Proteins associated with reproductive disorders in testes of human erythropoietin gene-harboring transgenic boars

To investigate reproductive disorder in human erythropoietin (EPO)-expressing pig, we performed comparative proteomic analyses of testicular tissues from human erythropoietin (hEPO) gene-harboring transgenic pigs and wild type pigs born from natural conception. In hEPO TG pigs, we found relatively low sperm motility and higher death rate indicating impaired sperm development. Consistently, plasma concentration of testosterone was significantly lower in the transgenic post-pubertal boars compared with wild type boars. Normalized protein spots showing higher than 2-fold differential expression intensity in two-dimensional polyacrylamide gel electrophoresis were selected for matrix associated laser desorption/ionization time-to-flight mass spectrometry analysis. Specific proteins were identified by searching the NCBI protein sequence databases. Among 55 proteins selected, 12 proteins were identified as those differentially expressed between transgenic and wild type pigs. Three downregulated proteins (β-globin, carbonyl reductase 1, and peroxiredoxin 6) and nine upregulated proteins (cytoskeletal β-actin, α 2,3-sialyltransferase, apolipoprotein A-I, tubulin α-1A chain, tropomodulin 3, thioredoxin, heat shock Protein 70.2, ch4/domains of swine IgM, and albumin), all of which are closely related to apoptosis and cytoskeletal development, were found in the transgenic boar testes. Terminal deoxynucleotidyltransferase-mediated dUTP nick end labeling assay confirmed the increased occurrence of apoptosis in the transgenic boar testes compared with the wild type boar testes. Reproductive defects of the hEPO-expressing transgenic pigs may be caused by the abnormal expression of the genes identified in this study.     

Identification of cytochrome c oxidase subunit 6A1 as a suppressor of Bax-induced cell death by yeast-based functional screening

Human cytochrome c oxidase subunit VIa polypeptide 1 (COX6A1) was identified as a novel suppressor of Bcl-2-associated X protein (Bax)-mediated cell death using yeast-based functional screening of a mammalian cDNA library. The overexpression of COX6A1 significantly suppressed Bax- and N-(4-hydroxyphenyl)retinamide (4-HPR)-induced apoptosis in yeast and human glioblastoma-derived U373MG cells, respectively. The generation of reactive oxygen species (ROS) in response to Bax or 4-HPR was inhibited in yeast and U373MG cells that expressed COX6A1, indicating that COX6A1 exerts a protective effect against ROS-induced cell damage. 4-HPR-induced mitochondrial translocation of Bax, release of mitochondrial cytochrome c, and activation of caspase-3 were markedly attenuated in U373MG cells that stably expressed COX6A1. Our results demonstrate that yeast-based functional screening of human genes for inhibitors of Bax-sensitivity in yeast identified a protein that not only suppresses the toxicity of Bax in yeast, but also has a potential role in protecting mammalian cells from 4-HPR-induced apoptosis.     

Methylation status of putative differentially methylated regions of porcine IGF2 and H19

This study was designed to identify the putative differentially methylated regions (DMRs) of the porcine imprinted genes insulin-like growth factor 2 and H19 (IGF2-H19), and to assess the genomic imprinting status of IGF2-H19 by identifying the methylation patterns of these regions in germ cells, and in tissues from porcine fetuses, an adult pig, as well as cloned offspring produced by somatic cell nuclear transfer (SCNT). Porcine IGF2-H19 DMRs exhibit a normal monoallelic methylation pattern (i.e., either the paternally- or the maternally derived allele is methylated) similar to the pattern observed for the same genes in the human and mice genomes. Examination of the methylation patterns of the IGF2-H19 DMRs revealed that the zinc finger protein binding sites CTCF1 and 2 did not exhibit differential methylation in both control and cloned offspring. In contrast, the CTCF3 and DMR2 loci of the IGF2 gene showed abnormal methylation in cloned offspring, but a normal differential or moderate methylation pattern in tissues from control offspring and an adult pig. Our data thus suggest that regulation of genomic imprinting at the porcine IGF2-H19 loci is conserved among species, and that the abnormal methylation pattern in the regulatory elements of imprinted genes may lead to an alteration in the coordinated expression of genes required for successful reprogramming, which, in consequence, may contribute to the low efficiency of porcine genome reprogramming induced by nuclear transfer.     

Up-regulation of aldose reductase expression mediated by phosphatidylinositol 3-kinase/Akt and Nrf2 is involved in the protective effect of curcumin against oxidative damage

Up-regulation of aldose reductase (AR) by reactive oxygen species (ROS) and aldehyde derivatives has been observed in vascular smooth muscle cells. However, the pathophysiological consequences of the induction of AR in vascular tissues are not fully elucidated. Herein we report that an herb-derived polyphenolic compound, curcumin, elicited a dose- and time-dependent increase in AR expression. Inhibition of phosphatidylinositol 3-kinase (PI3K) and p38 mitogen-activated protein kinase (MAPK) significantly suppressed the curcumin-augmented mRNA levels and promoter activity of the AR gene. Luciferase reporter assays indicated that an osmotic response element in the promoter was essential for the responsiveness to curcumin. Curcumin accelerated the nuclear translocation of nuclear factor-erythroid 2-related factor 2 (Nrf2), and overexpression of Nrf2, but not the dominant negative Nrf2, enhanced the promoter activity of the AR gene. Cells preincubated with curcumin demonstrated resistance to ROS-induced apoptotic death. These effects were significantly attenuated in the presence of AR inhibitors or small interfering RNAs, indicating a protective role for AR against ROS-induced cell damage. Taken together, the activation of PI3K and p38 MAPK by curcumin augmented the expression of the AR gene via Nrf2, and increased AR activity may be an important cellular response against oxidative stress.     

Development of a positive method for male stem cell-mediated gene transfer in mouse and pig

Classical approaches for producing transgenic livestock require labor-intensive, time-consuming, and expensive methods with low efficiency of transgenic production. A promising approach for producing transgenic animals by using male stem cells was recently reported by Brinster and Zimmermann (1994: Proc Natl Acad Sci 91:11298-11302) and by Brinster and Avarbock (1994: Proc Natl Acad Sci USA 91:11303-11307). However, in order to apply this technique to producing transgenic animals, some difficulties have to be overcome. These include a satisfactory method for short-term in vitro culture for drug selection after transfection with exogenous DNA, and methods for the use of livestock such as pigs. We developed a new method for transferring foreign DNA into male germ cells. Mice and pigs were treated with busulfan, an alkylating agent, to destroy the developing male germ cells, and liposome/bacterial LacZ gene complexes were introduced into each seminiferous tubule by using a microinjection needle. As a control, lipofectin was dissolved in phosphate-buffered saline at a ratio of 1:1, and then injected into seminiferous tubules. In mice, 8.0–14.8% of seminiferous tubule expressed the introduced LacZ gene, and 7–13% of epididymal spermatozoa were confirmed as having foreign DNA by polymerase chain reaction. The liposome-injected testes were all negative for X-gal staining. These results indicate that some spermatozoa were successfully transformed in their early stages by liposome/DNA complexes. In pigs, foreign DNA was also incorporated efficiently into male germ cells, and 15.3–25.1% of the seminiferous tubules containing germ cells expressed the LacZ gene. The data suggest that these techniques can be used as a powerful tool for producing transgenic livestock. Mol. Reprod. Dev. 46:515–526, 1997. © 1997 Wiley-Liss, Inc.