实验海洋生物学重点实验室知识库

性腺体细胞是性腺的主要组成部分，在鱼类性腺分化和发育中的重要作用甚至主导作用已引起关注，但相关研究仅限少数鱼种，也缺乏作用机制的系统分析。本实验以具有雌雄生长差异的重要海水养殖鱼类牙鲆（Paralichthys olivaceus）为研究对象，从性腺体细胞角度，借助组织细胞学观察和性别相关基因（amh、oct4、cyp17a1和3β-hsd）的表达，对雌雄性腺分化与发育过程中的特征及其增殖变化规律进行了分析；结合减数分裂标记基因（scp3和dazl）与性类固醇激素合成相关基因（cyp19a1a和cyp11b）的表达图示，明确了生殖细胞与体细胞的作用时序；同时，通过白消安抑制牙鲆生殖细胞，初步探讨了生殖细胞对体细胞的作用。研究结果将为明确性腺体细胞在牙鲆等鱼类的性腺分化与发育中的作用及机制提供基础。主要研究结果如下：

在牙鲆性腺分化开始前，体细胞分布于细线状的原始性腺周围。随着原始性腺的发育，锥形的塞托利（Sertoli）细胞和椭圆形的间质（Leydig）细胞逐渐分布于原始生殖细胞周围。当卵巢腔以及输精管原基形成以及性腺分化完成时，卵巢由卵原细胞与体细胞（颗粒细胞和鞘膜细胞）组成，而精巢由精原细胞与体细胞（Sertoli和Leydig细胞）组成，且精巢和卵巢的生殖细胞和体细胞数量在性腺分化后出现快速增加的现象。性腺经过继续发育分别形成精小叶和产卵板结构，至V期成熟时，精小叶发生联合，卵巢则会形成双层的滤泡细胞结构。透射电镜的亚显微结构分析显示，II期卵巢的颗粒细胞呈椭圆或近圆形，细胞中充斥着染色质；鞘膜细胞则为狭长的椭圆或锥形，主要由细胞核占据，颗粒细胞与鞘膜细胞围绕在卵母细胞周围形成滤泡细胞。III期精巢的Leydig细胞呈椭圆形，充斥在生殖细胞或者精小叶的间隙中；Sertoli细胞则为锥形或长椭圆形，参与构成精小叶，为生精小叶提供机械支持。

实时定量PCR（qPCR）及原位杂交（ISH）结果表明，在雄性分化前，amh高表达，且主要定位于生殖细胞周围的Sertoli细胞中，但在卵巢分化前和分化过程中均没有表达，提示amh可能启动了雄性分化；在性腺I-V期发育过程中，qPCR、ISH和免疫组化（IHC）结果表明，amh也主要在精巢精小叶外周的Sertoli细胞中表达，在II期的表达最高。3β-hsd、oct4和cyp17a1在卵巢分化早期表达量较高；在I-V性腺发育期中，也主要在卵巢中表达，定位于I和II时相的卵母细胞；而在精巢发育各时期中，仅在精巢精小叶外周的Sertoli细胞中有微弱的表达，说明它们在卵巢分化及卵子发生过程中有重要作用。所以，amh可以作为牙鲆精巢Sertoli细胞的分子标记，而3β-hsd、oct4和cyp17a1可以做为牙鲆卵巢卵母细胞的分子标记。

利用减数分裂标记基因scp3 和dazl来检测生殖细胞减数分裂的起点，为明确牙鲆生殖细胞与性腺体细胞的作用时序提供依据。研究显示，scp3和dazl基因的表达上调点均在精巢和卵巢分化之后出现，暗示在牙鲆雌雄性腺分化后，生殖细胞才开始进行减数分裂。进一步分析了性类固醇激素合成相关基因cyp19a1a与cyp11b2的表达图示，前者在性腺分化后表达升高，而cyp11b2除了在原始性腺时期表达量略高，在性腺分化过程一直维持较低水平。但性激素睾酮（T）和雌二醇（E2）水平却在性腺分化启动前和分化过程中都有升高的现象：雌性分化过程中，T和E2水平分别在全长60 mm（total length，TL）时最高；在雄性分化之前，T和E2水平分别在40 mm TL时升高。这表明性激素水平似乎在性腺（包括生殖细胞和体细胞）的cyp19a1a与cyp11b2高表达之前就已经升高。RT-PCR结果显示，在I-V期雌雄性腺发育过程中，scp3和dazl均有表达；但scp3在II-V期精巢中的表达均显著高于卵巢（P < 0.05），且在其初级精母细胞有较强的表达信号；dazl在I、II和V期卵巢中的表达显著高于精巢（P < 0.05），且在I和II时相的卵母细胞中检测到较强信号。故scp3适合用于标记牙鲆精巢减数分裂的过程，而dazl更适合用于指示其卵巢减数分裂的过程。

白消安（1，4-丁二醇二甲磺酸酯）在一些物种中可以抑制生殖细胞的增殖。在鱼类中的研究主要集中在生殖细胞抑制和移植方面，经过白消安处理后，对鱼类生殖细胞产生影响，并导致其短暂或永久性不育，但对性腺体细胞的影响还没有得到关注。本研究发现，牙鲆成体腹腔注射80（80 B）或120（120 B）mg/kg体重的白消安后，其雌雄个体的双侧性腺均出现萎缩，卵巢颜色发生变化并且与内脏粘连。组织切片观察和分析表明，两种浓度下，雄鱼精巢精原细胞排列疏松、精原细胞胞核变大、细胞质变小，其数量显著少于对照组，而体细胞数显著多于对照组（P < 0.05）；但是，雌鱼卵巢卵母细胞数和体细胞数则均少于对照组（P < 0.05），显示了白消安对生殖细胞影响的性别差异性。qPCR分析发现，相对于对照组，两个处理组的雌鱼卵巢中生殖细胞相关基因vasa表达无显著差异，而精巢vasa的表达显著降低（P < 0.05），这又表明了白消安的毒性可能是性别特异性的，对雄性的影响比雌性的大。进一步通过ISH分析了vasa与体细胞相关基因cyp19a1a和amh在这两个处理组和对照组中的差异表达，结果显示，vasa和cyp19a1a在两个处理组的雌鱼卵巢卵母细胞中有较弱表达信号，而cyp19a1a在鞘膜细胞中仍然表达；雄鱼精巢中，vasa在精原细胞和破坏的精原细胞中表达信号均较弱，80 B处理组中，amh在精原细胞的表达量较低，而120 B处理组的精原细胞和破坏的精原细胞中都有较高的表达信号。这表明白消安导致精原细胞破坏后变成类似体细胞样的细胞。同时，对分化期牙鲆鱼苗进行160 mg/kg体重的白消安处理，初步实验显示在处理后的性腺中原始生殖细胞数目显著减少（P < 0.05）。由上述结果表明，白消安可以影响牙鲆性腺分化与发育，特别是有效地抑制精巢精原细胞的发育。

Gonadal somatic cells are the main component of fish gonad. Their important and even leading roles during gonadal differentiation and development of fish have attracted much attention. However, the study on them is limited to a few fish species. Their function in sex determination and differentiation is still unclear. Olive flounder (Paralichthys olivaceus) is an important maricultured fish and females grow much faster than males. This research mainly studied function of somatic cells during the flounder gonadal differentiation and development. Initially, characteristics and changing trends of proliferation of somatic cells during the gonadal differentiation and development processes were studied by using histological observation and expression patterns of the related genes including amh, oct4, cyp17a1, and 3β-hsd. And then, the order of action of somatic cells and germ cells was analyzed according to expression of the meiosis marker genes scp3 and dazl, and the genes related to sex steroid hormone synthesis cyp19a1a and cyp11b. At the same time, the effect of germ cells on somatic cells was evaluated with busulfan treatment. The results will provide a basis for clarifying roles of somatic cells in gonadal differentiation and development of the flounder and other fish. The main results are as follows:

Before the gonadal differentiation, somatic cells were distributed around the filamentous primitive gonads. With development of the gonads, conical Sertoli cells and oval Leydig cells were gradually distributed around primordial germ cells (PGCs). After formation of ovarian cavity and vas deferens primordia as well as completion of the gonadal differentiation, the ovary was composed of oogonia and somatic cells including granulosa cells and theca cells, and the testis was composed of spermatogonia and somatic cells such as Sertoli and Leydig cells. Numbers of germ cells and somatic cells in the testis and ovary increased rapidly after the gonadal differentiation. Then seminal lobule in the testis and spawning plate in the ovary were formed, respectively. When the gonads developed to stage V, seminal lobule of the testis united, and the ovary formed a double-layer follicular cell structure. Ultrastructural observation under transmission electron microscope showed that granulosa cells of the ovary at stage II were oval or nearly round filled with chromatin, and theca cells were a long and narrow ellipse or cone shaped, mainly occupied by nucleus. Both often surrounded oocytes to form follicular cells. Leydig cells of the testis at stage II were oval in shape and filled between germ cells and seminal lobule, while Sertoli cells were cone-shaped or oblong in shape, participating in the formation of structure of seminal lobule and providing support for seminal lobule.

During the gonadal differentiation, the results of real-time quantitative PCR (qPCR) and in situ hybridization (ISH) showed that amh was highly expressed in the gonads before the testicular differentiation, and mainly located in Sertoli cells, but didn’t express in the gonads before and during ovarian differentiation, suggesting that amh may initiate testicular differentiation. The results of qPCR, ISH, and immunohistochemical (IHC) showed that amh was mainly expressed in Sertoli cells around seminal lobules at stages I-V of the testis, and the highest expression of amh was detected in the testis at stage II. The 3β-hsd, oct4, and cyp17a1 were highly expressed at the early stage of the ovarian differentiation, and mainly expressed in the ovaries at stages I-V and located in oocytes at phases I and II, but only weakly expressed in Sertoli cells around seminiferous lobules at all stages of the testicular development, indicating that they play an important role in ovarian differentiation and oogenesis. So, amh can be used as a molecular marker of Sertoli cells in the testis, while 3β-hsd, oct4, and cyp17a1 are more suitable as molecular markers of oocytes in the ovary.

Expression of meiosis marker genes scp3 and dazl was detected to learn the starting point of meiosis of the flounder germ cells. The results showed that the up-regulated expression points of scp3 and dazl respectively appeared in the differentiated testis and ovary, suggesting that the flounder germ cells began meiosis only after the gonadal differentiation. Differential expression of genes related to sex steroid hormone synthesis cyp19a1a and cyp11b2 was further analyzed. The former presented higher expression in the differentiated gonads, while the latter was slightly higher expression in the primitive gonads, but maintained at a low level in the differentiation gonads. However, levels of sex hormones testosterone (T) and estradiol (E2) all increased before and during the gonadal differentiation. T and E2 levels at 60 mm total length (TL) were the highest during the female differentiation, and their levels increased at 40 mm TL before the male differentiation. These results indicated that sex hormone levels seemed to have increased earlier than expression of cyp19a1a and cyp11b2 in the gonads (including germ cells and somatic cells). RT-PCR results showed that scp3 and dazl were expressed in the flounder testes and ovaries at stages I - V, and expression of scp3 in the testes at stages II-V was significantly higher than that in the ovary (P < 0.05), and strong signals in primary spermatocytes of the testis were detected. While, expression of dazl in the ovary at stages I, II, and V was significantly higher than that in the testis (P < 0.05), and stronger signals were detected in oocytes of the ovary at stages I and II. Therefore, as molecular markers, scp3 is suitable for marking the meiosis process in the flounder testis, while dazl is more suitable for indicating the meiosis process of the ovary.

Busulfan, a cytotoxic alkylating agent, is used to inhibit germ cell proliferation in some animal species. In fish species, studies with busulfan have been focused on the inhibition and transplantation of germ cells, and find that buulfan has effects on germ cells and resultes in transient or permanent sterility. However, effects of busulfan on gonadal somatic cells have not been fully studied. In this study, after intraperitoneal injection with 80 (80B) or 120B (120B) mg/kg bufulfan in the adult flounder, both the bilateral gonads were atrophied, and the ovaries were discolored with adhesion to the visceral mass. Histological results indicated that germ cells in the gonads were detached, and there was a larger nucleus size and smaller cytoplasmic volume in spermatogonia. Numbers of spermatogonia and somatic cells in the testis were markedly less and greater, respectively (P < 0.05), while, in the ovary, numbers of oocytes and somatic cells were both less (P < 0.05). The qPCR analysis presented that, compared with the control groups, vasa expression in the ovary of the two treatment groups was not different, but its expression in the testis was markedly less (P < 0.05), indicating that the toxicity of busulfan may be sex-specific with there being greater effects in the males than the females. Furthermore, by using ISH,  in the flounder ovaries of the two treatment groups, relative abundance of vasa and cyp19a1a was very small in the cytoplasm of oocytes, while cyp19a1a was still present in theca cells. In the testis, abundance of vasa was markedly less (P < 0.05) with there being very little vasa in spermatogonia and disrupted spermatogonia. In the 80B treatment group, amh was in lesser abundance with there being very little amh in spermatogonia, however, with the 120B treatment there was a large amh abundance in spermatogonia with there being disruption of structure of these germ cells and Sertoli cells. Busulfan, therefore, might inhibit the development of spermatogonia and lead to spermatogonia forming somatic-like cells in the flounder testis. After the flounder larvae in gonadal differentiation period was treated with 160 mg/kg busulfan, numbers of the PGC were markly less (P < 0.05), and few PGCs were observed in the gonads. These results implied that busulfan could affect the flounder gonadal differentiation and development, especially the development of spermatogonia in the testis.

目 录

第一章 绪 论......................................................................................... 1

1.1 鱼类性腺分化的研究进展................................................................................. 2

1.1.1原始性腺....................................................................................................... 3

1.1.2 性腺分化...................................................................................................... 4

1.1.2.1 卵巢分化特征....................................................................................... 4

1.1.2.2 精巢分化特征....................................................................................... 5

1.1.3 性腺分化的相关基因.................................................................................. 5

1.1.3.1 卵巢分化的相关基因........................................................................... 8

1.1.3.2 精巢分化的相关基因......................................................................... 10

1.2 鱼类性腺体细胞研究进展............................................................................... 13

1.2.1 性腺体细胞的来源.................................................................................... 13

1.2.2 精巢的体细胞............................................................................................ 15

1.2.3  卵巢的体细胞.......................................................................................... 18

1.3 鱼类性腺体细胞与生殖细胞间的相互作用................................................... 19

1.3.1  体细胞对生殖细胞的调控...................................................................... 20

1.3.2  生殖细胞对体细胞的调控...................................................................... 22

1.4 本研究的目的及意义....................................................................................... 23

第二章 牙鲆性腺体细胞在性腺分化期及I-V发育期的增殖规律....... 25

2.1 引言................................................................................................................... 25

2.2 材料与方法....................................................................................................... 26

2.2.1 实验材料.................................................................................................... 26

2.2.1.1 实验鱼................................................................................................. 26

2.2.1.2 试剂与实验仪器................................................................................. 27

2.2.2 实验方法.................................................................................................... 27

2.2.2.1 性腺组织切片..................................................................................... 27

2.2.2.2 生殖细胞、体细胞计数..................................................................... 29

2.2.2.3 成体性腺透射电镜切片与观察......................................................... 29

2.2.2.4 总RNA提取与cDNA合成.............................................................. 30

2.2.2.5 RT-PCR............................................................................................... 32

2.2.2.6 实时荧光定量PCR............................................................................ 33

2.2.2.7 原位杂交............................................................................................. 37

2.2.2.8 Western Blot（WB）和免疫组化..................................................... 42

2.3 结果................................................................................................................... 44

2.3.1性腺体细胞在分化期的组织细胞学特征................................................. 44

2.3.2性腺体细胞、生殖细胞在分化期的增殖................................................. 46

2.3.3性腺体细胞在I - V期的变化特征........................................................... 47

2.3.4性腺体细胞在雌雄成体期的亚显微观察................................................. 49

2.3.5 雄性相关基因——amh............................................................................. 50

2.3.5.1 成体组织中的定量差异表达............................................................. 50

2.3.5.2  精巢分化期的时空表达................................................................... 51

2.3.5.3 性腺I - V期中的差异表达............................................................... 53

2.3.6 雌性相关基因——3β-hsd、oct4和cyp17a1.......................................... 56

2.3.6.1成体组织的RT-PCR结果.................................................................. 56

2.3.6.2 性腺分化过程中的表达..................................................................... 57

2.2.6.3性腺I-V期的表达.............................................................................. 61

2.4 讨论................................................................................................................... 66

2.4.1 性腺分化与发育过程中生殖细胞、体细胞增殖发育规律.................... 66

2.4.2 Amh在精巢分化与发育的Sertoli细胞中表达的变化........................... 67

2.4.3 3β-hsd、oct4和cyp17a1在卵巢分化与发育期体细胞中的差异表达.. 69

第三章 性腺体细胞与生殖细胞在牙鲆性腺分化中的作用时序......... 71

3.1 引言................................................................................................................... 71

3.2 材料与方法....................................................................................................... 72

3.2.1 实验材料.................................................................................................... 72

3.2.1.1 实验鱼................................................................................................. 72

3.2.1.2 试剂与实验仪器................................................................................. 72

3.2.2 实验方法.................................................................................................... 73

3.2.2.1 性腺组织切片..................................................................................... 73

3.2.2.2 生殖细胞计数..................................................................................... 73

3.2.2.3 总RNA提取与cDNA合成.............................................................. 73

3.2.2.4 RT-PCR............................................................................................... 73

3.2.2.5 QPCR 分析........................................................................................ 74

3.2.2.6 原位杂交............................................................................................. 75

3.2.2.7 性激素测定......................................................................................... 75

3.3 结果................................................................................................................... 76

3.3.1 Scp3和dazl的差异表达分析................................................................... 76

3.3.1.1  在牙鲆成体组织的表达差异........................................................... 76

3.3.1.2 性腺分化过程中的时空表达............................................................. 77

3.3.1.3性腺I-V期的时空表达...................................................................... 80

3.3.2卵巢减数分裂过程中scp3和dazl的表达分析...................................... 83

3.3.3雌激素/雌激素受体抑制剂处理对卵巢减数分裂相关基因表达的影响 85

3.3.4 Cyp19a和cyp11b在牙鲆性腺分化过程中的表达................................. 86

3.3.5 性腺分化过程中性激素变化水平............................................................ 88

3.4 讨论................................................................................................................... 89

3.4.1  牙鲆性腺体细胞与生殖细胞的作用时序.............................................. 89

3.4.2 Scp3和dazl在牙鲆雌雄性腺减数分裂过程中表达的比较................... 90

第四章 生殖细胞对体细胞作用的初步探讨....................................... 93

4.1 引言................................................................................................................... 93

4.2 材料与方法....................................................................................................... 94

4.2.1 实验材料.................................................................................................... 94

4.2.1.1 实验鱼................................................................................................. 94

4.2.1.2 试剂与实验仪器................................................................................. 94

4.2.2 实验方法.................................................................................................... 94

4.2.2.1 白消安的处理..................................................................................... 94

4.2.2.2 性腺组织切片..................................................................................... 95

4.2.2.3  生殖细胞与体细胞计数................................................................... 95

4.2.2.4 QPCR 与原位杂交............................................................................ 95

4.3 结果................................................................................................................... 97

4.3.1 白消安处理对性腺发育的影响................................................................ 97

4.3.2 白消安对性腺生殖细胞与体细胞的影响................................................ 98

4.3.3 白消安处理后性腺中相关基因的表达变化............................................ 99

4.3.4 白消安对性腺分化期生殖细胞的抑制.................................................. 103

4.4 讨论................................................................................................................. 104

4.4.1  白消安对牙鲆成体性腺发育影响的性别差异.................................... 104

4.4.2  生殖细胞抑制后性腺体细胞的变化.................................................... 105

结论..................................................................................................... 107

参考文献................................................................................................................. 109

致 谢........................................................................................................................ 127

作者简历及攻读学位期间发表的学术论文与研究成果......................... 129