雞胚胎早期性腺型態發育始於配種後6.5天，為了檢測雄性雞胚早期性別發育相關之轉錄體，本研究建立了第三天 (E3, Hamburger and Hamilton Stage 20) 雞胚雄性削減雌性互補去氧核醣核酸資料庫，並得到全部包括89個已知及127個未知和雄性發育相關的轉錄體。本研究選出其中35個可能和性別發育相關的轉錄體，之後以即時反轉錄聚合酶連鎖反應分析出有25個轉錄體在雄性第三天胚胎表現量顯著高於雌性胚胎。在這25個轉錄體中有12個是位於性染色體Z上。另外在雞隻孵化出來72週齡時，有20個轉錄體在睪丸表現量顯著高於卵巢，而有四個轉錄體在雄性腦部表現量顯著高於雌性。全胚胎及切片原位雜交方法證實了riboflavin kinase (RFK), WD repeat domain 36 (WDR36) 以及EY505808核醣核酸表現量在第七天雄性胚胎性腺表現量較高，同時利用西方墨點法實驗證明了RFK, WDR36蛋白質表現量在第七天雄性胚胎性腺表現量較高。在第四天的雞胚處理aromatase抑制劑formestane後影響了第七天的雞胚coatomer protein complex (subunit beta 1), solute carrier family 35 member F1, LOC427316以及EY505812轉錄體表現量，這個結果和doublesex and mab-3 related transcription factor 1基因情形是一樣的。另外在處理formestane後有15個雄性發育相關轉錄體和性別有交互效應現象。總之，本研究鑑定了可能的雄性雞胚胎早期發育之轉錄體，部分轉錄體亦有機會受到性賀爾蒙的調控。

From the sexual preselection point of view, understanding sex determination/differentiation mechanisms in the bird is critical in both evolutionary and industrial applications. The chicken embryo provides an unique vertebrate model in the field of development biology. Morphological sex development in the chick gonad starts at 6.5 embryonic day (E6.5), however, genetic sex determination and development should occur earlier. In order to comprehend genes and their underlying mechanisms being involved in sex-determination/development during early embryogenesis, we not only made a male-subtract-female and a female-subtract-male cDNA library as early as embryonic day 3 (E3; Hamburger and Hamilton Stage 20), but also examined early transcripts related to male development in chicken embryo and their expression profiles in this study. A total of 89 and 127 candidates of male-development transcripts represented respectively for 83 known and 119 unknown non-redundant sequences, which were characterized in an E3 male- subtract-female complementary DNA library. In this study, thirty-five selected transcripts being validated by quantitative reverse transcription-polymerase chain reaction that the expression levels of 25 transcripts were higher in male E3 whole embryos than in females (P < 0.05). Notably, twelve of these transcripts mapped to the Z chromosome. At 72 weeks of age, twenty transcripts were expressed at higher levels in testes than in ovaries. Meanwhile, four transcripts were expressed at higher levels in brains of male than in brains of female chickens (P < 0.05). By using of methods of whole mount and frozen cross-section in situ hybridization, the expressions of riboflavin kinase (RFK), WD repeat domain 36 (WDR36) and EY505808 transcripts on E7 chicken male gonads were corroborated to be better than female gonads. This result was confirmed by using of western blotting analysis which also showed the expressions were specifically on gonads than other tissues. Treatment with an aromatase inhibitor formestane at E4 depicted the effect of the expression levels at E7 of the coatomer protein complex (subunit beta 1), solute carrier family 35 member F1, LOC427316 and EY505812 transcripts across both sexes (P < 0.05), which was similar to the observed gene expressions for both doublesex and mab-3 related transcription factor 1 gene. Additionally, the interaction effects of sex with formestane treatment were observed in 15 candidate male development transcripts (P < 0.05). This study demonstrated a panel of potentially candidate male development transcripts being identified during early chicken embryogenesis; some might be regulated by sex hormones.

目錄
頁碼
摘要
中文 --------------------------------------------------------------------- I
英文 --------------------------------------------------------------------- II

Chapter 1. Literature review
內文 --------------------------------------------------------------------- 1
附圖 --------------------------------------------------------------------- 11
附表 --------------------------------------------------------------------- 13

Chapter 2. Identification of early transcripts related to male development in chicken embryos during early embryogenesis
研究背景 --------------------------------------------------------------------- 17
研究目的 --------------------------------------------------------------------- 19
材料與方法 --------------------------------------------------------------------- 20
研究結果 --------------------------------------------------------------------- 28
討論 --------------------------------------------------------------------- 34
附圖 --------------------------------------------------------------------- 40
附表 --------------------------------------------------------------------- 59

參考文獻 --------------------------------------------------------------------- 68

附錄 --------------------------------------------------------------------- 77
發表文章 --------------------------------------------------------------------- 77

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