm, dominant follicles; ≥10 mm). One hundred and twenty eight genes are differentially expressed between these four groups, including genes involved in cell proliferation (0.00002), cell death (0.0006), cell‐to‐cell signaling (0.003), cell morphology (0.003), lipid metabolism (0.0004), and immune response (0.00007). The expression levels of 10 genes were confirmed using quantitative real time PCR. As expected, we identified new differentially regulated genes involved in the control of cell growth and apoptosis. We also discovered a potential role for immune cells, and in particular macrophages, in follicular selection. Mol. Reprod. Dev. 76: 351–366, 2009.

CORRELATION OF DEVELOPMENTAL DIFFERENCES OF NUCLEAR TRANSFER EMBRYOS CELLS TO THE METHYLATION PROFILES OF NUCLEAR TRANSFER DONOR CELLS IN SWINE

Methylation of DNA is the most commonly studied epigenetic mechanism of developmental competence and somatic cell nuclear transfer (SCNT). Previous studies of epigenetics and the SCNT procedures have examined the effects of different culture media on donor cells and reconstructed embryos, and the methylation status of specific genes in the fetus or live offspring. Here we used a microarray based approach to identify the methylation profiles of SCNT donor cells including three clonal porcine fetal fibroblast-like cell sublines and adult somatic cells selected from kidney and mammary tissues. The methylation profiles of the donor cells were then analyzed with respect to their ability to direct development to the blastocyst stage after nuclear transfer. Clonal cell lines A2, A7, and A8 had blastocyst rates of 11.7%a, 16.7%ab, and 20.0%b, respectively (ab P