Tamas Revay

Effect of histone deacetylase inhibitor valproic acid treatment on donor cell growth characteristics, cell cycle arrest, apoptosis, and handmade cloned bovine embryo production efficiency.

In this study, we tested the effects of valproic acid (VPA), a known histone deacetylase inhibitor (HDACi), on the growth characteristics, apoptosis, and cell cycle stages distribution of donor cells, as well as cloning efficiency, embryo development, and histone methylation. Our results showed that treatment of donor cells with VPA (2.5 mM, 5.0 mM, 7.5 mM, or 10 mM) for 24 h resulted in altered cell proliferation, extent of apoptosis and necrosis, and cell cycle stage distribution, whereas no changes in cell viability and chromosomal complements were observed. Measurement of relative gene expression using real-time PCR of a few developmentally important genes in treated donor cells showed decreased expression of HDAC1 and increased expression of BAX (p<0.05). No change in relative expression of HDAC2 and Bcl2 was noticed. Treatment of donor cells with VPA for 24 h before electrofusion significantly (p<0.05) increased the blastocyst formation rate of somatic cell nuclear transfer (SCNT) embryos compared to the control embryos. Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL)-positive nuclei in SCNT blastocysts derived from VPA-treated donor cells were significantly decreased compared to the control blastocysts (p<0.05). Immunolocalization studies revealed that the levels of histone H3 at lysine 9 (H3K9me3) were lower in VPA-treated donor cells derived cloned blastocysts than nontreated cloned embryos, and was at the level of in vitro fertilization (IVF) counterparts, although no effects of treatments were found in donor cells. Our study demonstrates that the use of VPA in SCNT has been beneficial for efficient reprogramming of donor cells. Its effect on histone methylation in cloned embryos correlates with their developmental potential and may be a useful epigenetic marker to predict the efficiency of SCNT.

A Large Expansion of the HSFY Gene Family in Cattle Shows Dispersion across Yq and Testis-Specific Expression

Heat shock transcription factor, Y-linked (HSFY) is a member of the heat shock transcriptional factor (HSF) family that is found in multiple copies on the Y chromosome and conserved in a number of species. Its function still remains unknown but in humans it is thought to play a role in spermatogenesis. Through real time polymerase chain reaction (PCR) analyses we determined that the HSFY family is largely expanded in cattle (∼70 copies) compared with human (2 functional copies, 4 HSFY-similar copies). Unexpectedly, we found that it does not vary among individual bulls as a copy number variant (CNV). Using fluorescence in situ hybridization (FISH) we found that the copies are dispersed along the long arm of the Y chromosome (Yq). HSFY expression in cattle appears restricted to the testis and its mRNA correlates positively with mRNA markers of spermatogonial and spermatocyte cells (UCHL1 and TRPC2, respectively) which suggests that HSFY is expressed (at least in part) in early germ cells.

GTG Mutation in the Start Codon of the Androgen Receptor Gene in a Family of Horses with 64,XY Disorder of Sex Development

Genetic sex in mammals is determined by the sex chromosomal composition of the zygote. The X and Y chromosomes are responsible for numerous factors that must work in close concert for the proper development of a healthy sexual phenotype. The role of androgens in case of XY chromosomal constitution is crucial for normal male sex differentiation. The intracellular androgenic action is mediated by the androgen receptor (AR), and its impaired function leads to a myriad of syndromes with severe clinical consequences, most notably androgen insensitivity syndrome and prostate cancer. In this paper, we investigated the possibility that an alteration of the equine AR gene explains a recently described familial XY, SRY + disorder of sex development. We uncovered a transition in the first nucleotide of the AR start codon (c.1A>G). To our knowledge, this represents the first causative AR mutation described in domestic animals. It is also a rarely observed mutation in eukaryotes and is unique among the >750 entries of the human androgen receptor mutation database. In addition, we found another quiet missense mutation in exon 1 (c.322C>T). Transcription of AR was confirmed by RT-PCR amplification of several exons. Translation of the full-length AR protein from the initiating GTG start codon was confirmed by Western blot using N- and C-terminal-specific antibodies. Two smaller peptides (25 and 14 amino acids long) were identified from the middle of exon 1 and across exons 5 and 6 by mass spectrometry. Based upon our experimental data and the supporting literature, it appears that the AR is expressed as a full-length protein and in a functional form, and the observed phenotype is the result of reduced AR protein expression levels.

Thyroid hormones alter the transcriptome of in vitro-produced bovine blastocysts.

Thyroid hormones (THs) have been shown to improve in vitro embryo production in cattle by increasing blastocyst formation rate, and the average cell number of blastocysts and by significantly decreasing apoptosis rate. To better understand those genetic aspects that may underlie enhanced early embryo development in the presence of THs, we characterized the bovine embryonic transcriptome at the blastocyst stage, and examined differential gene expression profiles using a bovine-specific microarray. We found that 1212 genes were differentially expressed in TH-treated embryos when compared with non-treated controls (>1.5-fold at P < 0.05). In addition 23 and eight genes were expressed uniquely in control and treated embryos, respectively. The expression of genes specifically associated with metabolism, mitochondrial function, cell differentiation and development were elevated. However, TH-related genes, including those encoding TH receptors and deiodinases, were not differentially expressed in treated embryos. Furthermore, the over-expression of 52 X-chromosome linked genes in treated embryos suggested a delay or escape from X-inactivation. This study highlights the significant impact of THs on differential gene expression in the early embryo; the identification of TH-responsive genes provides an insight into those regulatory pathways activated during development.

Somatic Mosaicism in Bulls Estimated from Genome-Wide CNV Array and TSPY Gene Copy Numbers.

Somatic mosaicism has become a focus in human research due to the implications of individual genetic variability in disease. Here, we assessed somatic copy number variations (CNVs) in Holstein bulls in 2 respects. We estimated genome-wide CNVs and assayed CNVs of the TSPY gene, the most variable bovine gene from the Y chromosome. Somatic tissues (blood, lung, heart, muscle, testis, and brain) of 4 bulls were arrayed on the Illumina Bovine SNP50k chip and qPCR tested for TSPY copy numbers. Our results showed extensive copy number divergence in tissues within the same animal as well as significant copy number alterations of TSPY. We detected a mean of 31 CNVs per animal among which 14 were of germline origin, as they were constantly present in all investigated tissues of the animal, while 18 were specific to 1 tissue. Thus, 57% of the total number of detected CNVs was the result of de novo somatic events. Further, TSPY copy number was found to vary significantly among tissues as well as among the same tissue type from different animals in a wide range from 7 to 224% of the calibrator. Our study shows significant autosomal and Y-chromosomal de novo somatic CNV in bulls.

Missense Mutation in the Ligand-Binding Domain of the Horse Androgen Receptor Gene in a Thoroughbred Family with Inherited 64,XY (SRY+) Disorder of Sex Development.

Disorders of sex development (DSD) have long been documented in domestic animal species including horses. However, there is only a single report of an androgen receptor (AR) mutation causative of such a DSD syndrome in a horse pedigree. Here, we present a new familial AR mutation in horses. A missense mutation (c.2042G>C) at AR exon 4 explains the segregation of the DSD in a Thoroughbred horse pedigree. The mutation, expected to affect the ligand-binding domain of the AR protein, led to complete androgen insensitivity of 64,XY SRY+, testicular DSD individuals. Additionally, the design of a PCR-RFLP technique provided an accurate molecular test for the identification of horses carrying the mutation.

Ovarian tumour growth is characterized by mevalonate pathway gene signature in an orthotopic, syngeneic model of epithelial ovarian cancer

Epithelial ovarian cancer (EOC) is the most lethal gynecological cancer and often is not detected until late stages when cancer cells transcoelomically metastasize to the abdomen and typically become resistant to therapy resulting in very low survival rates. We utilize an orthotopic, syngeneic mouse model to study late stage disease and have discovered that the tumor cells within the abdominal ascites are irreversibly re-programmed, with an increased tumorigenicity and resistance to apoptosis. The goal of this study was to characterize the reprogramming that occurred in the aggressive ascites-derived cells (28-2 cells) compared to the original cell line used for tumor induction (ID8 cells). Microarray experiments showed that the majority of genes upregulated in the 28-2 cells belonged to the mevalonate pathway, which is involved in cholesterol biosynthesis, protein prenylation, and activation of small GTPases. Upregulation of mevalonate appeared to be associated with the acquisition of a p53 mutation in the ascites-derived cells. Treatment with simvastatin to inhibit HMG CoA reductase, the rate limiting enzyme of this pathway, induced apoptosis in the 28-2 cell line. Rescue experiments revealed that mevalonate, but not cholesterol, could inhibit the simvastatin-mediated effects. In vivo, daily intraperitoneal simvastatin treatment significantly regressed advanced stage disease and induced death of metastatic tumor cells. These data suggest that ovarian cancer cells become reprogrammed, with genetic mutations, and upregulation of the mevalonate pathway, which facilitates the development of advanced stage disease. The use of statins to inhibit HMGCR may provide novel therapeutic opportunities for the treatment of advanced stage EOC.

The Porcine TSPY Gene Is Tricopy but Not a Copy Number Variant.

The testis-specific protein Y-encoded (TSPY) gene is situated on the mammalian Y-chromosome and exhibits some remarkable biological characteristics. It has the highest known copy number (CN) of all protein coding genes in the human and bovine genomes (up to 74 and 200, respectively) and also shows high individual variability. Although the biological function of TSPY has not yet been elucidated, its specific expression in the testis and several identified binding domains within the protein suggests roles in male reproduction. Here we describe the porcine TSPY, as a multicopy gene with three copies located on the short arm of the Y-chromosome with no variation at three exon loci among 20 animals of normal reproductive health from four breeds of domestic pigs (Piétrain, Landrace, Duroc and Yorkshire). To further investigate the speculation that porcine TSPY is not a copy number variant, we have included five Low-fertility boars and five boars with exceptional High-fertility records. Interestingly, there was no difference between the High- and Low-fertile groups, but we detected slightly lower TSPY CN at all three exons (2.56-2.85) in both groups, as compared to normal animals, which could be attributed to technical variability or somatic mosaicism. The results are based on both relative quantitative real-time PCR (qPCR) and droplet digital PCR (ddPCR). Chromosomal localization of the porcine TSPY was done using fluorescence in situ hybridization (FISH) with gene specific PCR probes.

Androgen Insensitivity Syndrome in a Family of Warmblood Horses Caused by a 25-bp Deletion of the DNA-Binding Domain of the Androgen Receptor Gene

Testicular feminization, an earlier term coined for describing a syndrome resulting from failure of masculinization of target organs by androgen secretions during embryo development, has been well documented not only in humans but also in the domestic horse. The pathology, actually referred to as androgen insensitivity syndrome (AIS), has been proposed to follow an X-linked recessive pattern of inheritance in some horse breeds already investigated. Affected individuals are characterized by a female phenotype but with a stallion genotype of 64,XY SRY+ constitution. We identified a Warmblood horse pedigree segregating AIS, where the molecular analyses of the androgen receptor gene in the family provided evidences that a 25-bp deletion of the DNA-binding domain is causative of this equine syndrome.

Azoospermia and Testicular Hypoplasia in a Boar Carrier of a Novel Y-Autosome Translocation

Few sex-autosome chromosome abnormalities have been documented in domestic animal species. In humans, Y-autosome chromosome abnormalities may occur at a rate of 1/2,000 live births, whereas in the domestic pig only 2 Y-autosome reciprocal translocations have been previously described. During a routine cytogenetic screening of young boars, we identified a new Y-autosome translocation carrier, which after puberty showed semen devoid of sperm and testicular hypoplasia with spermatogenesis arrest. Whole chromosome painting by FISH analysis corroborated the reciprocal nature of the chromosomal exchanges between the Y chromosome and SSC13. The possible causes for the observed meiotic arrest of the carrier are reviewed.