实验海洋生物学重点实验室知识库

   鱼类性腺分化和发育的研究在理论和实践上都具有重要意义。芳香化酶Cyp19a是性类固醇激素合成的关键酶之一，与其重要转录因子Foxl2、Dmrt1和Sox9a等均参与鱼类性腺分化和发育。但相关研究集中在mRNA水平，而作为其主要作用形式的蛋白水平研究则仅限于表达模式和真核细胞转染的转录调控分析，性腺分化中的相关研究也只见到斑马鱼、青鳉以及尼罗罗非鱼等少数鱼中的表达图示，因而它们在性腺分化和发育中的准确作用尚待明确。牙鲆（Paralichthys olivaceus）是我国、日本和韩国重要的海水养殖鱼类，其生长具有明显的性别二态性，故研究其性别差异与性腺分化具有重要价值。本研究以牙鲆为研究对象，获得了纯度高且具备生物学活性的Cyp19a、Foxl2、Dmrt1和Sox9a体外重组蛋白，通过注射分析了Foxl2和Dmrt1在性腺分化过程中的作用；同时，筛选获得了可用于牙鲆的Foxl2、Dmrt1和Sox9a特异性抗体，制备了Cyp19a特异性抗体，分析了它们在成体雌雄性腺中的表达特征。研究结果不仅有助于深入理解鱼类性腺分化和发育的调控机制，也将为水产养殖中的性别控制提供新途径。具体研究结果如下：

        1、牙鲆Cyp19a、Foxl2、Dmrt1和Sox9a蛋白体外重组及分析。根据牙鲆基因组和转录组数据中的cyp19a、foxl2、dmrt1和sox9a序列，通过克隆与测序进行验证，经序列比对获得ORF区。它们的氨基酸序列分析预测均含有保守的结构域，都是亲水蛋白，都不具有跨膜区和信号肽。利用同源重组构建了重组蛋白表达质粒，经体外重组和纯化获得重组蛋白，其中，重组蛋白Cyp19a、Foxl2和Dmrt1是包涵体蛋白，Sox9a是可溶性蛋白。用Ni²⁺亲和层析吸附法和梯度透析法对上述重组蛋白进行了纯化和包涵体蛋白复性，最终获得较高纯度的重组蛋白。

        2、牙鲆Cyp19a、Foxl2、Dmrt1和Sox9a特异性抗体的筛选、制备与雌雄性腺差异表达分析。提取牙鲆雌雄成体性腺组织总蛋白，通过Western blot（WB）对相关商用抗体进行筛选，获得可用于牙鲆的Foxl2、Dmrt1和Sox9a特异性抗体。同时，将上述重组蛋白Cyp19a注射至兔子体内，获得多克隆抗体，内源性验证显示该抗体具有特异性。WB结果分析表明，Cyp19a、Foxl2、Dmrt1和Sox9a存在性别二态性表达。其中，Cyp19a和Foxl2在牙鲆卵巢高表达，而Dmrt1和Sox9a在精巢高表达。进一步免疫组化表达定位分析显示，Foxl2主要表达在卵巢卵母细胞周围的颗粒细胞中，Dmrt1主要定位在精巢精原细胞周围的Sertoli细胞中，Sox9a则主要表达在精巢精母细胞和Sertoli细胞。

        3、复性后Cyp19a、Foxl2和Dmrt1重组包涵体蛋白及可溶性Sox9a重组蛋白生物活性验证。用含有10、100或600 μg重组Cyp19a、Foxl2、Dmrt1和Sox9a蛋白及Lipofectamine™ 3000转染试剂的L15培养基，分别孵育成体牙鲆卵巢和精巢。结果显示，这些重组蛋白均能借助体外转染试剂进入性腺组织，重组Foxl2、Dmrt1和Sox9a蛋白能够分别上调和下调性腺中靶基因cyp19a mRNA的转录水平（p < 0.05），而重组Cyp19a则分别上调和下调卵巢中17β-雌二醇（17β-E2）水平和精巢中11-酮基睾酮（11-KT）水平（p < 0.01），说明复性后的Cyp19a、Foxl2和Dmrt1蛋白及可溶性Sox9a蛋白具有生物活性。

        4、Foxl2、Dmrt1和Sox9a重组蛋白对成体牙鲆性腺发育影响的分析。将100 μg/g体重Foxl2、Dmrt1和Sox9a重组蛋白分别与in vivo-jetPEI转染试剂一起注射至牙鲆成体雌鱼腹腔，每3 d注射1次，共3次，第2、3次的Foxl2和Dmrt1组注射浓度同前，而Sox9a组的浓度增加至300 μg/g体重。第5 d样品的WB分析结果显示，这些重组蛋白可进入性腺组织，其靶蛋白Cyp19a表达水平出现上调或下调趋势，但不具有显著性。然而，实时定量PCR（qPCR）分析结果却显示，注射重组Foxl2蛋白，使得雌性相关基因（cyp19a、nr5a2、erα、erβ和fshr）表达量明显增加，雄性相关基因（sox9a、dmrt1和arβ）表达量显著降低（p < 0.05）；注射Dmrt1则得到相反的结果，雌性相关基因的表达下调，雄性相关基因的表达上调（p < 0.05）；注射Sox9a的结果与Dmrt1的趋势类似，但仅dmrt1、erα和erβ的变化存在显著性（p < 0.05）。注射后21 d的样品性腺组织切片结果显示，Foxl2组卵巢发育正常，但Dmrt1引起了卵巢卵母细胞的细胞核染色质凝缩。由此表明外源性别相关蛋白Foxl2和Dmrt1可以影响其靶基因cyp19a等相关基因mRNA水平的表达，Dmrt1甚至影响了卵巢组织的正常发育，但对其靶基因蛋白水平的影响较弱。

        5、Foxl2和Dmrt1重组蛋白在牙鲆性腺分化中的作用。分化前的雌核发育牙鲆幼鱼（全长1.2 – 1.5 cm（total length，TL）），每7 d用in vivo-jetPEI转染试剂转染Foxl2、Dmrt1和EGFP重组蛋白。首次注射浓度为10 μg/尾，后随幼鱼的生长成比例增加，Foxl2组在4.5 cm TL时注射浓度增加至30 μg/尾，Dmrt1组在7.0 cm TL时增加至100 μg/尾。根据10.0 cm TL鱼苗性腺的组织切片观察结果，未注射组、EGFP组和Foxl2组的雌性率均为100%；而Dmrt1组的雌性率为18%（雄性率为82%），精巢发育正常，但卵巢出现异常。采用酶联免疫吸附剂测定法（ELISA）对幼鱼性腺性激素水平进行检测，Foxl2组在6.0 cm TL处的17β-E2水平显著升高（p < 0.05）；而Dmrt1组17β-E2始终处于较低水平（p < 0.05），睾酮（T）、11-KT则处于较高水平（p < 0.05）。qPCR结果显示，Foxl2组的cyp19a表达显著升高（p < 0.05），而Dmrt1组的cyp19a表达在整个性腺分化过程均维持较低水平（p < 0.01）。与EGFP组相比，在3.0 cm TL时，重组Foxl2蛋白显著上调了foxl2、卵巢分化相关基因nr5a2（p < 0.01）和雌激素分泌相关受体基因（fshr、erα和erβ）（p < 0.001）的表达，下调了精巢分化相关基因（dmrt1和sox9a）（p < 0.05）和雄激素分泌相关受体基因（lhr和arβ）（p < 0.05）的转录水平；同时，重组蛋白Dmrt1显著下调卵巢分化相关基因（p < 0.01）和雌激素分泌相关受体基因（p < 0.001），上调精巢分化相关基因（p < 0.05）的转录水平，雄激素受体基因的转录水平也发生了变化，但没有显著差异。而在6.0 cm TL时，Dmrt1组依然维持相同的趋势，且雄激素分泌相关受体基因表达上调且具有极显著性（p < 0.01）。由此显示，重组蛋白Foxl2和Dmrt1注射可以引起幼鱼cyp19a表达变化，从而影响性激素合成，进而影响其相应受体基因的表达。Dmrt1组cyp19a表达的降低及T和11-KT水平的升高促进了雄性性别表型的形成。

        Study on fish gonadal differentiation and development is important both from academic and practical aspects. Cyp19a has been shown as one of the key enzymes in the synthesis of sex steroid hormones, and its important transcription factors Foxl2, Dmrt1, and Sox9a should be involved in fish gonadal differentiation and development. Compared to mRNA, researches on protein level are limited to expression patterns and transcription regulation analysis based on eukaryotic cell transfection method although above genes mainly function in the form of proteins. And related studies in gonadal differentiation also limit to expression patterns (Western blot (WB) or immunohistochemistry (IHC)) in a few species of fish such as zebrafish, medaka, and Nile tilapia. Nevertheless, the precise functions of these proteins in gonadal differentiation and development still require further exploration. Olive flounder Paralichthys olivaceus, an important mariculture fish in China, Japan, and Korea, shows sex-dimorphic growth. Therefore, the sex determination and differentiation mechanism of the flounder has garnered widespread concern. In this study, the bioactive high-purity recombinant proteins Cyp19a, Foxl2, Dmrt1, and Sox9a of the flounder were obtained in vitro. The roles of Foxl2 and Dmrt1 in the flounder gonadal differentiation were evaluated by intraperitoneal injection in juvenile fish. At the same time, Cyp19a, Foxl2, Dmrt1, and Sox9a protein expression characteristics in the gonads of the adult flounder were revealed with WB and IHC by using the selected specific commercial antibodies and the prepared specific antibody of the flounder Cyp19a. These findings could contribute to our understanding of the mechanism of fish gonadal differentiation and development, and provide a new way for sex control in aquaculture. The main results are introduced as follows:

        1. The Cyp19a, Foxl2, Dmrt1, and Sox9a proteins of the flounder were recombined in vitro and analyzed. According to the transcriptomic and genomic data, the open reading frame (ORF) sequences of the flounder cyp19a, foxl2, dmrt1, and sox9a were cloned and verified. Based on prediction of their amino acid sequences, the Cyp19a, Foxl2, Dmrt1, and Sox9a proteins lacked of transmembrane helices and were hydrophilic proteins, and all had conserved domains. The recombinant protein expression plasmids were constructed with homologous recombination, and the recombinant proteins were obtained with prokaryotic expression. Among them, the recombinant proteins Cyp19a, Foxl2, and Dmrt1 are insoluble proteins, and Sox9a is soluble protein. They were purified by using Ni²⁺ affinity chromatography column adsorption and insoluble proteins were renatured with gradient dialysis, and then high-purity recombinant proteins were obtained.

        2. Screening or preparation, and differential expression analysis of the Cyp19a, Foxl2, Dmrt1, and Sox9a specific antibodies for the flounder were performed. The immunoreactive bands of the WB results in the flounder gonads showed that the Foxl2, Dmrt1, and Sox9a commercial antibodies are specific. At the same time, above recombinant protein Cyp19a of the flounder was injected into rabbits to obtain the polyclonal antibody and endogenous verification showed that the anti-Cyp19a is specific. The protein expression profiles were then compared between the ovaries and testes of the adult flounders. All the flounder Cyp19a, Foxl2, Dmrt1, and Sox9a proteins showed sexually dimorphic expression, evidenced by the fact that the Cyp19a and Foxl2 were highly expressed in the ovary, while the Dmrt1 and Sox9a mainly in the testis. Further IHC analysis showed that the Foxl2 was mainly located in granulosa cells around oocytes in mosaic pattern, the Dmrt1 was observed in Sertoli cells surrounding spermatogonia of the testis, and Sox9a was mainly expressed in the testicular spermatocytes and Sertoli cells.

        3. Bioactivity verification of the recombinant insoluble proteins Cyp19a, Foxl2, and Dmrt1 with renaturation and soluble protein Sox9a was carried out. The flounder ovaries and testes were incubated with L15 medium containing 10, 100, or 600 μg recombinant Cyp19a, Foxl2, Dmrt1, or Sox9a protein and Lipofectamine^TM 3000 transfection reagent. The results showed that the recombinant proteins were able to smoothly enter the gonadal cells by using in vitro transfection reagent. The recombinant Foxl2, Dmrt1, and Sox9a proteins up-regulated and down-regulated transcription level of cyp19a, respectively (p < 0.05), and the recombinant Cyp19a protein up-regulated and down-regulated the level of 17β-estradiol (17β-E2) in the ovary and 11-ketotestosterone (11-KT) in the testis, respectively. These data proved that the recombinant flounder Cyp19a, Foxl2, Dmrt1, and Sox9a proteins have biological activity.

        4. Effects of the Foxl2, Dmrt1, and Sox9a recombinant proteins on the flounder gonadal development were analyzed. The 100 μg/g recombinant proteins Foxl2, Dmrt1, and Sox9a were respectively transfected with in vivo-jetPEI transfection reagent into the female flounder once every 3 days for 3 times. The concentrations of the Foxl2 and Dmrt1 groups at the second and third times were not changed, while the concentration of the Sox9a group was increased to 300 μg/g. The WB results of the samples on day 5 showed that these recombinant proteins could enter the gonadal tissue, and the expression level of the target protein Cyp19a showed upward or downward trend, but there was no significant difference. However, real-time quantitative PCR (qPCR) results showed that the recombinant protein Foxl2 significantly increased transcription levels of female-related genes (cyp19a, nr5a2, erα, erβ, and fshr), and significantly reduced transcription levels of male-related genes (sox9a, dmrt1, and arβ) (p < 0.05). The opposite results were obtained in the Dmrt1 group, with the female-related genes down-regulation and male-related genes up-regulation (p < 0.05). The results of the Sox9a group were consistent with trend of the Dmrt1 group, but only changes of dmrt1, erα and erβ were significant (p < 0.05). The observation of the gonadal tissue sections on day 27 under the microscope found that the ovarian development was normal in the Foxl2 group, but the Dmrt1 caused nuclear chromatin condensation in the ovarian oocytes. These results indicated that the exogenous sex-related proteins Foxl2 and Dmrt1 could affect the mRNA transcription levels of target gene cyp19a and other related genes. The Dmrt1 even affected the normal development of the ovary although there was little effect on the expression of protein level of the target gene.

        5. Roles of the Foxl2 and Dmrt1 recombinant proteins during the flounder gonadal differentiation were studied. The recombinant proteins Foxl2, Dmrt1, and EGFP were respectively transfected into the juvenile gynogenetic flounder by using in vivo-jetPEI transfection reagent every 7 days before the fish began the gonadal differentiation (1.2 - 1.5 cm total length (TL)). The initial injection concentration was 10 μg/fish and was increased proportionally with growth of the juveniles. For instance, the Foxl2 group was increased to 30 μg/fish at 4.5 cm TL and the Dmrt1 group was increased to 100 μg/fish at 7.0 cm TL. According to the histological section results at 10.0 cm TL, the female rates were all 100% in the untreated group, EGFP group, and Foxl2 group, while the female rate in the Dmrt1 group was 18% (male rate was 82%). In the last group, the testis could develop normally, while the ovary presented abnormally. Enzyme linked immunosorbent assay (ELISA) was used to detect the gonadal hormone levels of the juvenile flounder. Compared with the EGFP group, the level of 17β-E2 in the Foxl2 group was significantly higher at 6.0 cm TL (p < 0.05). However, in the Dmrt1 group, 17β-E2 was consistently at lower level (p < 0.05), and testosterone (T) and 11-KT were at higher level (p < 0.05). The qPCR results showed that the transcription levels of cyp19a in the Foxl2 group was significantly increased (p < 0.05), while in the Dmrt1 group, cyp19a transcription levels were significantly decreased (p < 0.01) and maintained low level during the whole gonad differentiation period. Compared with the EGFP group, at 3.0 cm TL, the recombinant protein Foxl2 up-regulated the transcription levels of foxl2 and gene related to ovarian differentiation nr5a2 (p < 0.01) and receptor genes related to estrogen secretion (fshr, erα, and erβ) (p < 0.001), and down-regulated the transcription levels of genes related to testicular differentiation (dmrt1 and sox9a) (p < 0.05) and genes related to androgen secretion (lhr and arβ) (p < 0.05). Meanwhile, the recombinant protein Dmrt1 significantly down-regulated genes related to ovarian differentiation (p < 0.01) and to estrogen secretion (p < 0.001), and significantly up-regulated genes related to testicular differentiation (p < 0.05). The transcription level of androgen receptor gene also changed, but there was no significant difference. At 6.0 cm TL, the Dmrt1 group still maintained the same trend, and the transcription level of gene related to androgen secretion (p < 0.01) significantly increased. Therefore, the recombinant proteins caused change in cyp19a, which in turn affected the levels of sex hormones and altered the transcription levels of endocrine-related genes on HPG axis of the flounder. The decreasing transcription level of cyp19a and the increasing levels of T and 11-KT promoted the formation of male phenotype.

第1章  绪论 1

1.1  鱼类性别决定 1

1.1.1  遗传因素 2

1.1.2  环境因素 4

1.1.3  内分泌调控 5

1.2  鱼类性腺分化 7

1.2.1  性腺分化的组织细胞学过程 7

1.2.2  性腺分化相关基因 9

1.3  鱼类性腺发育 16

1.3.1  性腺发育的生物学过程 16

1.3.2  性腺发育的分子生物学与内分泌学研究 17

1.4  性别相关蛋白在鱼类性腺分化和发育中的相关研究进展 18

1.5  立项依据和目的意义 21

第2章  Cyp19a、Foxl2、Dmrt1和Sox9a蛋白体外重组及其特异性抗体筛选与制备 23

2.1  研究背景 23

2.2  材料与方法 24

2.2.1  实验鱼与样品采集 24

2.2.2  实验试剂和仪器 24

2.2.3  总RNA提取及cDNA合成 26

2.2.4  总蛋白提取 27

2.2.5  生物信息学分析 27

2.2.6  体外原核表达重组 28

2.2.7  重组蛋白纯化 34

2.2.8  目的蛋白的透析复性 35

2.2.9  Cyp19a多克隆抗体制备 36

2.2.10  抗体特异性检测 38

2.3  实验结果 38

2.3.1  蛋白结构及生化特性预测 38

2.3.2  重组蛋白的表达与纯化 40

2.3.3  抗体特异性检测 42

2.3.3.1  Cyp19a多克隆抗体制备及其特异性检测 42

2.3.3.2  Foxl2、Dmrt1和Sox9a商业抗体的特异性检测 44

2.4  讨论 45

第3章  Cyp19a、Foxl2、Dmrt1和Sox9a对牙鲆性腺发育的影响 49

3.1  研究背景 49

3.2  材料与方法 50

3.2.1  实验鱼 50

3.2.2  实验试剂和仪器 50

3.2.3  性腺组织切片 51

3.2.4  免疫组化 52

3.2.5  组织孵育 53

3.2.6  腹腔注射 53

3.2.7  总RNA提取及cDNA合成 54

3.2.8  总蛋白提取 54

3.2.9  WB检测 54

3.2.10  性激素水平测定 54

3.2.11  定量表达分析 55

3.2.12  数据分析 57

3.3  实验结果 57

3.3.1  Foxl2、Dmrt1和Sox9a在雌雄性腺中表达定位 57

3.3.2  Cyp19a、Foxl2、Dmrt1和Sox9a重组蛋白具备生物活性 59

3.3.3  外源Foxl2、Dmrt1和Sox9a对性腺发育的影响 63

3.4  讨论 66

第4章  Foxl2和Dmrt1蛋白在牙鲆性腺分化中功能初探 71

4.1  研究背景 71

4.2  材料与方法 72

4.2.1  实验鱼、注射与样品采集 72

4.2.1.1  实验鱼 72

4.2.1.2  重组蛋白注射 72

4.2.1.3  样品采集 72

4.2.2  实验试剂和仪器 72

4.2.3  性腺组织切片 73

4.2.4  性激素水平测定 73

4.2.5  免疫组化 73

4.2.6  总RNA提取及cDNA合成 73

4.2.7  总蛋白提取 73

4.2.8  WB检测 73

4.2.9  定量表达分析 73

4.2.10  数据分析 73

4.3  实验结果 73

4.3.1  外源Foxl2和Dmrt1对牙鲆性腺分化的影响 74

4.3.2  外源Foxl2和Dmrt1对性激素水平的影响 75

4.3.3  外源Foxl2和Dmrt1对cyp19a及相关基因表达的影响 76

4.4  讨论 80

第5章  结论与展望 85

参考文献 87

致 谢 109

作者简历及攻读学位期间发表的学术论文与研究成果 110