夏季中，環境的高溫會造成公豬精液品質的下降。目前，有關公豬繁殖之功能性基因體研究，特別是針對熱休克之影響並不很清楚。為了研究影響精液品質之機制，利用高通量基因晶片的方法進行大量篩選差異表現基因，找出潛在的生物標記。在原始精細胞中，受到熱緊迫之早期表現基因對於精子的發育可能扮演重要角色。這些基因可能與精子之耐熱性相關，可作為生物標記輔助育種之參考標的。
本研究主要發現和結論歸納如下：
1.利用實驗設計的方法，找出一種優化的 PCR 反應條件以及一種高效性的PCR 增強劑。在此二種改進配方的組合下，有效地將整體 PCR 反應成功率，從原來的 PCR 反應條件下的 80 %增加到 97 %。
2.建立了一種含有 9,944 個獨立探針之公豬睾丸 cDNA 微矩陣晶片。以部分純化之公豬生殖細胞模式研究中，此 cDNA 微矩陣可成功運用來篩選熱緊迫反應之差異表現基因。
3.以公豬生殖細胞模式研究熱緊迫，細胞在受到短暫且緩和之熱緊迫後(39ºC，1 小時)，共有 380 個基因(5 %)受到明顯之調控，其中 326 個基因的表現量受到調升，54 個則是調降。進一步以即時定量 PCR 驗證，確認有 3 個基因調升大於 1.5 倍：heat shock 105 kDa/110 kDa protein 1 (Hsph1 or Hsp105)、heat shock 70 kDa protein 4-like (Hspa4l)以及 THAP domain containing 4 (Thap4)。
4.在 Hsp105 基因上游的 1.6-kb 啟動子區中，發現一個單核苷酸多型態(SNP)；該 SNP 可為 C 或 T。此 SNP 的基因型與成熟公豬正常形態精子之百分率具有中度相關性，基因型 TC 優於 CC 型，具有作為選育公豬之參考價值。
5.此 SNP 位點上的改變(T 或 C)對特定蛋白質之結合能力具有極大之差別。這個 SNP 位點可能代表一種被動式的基因調控模式，藉由增加此位點與特定蛋白質(轉錄因子)結合之多樣性，達到調控基因之目的，進而增加精子在不同的生理狀況下之耐受度或適應性。

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.

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