精子生成需要經過複雜精細的epigenetic調控，才能製造出正常的精蟲。DNA甲基轉位酶3 (DNMT3)，主要功能在將DNA未修飾的胞嘧啶加上甲基修飾，目前已發現有三個成員，分別為DNMT3A、DNMT3B與DNMT3L，其中DNMT3L雖沒有甲基轉位酶活性，有許多研究顯示與製造精子過程有密切關聯。無精症可概略區分為阻塞型與非阻塞型。阻塞型有正常的製造精子功能，因不同原因無法運輸精蟲至精液中；非阻塞型則是製造精子功能受損，最嚴重則是細精管內沒有生殖細胞存在。因此我們針對不孕症門診治療的無精症病患，利用RT-PCR、qPCR與免疫組織化學染色，來研究DNMT3家族在睪丸是否因病情而有不同表現。結果在17位非阻塞型無精症患者，只有5位 (29.4%) 偵測到DNMT3L的表現； 而在20位阻塞型患者，則全部都有表現。另外在DNMT3A與DNMT3B的表現上，則看不出有明顯差異。DNMT3L的免疫組織化學染色，主要在精原細胞與精母細胞的細胞核看到染色。會造成這兩型病患的差異表現，應是生殖細胞存在與否所致。人工生殖技術的進步，讓睪丸內尚有精原幹細胞的非阻塞型無精症男性，有希望能擁有親生後代；利用本實驗發現的DNMT3L表現差異，可以當作判斷能否進行生殖細胞培養的參考。

Delicate epigenetic modifications are essential for production of spermatids during spermatogenesis. DNA methyltransferase 3 (DNMT3), the enzymes involved in adding a methyl group to unmodified DNA, contains three members: DNMT3A, DNMT3B and DNMT3L. The latter lacks methyltransferase activity, but was closely associated with spermatogenesis in many reports. According to the presentation of mature spermatids in testis, azoospermia could be separated into obstructive and non-obstructive categories. Non-obstructive azoospermia is spermatogenesis defective, germ cells absent in seminiferous tube is the most serious type. Therefore, we would like to find out if there are differential expression of DNMT3 family transcripts in testes of azoospermia patients from infertility clinic. Using RT-PCR and qPCR, we found only 5 (29.4%) expressed DNMT3L in 17 non-obstructive patients, whereas all 20 obstructive patients expressed. Both groups were similar in expression levels of DNMT3A and DNMT3B. Nuclei of spermatogonia and spermatocyte were the main immunohisto-chemical localization of DNMT3L protein. Lost of germ cells should be the cause of undetectable DNMT3L expression in azoospermia patients. By this founding, it could serve as an indicator for ability of male germ cell culture in further applications of assisted reproduction.

中文摘要............................................. I
英文摘要............................................. II
英文縮寫............................................. III

壹. 緒言.............................................. 1
一. DNA甲基化..................................... 1
二. DNA甲基轉位酶................................. 1
三. The DNMT3-like protein, DNMT3L................. 2
四. 精子生成 (Spermatogenesis)..................... 3
五. DNA甲基化與精子生成............................ 3
六. 男性不孕症...................................... 4
七. 實驗目的........................................ 6
貳. 材料與方法......................................... 7
一. 材料........................................... 7
二. 實驗方法....................................... 7
1. 睪丸組織RNA萃取............................ 7
2. 反轉錄mRNA成cDNA........................... 8
3. PCR聚合酶連鎖反應........................... 8
4. 定量聚合酶連鎖反應 qPCR..................... 9
5. 免疫組織化學染色............................ 9
参.結果............................................... 11
一. DNMT3L的差異表現............................... 11
二. 定量DNMT3家族的表現........................... 12
三. DNMT3L免疫組織化學染色情形.................... 12
肆.討論............................................... 14
伍.結論................................................ 21
參考文獻............................................... 22
表..................................................... 26
圖..................................................... 30

楊友仕等著,生殖內分泌及不孕症槪要,1999
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