本研究針對經鑑別為家禽性別決定候選基因DMRT1, FET1, FOXL2, LHX9, HINT1, SMC2L1與SOX9在雞胚性腺發育早期，以定量、及時反轉錄PCR方式進行各性別決定候選基因表現量變曲線（profile）之分析，以釐清此些基因間表現相關與位階性；目前研究的結果，可證實DMRT1在雄性胚胎中，在諸多候選基因，表現時間最早，4天已有表現量，因此雄化扮演很重要的角色，而FET1, FOXL2, LHX9與HINT1在雌性則可以影響胚胎的雌化，但在SMC2L1與 SOX9的基因表現在雄性與雌性中沒有太大的差異。 此外，本研究建立兩個胚胎早期，先將胚胎之遺傳性別予以區分後，利用雄性胚胎減除雌性胚胎cDNA方法，以及雌性胚胎減除雄性胚胎cDNA方法，建立兩個基因庫，期尋求在3.5天的雞胚胎中，表現於雄性或雌性的性別差異基因，所建立的基因庫中，目前完成序列比對結果發現一些與哺乳動物同源的基因與蛋白質，其中出現在雄性減雌性基因庫的一個steroid 5α-reductase (SRD5A1) 蛋白質，被認為與雄性性別分化有關，將對此蛋白質作進一步分析，瞭解其在雞胚發育所扮演的角色。

To establish the gene expression profile and cascade subsequently on avian sex-determining candidate genes, seven avian sex-determining candidate genes including DMRT1, FET1, FOXL2, LHX9, HINT1, SMC2L1 and SOX9 were analyzed at early embryogenesis. Quantitative reverse transcription PCR (Quantitative RT-PCR) technology was used to establish the gene expression profiles among these genes at four, five, six and seven days of embryos. The results of quantitative RT-PCR reveal that the DMRT1 was expressed in chicken embryos of both sexes. DMRT1 gene expressions were up-regulated at four, five and six days of chicken embryos. DMRT1 expression increased at 5-Dpc. of male embryos, however, expression was not signification different in females embryos. Gene expression of FET1, FOXL2, LHX9 and HINT1 were higher in females than in males. The SMC2L1 and SOX9 were expressed in both sexes. Also, to identify the novel sex-determination genes in early chicken subtractive embryos, cDNA libraries from male-minus-female and female-minus-male 3.5 Dpc. embryos cDNA were established. Gene annotation was carried out by data-mining in public databases, GeneBank (NCBI, USA) and TIGR gene indices (The Institute for Genome Research, USA). A total 548 of colonies in male-minus-female library and 79 sequences were annotated. However, a total of 589 of colonies in female-minus-male library and 16 sequences were annotated. Sequences were homologous to the steroid 5α-reductase protein (SRD5A1) using BLASTx in male-minus-female subtractive library. The SRD5A1 may play a sex-differentiation role in male chicken. We need more study to know function of steroid 5α-reductase protein in future.

TABLE OF CONTENTS......................................................................................I CHAPTER 1. LITERATURE REVIEW.............................................................3 1.1. Sex Determination in Vertebrates...................................................................................................3 1.2. Genes Involved in Gonadal Differentiation across Vertebrates...................................................5 1.3. Sex Determination in Birds...........................................................................................................10 1.3.1. Avian Sex Chromosomes.........................................................................................................10 1.3.2. Genes Involved in sex determination in Birds..........................................................................11 1.3.2.1. Anti-müllerian hormone (AMH).......................................................................................13 1.3.2.2. Cytochrome P450, family 19, subfamily A, polypeptide 1 (CYP19A1)...........................14 1.3.2.3. Dosage-sensitive sex reversal (DSS)................................................................................15 1.3.2.4. Female expressed hormone (FET1)..................................................................................16 1.3.2.5. Forkhead box L2 (FOXL2)...............................................................................................17 1.3.2.6. GATA4-binding protein (GATA4).....................................................................................17 1.3.2.7. LIM homobox 9 (LHX9)..................................................................................................18 1.3.2.8. Nuclear receptor subfamily 5, group A, member 1 (NR5A1)...........................................18 1.3.2.9. Wilm’s tumor 1 (WT1)......................................................................................................20 1.3.3. Identification of Sex-determining Candidate Genes in Birds...................................................20 CHAPTER 2. THE EXPRESSION PROFILING AND SPECULATED CASCADE ON AVIAN SEX-DETERMINING CANDIDATE GENES..................................29 2.1. Introduction...................................................................................................................................29 2.2. Materials and Methods..................................................................................................................29 2.2.1. Chicken embryos......................................................................................................................29 2.2.2. Total RNA extraction from chicken embryos...........................................................................30 2.2.3. Reverse Transcription PCR (RT-PCR).....................................................................................30 2.2.4. Identification of the genetic sex in chicken embryos...............................................................31 2.2.5. Quantitative RT-PCR................................................................................................................31 2.2.5.1. Optimization of primer concentrations.............................................................................31 2.2.5.2. Statistical analysis............................................................................................................33 2.3. Results............................................................................................................................................35 2.3.1. Total RNA extraction from chicken embryos and identification of the genetic sex in chicken embryos................................................................................................................................................35 2.3.2. Quantitative RT-PCR...............................................................................................................35 2.4. Discussion......................................................................................................................................41 CHAPTER 3. IDENTIFICATION OF CANDIDATE SEX DIFFERENTIATION GENES USING SUBTRACTIVE CDNA LIBRARIES IN THE EARLY CHICKEN EMBRYOGENESIS..........................................................................................43 3.1. Introduction...................................................................................................................................43 3.2. Materials and methods..................................................................................................................43 3.2.1. Isolation of poly A+ mRNA from total RNA..........................................................................43
3.2.2. cDNA synthesis......................................................................................................................46 3.2.3. Purification of double-stranded cDNA...................................................................................48 3.2.4. Rsa I digestion.......................................................................................................................48 3.2.5. Adaptor ligation.....................................................................................................................49 3.2.6. First hybridization..................................................................................................................49 3.2.7. Second hybridization..............................................................................................................50 3.2.8. PCR Amplification.................................................................................................................50 3.2.9. Adding 3´ A-Overhangs.........................................................................................................52 3.2.10. TA cloning and E.coli transformation....................................................................................52 3.2.11. Purification of plasmid...........................................................................................................53 3.2.12. Auto-sequencing....................................................................................................................53 3.3. Results.......................................................................................................................................54 3.3.1. Isolation of poly A+ mRNA from total RNA............................................................................54 3.3.2. cDNA synthesis and identification of the genetic sex in chicken embryos..............................55 3.3.3. PCR Amplification..................................................................................................................57 3.3.4. TA cloning and E.coli transformation......................................................................................58 3.3.5. cDNA annotation.....................................................................................................................59 3.4. Discussion......................................................................................................................................60 REFERENCES.................................................................................................94 APPENDIX.....................................................................................................100

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