Combined in vitro fertilization and culture (IVF/IVC) in mouse for reprotoxicity assessment of xenobiotic exposure.

Litter size and other conventional measures in rodents are common end-points in the assessment of xenobiotics for reprotoxic effects. However, since litter size may be normal despite reduced semen quality, we established and tested a mouse in vitro fertilization/in vitro culture (IVF/IVC) system to assess other aspects of reprotoxicity of xenobiotic exposure. Two pesticides, vinclozolin (V) and chlormequat (C), were added to feed in low (40 and 900 ppm, respectively) and high (300 and 2700 ppm, respectively) doses and compared to control (nil pesticide). Exposed males were used for natural mating to evaluate litter size and then used for IVF/IVC and sperm evaluation. The IVF/IVC system detected significant adverse effect of high dose of vinclozolin on blastocyst formation, which was not detected by conventional measures such as litter size or sperm motility and viability. We conclude that assessment based on IVF/IVC measures may complement litter size and other conventional end-points.

Molecular cloning and characterization of porcine Na⁺/K⁺-ATPase isoform α4.

Na+/K+-ATPase is responsible for maintaining electrochemical gradients of Na+ and K+, which is essential for a variety of cellular functions including neuronal activity. The α-subunit of the Na+/K+-ATPase is composed of four different polypeptides (α1-α4) encoded by different genes. Na,K-ATPase α4, encoded by the ATP1A4 gene, is expressed in testis and in male germ cells of humans, rats and mice. The α4 polypeptide has an important role in sperm motility, and is essential for male fertility. Here we present the RT-PCR cloning and characterization of the porcine ATP1A4 cDNA coding for Na⁺/K⁺-ATPase polypeptide α4. The Na⁺/K⁺-ATPase polypeptide α4, consisting of 1030 amino acids, displays a high homology with its human counterpart (86%). Phylogenetic analysis demonstrated that porcine Na⁺/K⁺-ATPase polypeptide α4 is closely related to other mammalian counterparts. In addition, the genomic structure of the porcine ATP1A4 gene was determined, and the intron-exon organization was found to be similar to that of the human ATP1A4 gene. The promoter sequence for the porcine ATP1A4 gene was also identified. Investigation of the genetic variation in the porcine ATP1A4 gene revealed a missense A/G SNP in exon 18. This A/G polymorphism results in a substitution of a methionine to a glycine residue (M888G). A very high overall DNA methylation rate of the ATP1A4 gene, 70-80%, was observed in both brain and liver. Expression analysis demonstrated that the porcine ATP1A4 gene is predominantly expressed in testis. The sequence of the porcine ATP1A4 cDNA encoding the Na⁺/K⁺-ATPase α4 protein has been submitted to GenBank under the accession number GenBank Accession No. MG587082.

A-to-I RNA editing of the IGFBP7 transcript increases during aging in porcine brain tissues.

The IGFBP7 gene encodes insulin-like growth factor protein 7. IGFPB7 is involved in diverse biological functions including cell growth regulation, senescence and apoptosis, and also acts as a tumor suppressor in multiple cancers. The IGFBP7 mRNA is subject to A-to-I RNA editing mediated by adenosine deaminases acting on RNA 1 and 2 (ADAR1 and ADAR2). In the current study we have examined molecular characteristics of the porcine IGFBP7 gene, and determined the mRNA editing in different tissues. The A-to-I RNA editing of human IGFBP7 in positions Arg78 and Lys95 was shown to be conserved in the porcine homologue. In addition, a novel editing site was discovered in position Lys97 in the porcine IGFBP7 transcript. A differential editing was demonstrated at the three positions in the IGFBP7 transcript with very high degrees of editing in frontal cortex, cerebellum and lung. Interestingly, the degree of editing increased during aging in porcine frontal cortex and cerebellum. The IGFBP7 gene was mapped to pig chromosome 8. The porcine IGFBP7 gene was found to be ubiquitously expressed in examined organs and tissues. The methylation status of the IGFBP gene was examined in brain and liver by bisulfate sequencing and a high degree of methylation was found in the two tissues, 52% and 54%, respectively.