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研究生：	李奕漢 Li, Yi-Han
論文名稱：	研究第九號纖維母細胞生長因子在雄性性腺發育所扮演的角色 Study the role of FGF9 in male sex determination
指導教授：	孫孝芳 Sun, H. Sunny
學位類別：	博士 Doctor
系所名稱：	醫學院 - 基礎醫學研究所 Institute of Basic Medical Sciences
論文出版年：	2017
畢業學年度：	106
語文別：	英文
論文頁數：	73
中文關鍵詞：	第九號纖維母細胞生長因子、雄性性別決定、雌性性別反轉、SRY基因轉錄調控、胚胎發育
外文關鍵詞：	FGF9, male sex determination, female-to-male sex-reversal, SRY transcription regulation, embryonic development
相關次數：	點閱：104 下載：2
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FGF9是一種自分泌/旁泌性生長因子並且在胚胎和器官發育，性別決定及許多生理過程中扮演了十分重要的角色。FGF9/Fgf9表達在SRY/Sry性別決定基因表達之後快速上升，Fgf9基因敲除小鼠出現雄性性別反轉的現象，此外FGF9異常表達也與人類性發育障礙有關聯。然而，FGF9基因是否具有性別決定功能，以及在男性睾丸發育過程中FGF9/Fgf9的表達調控機制尚待闡明。在這項研究中，我們假設在男性睾丸發育過程中，FGF9/Fgf9是受到轉錄因子SRY/Sry所調控，且高度表達的FGF9是足以觸發雌性性別反轉。我們利用體外培養試驗發現，在XX性腺中，外源性FGF9的確可以轉變細胞特性從雌性變為雄性以及誘導出類睾丸結構。接著為了研究SRY/Sry調控FGF9/Fgf9表達機制，我們先利用生物資訊分析發現在人類及小鼠FGF9/Fgf9啟動子區域都找到了一些SRY/Sry結合位點。之後利用報導基因實驗證明老鼠的Sry和Sf1共同促進Fgf9的的啟動子活性。更進一步，我們找出人類SRY基因直接透過FGF9 -833/-821啟動子區域來增加FGF9啟動子活性和表現。綜合以上結果，我們的研究指出FGF9具有作為性別決定基因功能的能力以及SRY/Sry會直接透過轉錄調控來控制FGF9/Fgf9的表現。

Fibroblast growth factor 9 (FGF9) is an autocrine/paracrine growth factor that plays critical roles in embryonic and organic development, sexual determination, and various physiological processes. FGF9/Fgf9 is expressed soon after sex-determining region Y (SRY/Sry) expression during male sex determination and Fgf9 knockout mice exhibit male-to-female sex reversal. Aberrant expression of FGF9 correlates with human disorders of sex development (DSDs). However, whether FGF9 is sufficient to trigger male sex development on its own, and how FGF9/Fgf9 is controlled during early embryonic development not completely understood. In this study, we hypothesize that the expression of FGF9 is sufficient to trigger male sex development and FGF9/Fgf9 expression is regulated by SRY/Sry during testes development. We applied the ex vivo 3D culture model and found that exogenous FGF9 switches cells from female-to-male and induces testis cord like structure in XX gonads. To study the SRY/Sry regulation on FGF9/Fgf9 expression, we found several putative SRY/Sry binding elements by using bioinformatics analysis, located in the FGF9/Fgf9 promoter region of both humans and mice. Reporter gene assays show that mouse Sry and Sf1 cooperatively increase Fgf9 promoter activity at the -1150/-998 position. Moreover, we found that human SRY directly interacts with the FGF9 -833/-821 promoter region and upregulates promoter activity and expression. Taken together, our study shows, for the first time, that FGF9 is sufficient to trigger male sex determination and the expression of FGF9/Fgf9 is directly controlled by SRY/Sry through transcriptional activation.

中文摘要	I
Abstract	II
誌謝	III
Table of Contents	V
List of Tables	IX
List of Figures	X
Chapter 1. Introduction	12
1 Mammalian Sex Development	12
2 Male Sex Determination	13
3 The SRY/Sry transcription regulation network	14
4 FGF9 in Male Sex Development	15
5 Human Disorders of Sex Development	16
6 Rationale and Specific aims	17
Chapter 2. Materials and Methods	19
1 Mice	19
2 Primary gonads ex vivo 3D culture	19
3 Immunofluorescence (IF) assay	20
4 Cell lines and Cell Culture	21
5 Transcription factor binding site prediction	21
6 Plasmid constructs	22
7 Transient transfection and luciferase reporter assay	23
8 Western blot analysis	23
9 Total RNA extraction, reverse-transcription, and quantitative real-time PCR	24
10 FGF9 Enzyme-linked immunosorbent assay (ELISA)	25
11 Chromatin immunoprecipitation (ChIP) assay	26
12 Electrophoretic mobility-shift assay (EMSA)	26
13 Statistical analysis	27
Chapter 3. Results	29
1 FGF9 is sufficient to promote testis-cord formation in XX gonads within a critical time window of the early embryonic stage	29
1.1 Establish the ex vivo 3D culture system for gonad culture	29
1.2 Testis-cord formation in XY gonads regardless development stages and recombinant FGF9 treatment	29
1.3 Exogenous FGF9 is sufficient to trigger male testis-cord formation in XX 12 and 16ts Gonads	30
1.4 Exogenous FGF9 switches the cell fate from female-to-male by increasing Sox9 and suppressing Foxl2 expression in XX gonads	30
1.5 Exogenous FGF9 promotes male sexual fate in XX gonads within critical time window	31
2 Mouse Sry and Sf1 cooperatively increase Fgf9 promoter activity	32
2.1 Mouse Sry upregulates Fgf9 promoter activity through Sry responsive element in the (mSRE) -1,010/-998 region	32
2.2 Mouse Sry and Sf1 cooperatively increases Fgf9 promoter activity	34
2.3 Mouse Sf1 upregulates Fgf9 promoter activity by interacting with the Fgf9 -1150/-1141 region	34
3 Human SRY directly binds to FGF9 promoter and upregulates FGF9 expression by transcriptional activation	35
3.1 Human SRY increases endogenous FGF9 mRNA and Protein in NT2/D1 cells	35
3.2 Human SRY upregulates FGF9 promoter activity in HEK293 and COS7 cells through the hSRE -833/-821 region	36
3.3 Endogenous SRY binds to FGF9 promoter hSRE -833/-821 region in NT2/D1 cells	37
3.4 Endogenous human SRY binds to FGF9 promoter hSRE -833/-821 region in vitro	37
3.5 Human SRY directly binds to FGF9 promoter at hSRE -833/-821 region and upregulates FGF9 promoter activity and expression	38
3.6 Human SF1 does not regulate FGF9 promoter activity and expression	39
Chapter 4. Discussion	60
1 FGF9 plays an essential and sufficient role in male testes development	60
References	69

 

                                    

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校內：2023-01-05公開
校外：2023-01-05公開

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