小白鼠儲精囊自體抗原啟動子之研究

Abstract

儲精囊等雄性附屬性腺分泌液占精液體積的二分之一以上，其中含有許多影響生殖力的因數。小白鼠儲精囊自體抗原（SVA）純化自小白鼠儲精囊分泌液，已知SVA可與精蟲結合並影響其活動力，且其基因表現受雄性素調控，故SVA在生殖功能上可能具有一定的重要性。 由比對分析的結果，在SVA基因序列上發現十個與雄性素反應單元（ARE）保守序列相似性大於50％的區域。我們建構了8個包含不同片段ARE-like elements的報導質體，以攝護腺癌細胞LNCaP作為表現系統，針對其中五個ARE-like elements，探討與雄性素所調控基因表現之間的關係。結果發現，當ARE-a, ARE-b, TATA-Box同時存在報導基因上游時，基因表現可受雄性素刺激達2.5倍；而當ARE-c, ARE-d, TATA-Box同時存在報導基因上游時，基因表現可受雄性素刺激達4.8倍。 另外，我們使用Electrophoresis Mobility Shift Assay (EMSA)分析雄性素受體與SVA?動子的結合區域，結果發現儲精囊細胞核萃取物與SVA基因上-250?＋8的區域有結合，且結合的程度隨著核萃取物濃度增加而增加。相較於重組的大鼠雄性素受體DNA結合區域（rAR(DBD)）與此段序列的結合，儲精囊核萃取物的結合程度明顯大了許多，因而猜測有某些儲精囊特有的因數與之結合。 本論文雖未能完整的解答雄性素與SVA基因表現之間的關係，所提供初步的研究結果可作為未來研究的參考。

Semen comes mainly from the secretions of seminal vesicle and other accessory glands. An autoantigen, the seminal vesicle autoantigen (SVA), has been purified from seminal vesicle secretion. It is a 19 kDa androgen-dependent glycoprotein. The preliminary data from our laboratory indicate that SVA is a sperm mobility inhibitor with the ability to suppress the BSA-induce sperm capacitation. By comparison, ten potential androgen response elements (AREs) have been mapped on the gene. In this work, we ligated different fragments of SVA promoter and intronic regions on the upstream of a reporter gene chloramphenicol acetyltransferase (CAT). Eight plasmids were constructed and their transcriptional activities were evaluated by CAT assay. Including ARE-a, ARE-b, and TATA-box together, the plasmid could enhance 2.5 folds of CAT expression under induced by 10 nM R1881. Under the same condition, ARE-c, ARE-d, and TATA-box were cooperated to enhance 4.8 folds of CAT expression. Our preliminary study suggested that the protein(s) from nuclear extract of seminal vesicle could bind to -250?+8 region of SVA gene.