Studies on the Heat Shock Response of Porcine Reproduction Related Cells with cDNA Microarray

• Main Authors: Bing-Huang Gau, 高炳煌
• Other Authors: I-Ming Chu
• Format: Others
• Language: en_US
• Published: 2008
• Online Access: http://ndltd.ncl.edu.tw/handle/59623893999593659063

博士 === 國立清華大學 === 化學工程學系 === 96 === Semen quality of boars is reduced by high environmental temperatures during the summer. The functional genomics of pig reproduction under heat shock is not clear. To investigate the mechanism contributing to the quality of sperm, a high-throughput cDNA microarray-assisted method was conducted to screen differentially expressed ranscripts as potential biomarkers. Early responding genes may play an important role in the development of sperm. Therefore, these genes are regarded as potential biomarkers relevant to heat tolerance, and may be applicable in bio-marker assisted breeding. The major findings of this study are summarized as follows: 1.Using Design of Experiment (DOE), an optimized PCR reaction and a high-potent PCR enhancer solution were established. The combinatorial PCR reaction increases the overall success rate of PCR to 97 % compared to a general PCR reaction of 80 %. 2.A porcine testicular cDNA microarray, with 9,944 unique probes, was established. This cDNA microarray was adequate to screen the differentially expressed genes in enriched germ cells responding to moderate heat stress. 3.Under heat shock (at 39 ºC for 1 h), 380 transcripts (5 %) of qualified probes revealed significant gene expression in primitive germ cells: 326 were upregulated and 54 were downregulated. Confirmed by quantitative PCR, three transcripts were upregulated > 1.5-fold: heat shock 105 kDa/110 kDa protein 1 (Hsph1 or Hsp105); heat shock 70 kDa protein 4-like (Hspa4l); and THAP domain containing 4 (Thap4). 4.A single nucleotide polymorphism (SNP), C or T, was found in the 1.6-kb promoter region upstream the Hsp105 gene. This SNP site is moderate correlated to the percentage of sperm with normal morphology. The normal percentage of sperm of boars with TC genotypes is superior to those with CC genotypes. This site has potential to be a quantitative trait locus for the breeding of boars. 5.A dramatic difference was found in the protein-DNA association between T and C moiety at the SNP site. The SNP site in the promoter region may possess a passive control of gene expression by increasing the flexibility of the binding site for different transcription factors under different physiological conditions.