• Reproductive and Developmental Biology
• /
• v.33 no.1
• /
• pp.49-54
• /
• 2009

The current study was designed to evaluate the effects of the reactive oxygen species (ROS) generated with a xanthine (X) and xanthine oxidase system (XO) on sperm function and DNA fragmentation in porcine spermatozoa. ROS were produced by a combination of $1,000{\mu}M$ X and 50 mU/ml XO. The ROS scavengers such as superoxide dismutase (SOD) (200 U/ml) and catalase (CAT) (500 U/ml) were also tested. Spermatozoa were incubated for 2 hours in BWW medium with a combination of X-XO supplemented with or without antioxidants at $37^{\circ}C$ under 5% $CO_2$ incubator. Ca-ionophore-induced acrosome reaction, the proportion of swollen spermatozoa under hypo-osmotic condition, malondialdehyde formation for the analysis of lipid peroxidation, and the proportion of DNA fragmentation were determined after 2 hours incubation. The action of ROS on porcine spermatozoa resulted in decreased Ca-ionophore-induced acrosome reaction and membrane integrity, increased the formation of malondialdehyde, and the proportion of sperm with DNA fragmentation(p<0.05). The toxic effects caused by ROS were completely alleviated by CAT in terms of sperm function and characteristics, however SOD did not serve the same scavenger effect as CAT. To conclude, the ROS can cause significant damage to porcine sperm functions and characteristics, which can be minimized by the use of antioxidants.

• Reproductive and Developmental Biology
• /
• v.33 no.2
• /
• pp.71-76
• /
• 2009

Salivary lipocalin (SAL1) is a member of the lipocalin protein family that has a property to associate with many lipophilic molecules. The importance of SAL1 during pregnancy in pigs has been suggested by our previous study which has shown that SAL1 is expressed in the uterine endometrium in a cell type- and implantation stage-specific manner and secreted into the uterine lumen. However, function of SAL1 in the uterus during pregnancy in pigs is not known. To understand SAL1 function in the uterus during pregnancy, we generated recombinant porcine SAL1 protein in an insect cell line. Porcine SAL1 cDNA was cloned into a baculovirus expression vector using RT-PCR and total RNA from uterine endometrium on day 12 of pregnancy, and the expression vector was used to generate recombinant Bacmid containing the SAL1 gene. The recombinant Bacmid was then transfected Sf9 cell to produce recombinant baculovirus. By infecting Sf9 cell with recombinant baculovirus, we established a SAL1-expressing insect cell expression system. Immunoblot analysis confirmed SAL1 expression in the infected cells. Recombinant SAL1 produced by the Sf9 cell line will be useful for understanding physiological function of SAL1 during pregnancy in pigs.

• Reproductive and Developmental Biology
• /
• v.38 no.2
• /
• pp.85-91
• /
• 2014

Despite many researches related with in-vitro culture of porcine spematogonial stem cells (SSCs), adherent culture system widely used has shown a limitation in the maintenance of porcine SSC self-renewal. Therefore, in order to overcome this obstacle, suspension culture, which is known to have numerous advantage over adherent culture, was applied to the culture of porcine SSCs. Porcine SSCs retrieved from neonatal testes were suspension-cultured for 5 days or 20 days, and characteristics of suspension-cultured porcine SSCs including proliferation, alkaline phosphatase (AP) activity, and self-renewal-specific gene expression were investigated and compared with those of adherent-cultured porcine SSCs. As the results, the suspension-cultured porcine SSCs showed entirely non-proliferative and significantly higher rate of AP-positive cells and expression of self-renewal-specific genes than the adherent-cultured porcine SSCs. In addition, long-term culture of porcine SSCs in suspension condition induced significant decrease in the yield of AP staining-positive cells on post-day 10 of culture. These results showed that suspension culture was inappropriate to culture porcine SSCs, because the culture of porcine SSCs in suspension condition didn't stimulate proliferation and maintain AP activity of porcine SSCs, regardless of culture periods.

• Reproductive and Developmental Biology
• /
• v.38 no.2
• /
• pp.79-84
• /
• 2014

MicroRNAs (miRNAs) are approximately 22 nucleotides of small noncoding RNAs that control gene expression at the posttranscriptional level through translational inhibition and destabilization of their target mRNAs. The miRNAs are phylogenetically conserved and have been shown to be instrumental in a wide variety of key biological processes including cell cycle regulation, apoptosis, metabolism, imprinting, and differentiation. Recently, a paper has shown that expression of the miRNA-302/367 cluster expressed abundantly in mouse and human embryonic stem cells (ESCs) can directly reprogram mouse and human somatic cells to induced pluripotent stem cells (iPSCs) efficiently in the absence of any of the four factors, Oct4, Sox2, c-Myc, and Klf4. To apply this efficient method to porcine, we analyzed porcine genomic sequence containing predicted porcine miRNA-302/367 cluster through ENSEMBL database, generated a non-replicative episomal vector system including miRNA-302/367 cluster originated from porcine embryonic fibroblasts (PEF), and tried to make porcine iPSCs by transfection of the miRNA-302/367 cluster. Colonies expressing EGFP and forming compact shape were found, but they were not established as iPSC lines. Our data in this study show that pig miRNA-302/367 cluster could not satisfy requirement of PEF reprogramming conditions for pluripotency. To make pig iPSC lines by miRNA, further studies on the role of miRNAs in pluripotency and new trials of transfection with conventional reprogramming factors are needed.

• Reproductive and Developmental Biology
• /
• v.32 no.2
• /
• pp.97-104
• /
• 2008

Plasminogen activators (PAs) are serine protease that cleave plasminogen to form the active protease plasmin. PA/plasmin system playa role in mammalian fertilization and motility and acrosome reaction of sperm. The present study was undertaken to identify PAs in porcine gametes and investigate a possible role of plasminogen in in vitro fertilization in the pig. When boar spermatozoa were preincubated in a fertilization medium (mTBM) for 0, 2, 4 or 6 h, the activity of tPA-PAI ($110{\sim}117\;kDa$), tPA ($62{\sim}70\;kDa$), and uPA ($34{\sim}38\;kDa$) was observed in the sperm incubation medium and sperm sample. PA activities in the sperm incubation medium significantly (p<0.05) increased according to increasing incubation times, while PA activities in sperm significantly (p<0.05) decreased at the same times. In addition, the rate of acrosome reaction in spermatozoa increased by increasing culture times. When oocytes were separated from porcine cumulus-oocytes complexes at 0, 22 or 44 h of maturation culture, no PA activities were observed in cumulus free-oocyte just after aspiration from follicles. However, the activity of tPA-PAI ($108{\sim}113\;kDa$) and tPA ($75{\sim}83\;kDa$) was observed at 22 h of in vitro culture and significantly (p<0.05) increased as the duration of the culture increased. On the other hand, when porcine oocytes were activated by sperm penetration or calcium ionophore, plasminogen significantly (p<0.05) increased ZP dissolution time (sec) in activated oocytes by sperm penetration. These results suggest that supplementation of plasminogen to fertilization medium may playa positive role in the improvement of in vitro fertilization ability in the pig.

• Reproductive and Developmental Biology
• /
• v.41 no.3
• /
• pp.51-55
• /
• 2017

The production of therapeutic protein from transgenic domestic animal is the major technology of biotechnology. Insulin-like growth factor-1 (IGF-1) is known to play an important role in the growth of the animal. The objective of this study is construction of knock-in vector that bovine IGF-1 gene is inserted into the exon 7 locus of ${\beta}$-casein gene and expressed using the gene regulatory DNA sequence of bovine ${\beta}$-casein gene. The knock-in vector consists of 5' arm region (1.02 kb), bIGF-1 cDNA, CMV-EGFP, and 3' arm region (1.81 kb). To express bIGF-1 gene as transgene, the F2A sequence was fused to the 5' terminal of bIGF-1 gene and inserted into exon 7 of the ${\beta}$-casein gene. As a result, the knock-in vector is confirmed that the amino acids are synthesized without termination from the ${\beta}$-casein exon 7 region to the bIGF-1 gene by DNA sequence. These knock-in vectors may help to create transgenic dairy cattle expressing bovine bIGF-1 protein in the mammary gland via the expression system of the bovine ${\beta}$-casein gene.

• Asian-Australasian Journal of Animal Sciences
• /
• v.21 no.2
• /
• pp.225-231
• /
• 2008

Oxidative stress plays a crucial role in the laying stage which is a critical period for chick survival. We investigated the relationship of neonatal chick performance, brain antioxidant status and vitamin E supplementation level in hens. Starting at 17 weeks, hens were randomly divided into five groups. The control group received a basal diet without supplemental vitamin E (VE, dl-${\alpha}$-tocopherol acetate). Other groups received the same basal diet supplemented with vitamin E (40, 80, 120 and 160 mg/kg) through growth to egg production. Hens were artificially inseminated at 28 weeks of age and egg yolks were collected at day two. All remaining eggs were hatched. Yolk vitamin E content, hatchability and fertility of eggs were evaluated. Brains of the newly hatched chicks were further evaluated for their oxidative stress status, antioxidative status and vitamin E levels. Increased reproductive performance was observed in fertility and hatchability in the group supplemented at 40 mg/kg. Egg yolk and neonatal brain ${\alpha}$-tocopherol was highest in eggs from hens fed 120 mg/kg and 80 mg/kg supplemental vitamin E, respectively. Brain MDA, ROS and iron levels were significantly higher in unsupplemented hens (p<0.01). SOD activity was significantly higher in the group supplemented at 160 mg/kg than in all other groups. We concluded that maternal supplementation of vitamin E had beneficial effects on fertility, hatchability of eggs, neonatal brain oxidative status and SOD activity.

• Korean Journal of Psychosomatic Medicine
• /
• v.7 no.2
• /
• pp.149-157
• /
• 1999

Food intake and body weight are determined by a complex interaction of regulatory pathways. Leptin, the product of the ob gene, is a recently discovered hormone secreted by adipocytes, that signals the amount of adipose tissue energy stores to the brain and exerts major effects on energy homeostasis and neuroendocrine function. In addition, leptin has recently been shown to affect reproductive function in rodents and humans. The study of leptin and its effectors in the hypothalamus may provide important insights with respect to the interplay of several hypothalamic neuropeptides in regulating feeding as well as the interaction of genetics and environment in the regulation of energy homeostasis. In this review we summarise the action of leptin in the regulation of food intake and highlight a working model of the effects of environmental factors on the leptin system.

• Korean Journal of Veterinary Service
• /
• v.39 no.1
• /
• pp.41-49
• /
• 2016

In the present study, fast and robust methods for the next generation sequencing (NGS) were developed for analysis of PRRSV full genome sequences, which is a positive sensed RNA virus with a high degree of genetic variability among isolates. Two strains of PRRSVs (VR2332 and VR2332-R) which have been maintained in our laboratory were used to validate our methods and to compare with the sequence registered in GenBank (GenBank accession no. EF536003). The results suggested that both of strains had 100% coverage with the reference; the VR2332 had the coverage depth from minimum 3 to maximum 23,012, for the VR2332-R from minimum 3 to maximum 41,348, and 22,712 as an average depth. Genomic data produced from the massive sequencing capacities of the NGS have enabled the study of PRRSV at an unprecedented rate and details. Unlike conventional sequence methods which require the knowledge of conserved regions, the NGS allows de novo assembly of the full viral genomes. Therefore, our results suggested that these methods using the NGS massively facilitate the generation of more full genome PRRSV sequences locally as well as nationally in regard of saving time and cost.

• Korean Journal of Veterinary Research
• /
• v.48 no.3
• /
• pp.347-355
• /
• 2008

Perfluorooctanoic acid (PFOA), a member of the perfluoroalkyl acids that have wide commercial applications, is persistent organic pollutants widely spread throughout the environment and human population. But little is known about the adverse biological effects of the PFOA. In the present study, the toxicological effects of PFOA were investigated in rats. Sprague-Dawley rats (N = 10 in each group) were orally administered with PFOA in drinking water for 4 weeks (0, 100, 200, or 400 ppm in male, and 0, 200, 400, or 800 ppm in female). Three female rats given 800 ppm died during the study. PFOA treatment decreased the body weight gain and increased the liver weights in both genders. Serum biochemical investigations revealed significant increases in the aspartate aminotransferase, alanine transaminase, alkaline phosphatase, blood urea nitrogen, total cholesterol, and total bilirubin in male but in female. Serum estradiol (E2) levels were increased in all treated rats. Histopathologically, hepatocellular hypertrophy around central vein was noted in the liver of treated rats. No significant histopathological changes were noted in other organs. In conclusion, PFOA induced toxicological changes in the liver and increased serum E2 level which was not related to histopathological changes of endocrine and reproductive system.