实验海洋生物学重点实验室知识库

  生殖细胞将遗传信息传递给下一代，对种族延续十分重要。近年来，鱼类生殖细胞移植技术得到快速发展，结合冷冻保存技术，在濒危物种保护和代理养殖方面展现广阔的空间。然而，目前关于鱼类生殖细胞发生发育及移植方面研究大多集中在模式及淡水鱼类，对于海洋经济鱼类涉及较少。大菱鲆（Scophthalmus maximus）、夏鲆（Paralichthys dentatus）、牙鲆（Paralichthys olivaceus）以及许氏平鲉（Sebastes schlegelii）等鱼类是我国重要海洋经济养殖品种，随着长期的人工养殖，逐渐出现了种质资源退化，遗传多样性降低，生殖力低下等问题。本研究以上述鲽形目及鲉形目鱼类为研究对象，开展生殖细胞起源模式及移植技术等研究。促进对鱼类生殖细胞起源、分化、成熟机制的认知，为种质资源的保存和高效开发利用开辟新途径。具体研究结果如下：

1.许氏平鲉生殖细胞的起源和发育

   借助许氏平鲉vasa基因原位杂交，分析其在生殖系的表达。vasa RNA在分裂期均匀分布于卵裂球；信号在原肠早期出现聚集，胚盾区域似乎比其它区域强，直到原肠晚期，一些vasa阳性细胞首次出现在外包底部，此时原始生殖细胞（PGCs）形成；PGCs聚集在狭窄早期神经胚的背侧，沿体轴的前部随机排列，并向后移动，于体节期松散排列在胚体两侧；PGCs在心跳期形成两个簇，腹部两侧对齐，继续轴向迁移，最后于孵化期聚集在肠的背部, 脊索的腹侧。仔鱼出生后，PGCs逐渐被体细胞完全包围，沿中肾管迁移，并于第10天迁移至原始生殖嵴，形成原始性腺。追踪整个性腺发育过程，vasa持续表达在早期生殖细胞。本研究首次揭示海洋胎生鱼类生殖细胞的起源迁移模式，与生殖质（vasa RNA）失去早期定位的真真骨鱼类（Euteleostei）青鳉相似，不同于生殖质早期定位在卵裂沟的骨鳔鱼类（Ostariophysans）斑马鱼。

2. 硬骨鱼类vasa/nanos3 3'UTR功能分析

   母源基因的3'UTR具有定位功能，融合荧光蛋白，可以体内追踪鱼类PGCs。构建包括大菱鲆、许氏平鲉在内几种常见硬骨鱼类vasa/nanos3 3'UTR嵌合mRNA，注入了1-2细胞受精卵，实现了大菱鲆等鱼类PGCs的活体标记。这种简单、快速、无基因污染的标记方法适用于经济鱼类。分析几种鱼类vasa 3'UTR中保守元件，探讨了参与定位的关键因素。具有早期定位的斑马鱼vasa 3'UTR含有丰富定位元件，可以标记多种鱼类PGCs。而失去早期定位的硬骨鱼类，如真鲷（Pagrus major）和海水青鳉（Oryzias melastigma）vasa 3'UTR不能标记斑马鱼和大菱鲆PGCs。但是许氏平鲉vasa 3'UTR因具有斑马鱼特有的3个重要保守元件（M10、M12、M19），可能参与斑马鱼PGCs标记。

3.鲽形目精原干细胞的移植

  从冷冻保存的牙鲆、夏鲆、大菱鲆整个精巢中分离纯化精原干细胞（SSCs），PKH-26标记，腹腔移植入孵化22 d内牙鲆三倍体仔鱼，探索亲缘关系由近及远（种内、属内、科间）的细胞移植。荧光跟踪发现供体细胞在移植后14天（14dpt）几乎都迁移到了原始生殖嵴，在50 dpt随机分布并整合到发育中的受体性腺；组织学展示在种内和属内移植中，受体分化为雄性和雌性嵌合体，而在科间移植中，受体分化为雄性和兼性嵌合体；原位杂交和免疫组化鉴定大菱鲆生殖细胞在三倍体牙鲆受体中存活及增殖，证明嵌合体除了包括牙鲆早期的卵母细胞和雄性生殖细胞外，还包括大菱鲆精原和精母细胞；随后，通过扫描电镜、PCR、人工授精进一步证实了供体来源精子及其功能。而且，兼性嵌合体可以成熟并产生大菱鲆功能性的精子。综合以上鉴定，移植效率为大菱鲆-牙鲆（33-50％），夏鲆-牙鲆（75-95％）和牙鲆-牙鲆（100％）。本研究首次突破了海洋鱼类科间移植，并缩短了大菱鲆精子的成熟时间。

4.鲉形目精原干细胞的移植

   从冷冻保存的许氏平鲉整个精巢中分离出SSCs，PKH-26标记，腹腔内移植入孵化5-10 d的同种仔鱼。荧光观察供体细胞在20dpt时迁移至生殖嵴附近（100%），在90dpt时已嵌入受体性腺（90%）。组织学显示受体性腺正常分化，形态与对照没有显著差异。移植1年后，微卫星鉴定29个受体，种内移植嵌合率达到82.76%。同时，分离鲉形目的其它物种朝鲜平鲉（Sebastes koreanus）、褐菖鲉（Sebastiscus marmoratus）SSCs移植许氏平鲉，探索胎生鱼类的种间移植。通过荧光追踪，发现供体细胞在15dpt迁移到生殖嵴，迁移效率60%以上。

     Germ cells transmit genetic information to the next generation, which is very important for the continuation of race. In recent years, fish germ cell transplantation technology has been rapidly developed. Combined with cryopreservation technology, it has shown a broad space in the protection of endangered species and surrogate broodstock. However, most of the current researches on fish germ cell development and transplantation focus on models and freshwater fishes, and rarely involve in marine economic fishes. Turbot (Scophthalmus maximus), Summer flounder (Paralichthys dentatus), Japanese flounder (Paralichthys olivaceus) and black rockfish (Sebastes schlegelii) are important marine economic cultured species in China. With the long-term artificial breeding, the degradation of germplasm resources, the decrease of genetic diversity and the low fertility gradually appear. In this study, we studied the origination pattern and transplantation technology of germ cells in the Pleuronectiformes and Scorpaeniformes. To promote the understanding of the origination, differentiation and maturation mechanism of fish germ cells, and open up new ways for the preservation and efficient utilization of germplasm resources. The specific research results are as follows:

1. Black rockfish germ cell origination and development

    The expression of black rockfish vasa gene in germline was analyzed by in situ hybridization. The vasa RNA evenly distributed in blastomere during cleavage stage. Signal concentration appeared at early gastrulation stage, and the areas of embryonic shield seemed to be darker than the rest. Until the late gastrulation stage, some vasa-positive cells firstly appeared at germ ring of epiboly bottom,  and the primordial germ cells (PGCs) formed. PGCs gathered on dorsal side of the narrow embryonic body at early neurula stage and randomly aligned along anterior of the body axes. Then, they moved posterior-ward and loosely lined along the anterior-posterior axis on both sides of the embryonic body at somite stage. PGCs formed two clusters and aligned bilaterally on the ventral side at heart beating stage. Finally, the PGCs localized at the dorsal sides of the gut, ventral side of notochord at hatching stage. After birth, PGCs were gradually surrounded by somatic cells and migrated along mesonephric duct. On the 10th day, PGCs migrated to the primordial genital ridge and formed the primordial gonads. During the entire gonadal development process, vasa expressed in early germ cells. This study firstly revealed the origination and migration pattern of germ cells in marine viviparous fish, which was similar to medaka of Euteleostei without early localization function of germplasm (vasa RNA), but different from zebrafish of Ostariophysans with germplasm early localized at cleavage furrows.

2. Function analysis of teleost vasa/nanos3 3’UTR

    The 3’UTR of the maternal gene has localization function, fused with fluorescent protein, and can track fish PGCs in vivo. Constructed vasa/nanos3 3’UTR chimeric mRNA of several common teleost, including turbot and black rockfish, and injected 1-2 cell fertilized eggs to realize the labeling of PGCs in turbot and other fishes in vivo. This simple, rapid, and non-gene pollution labeling method is suitable for commercial fishes. The conserved elements in vasa 3’UTR of several teleost were analyzed, and the key factors involved in localization were discussed. The zebrafish vasa 3’UTR with early localization was rich in localization elements, which could mark PGCs of various fishes. However, teleost that had lost their early localization, such as red seabream (Pagrus major) and marine medaka (Oryzias melastigma) vasa 3’UTR could not label the PGCs of zebrafish and turbot. However, black rockfish vasa 3’UTR had three important conserved elements (M10, M12, M19) of zebrafish, which might be involved in marking zebrafish PGCs.

3. Transplantation of spermatogonial stem cells within Pleuronectiformes

    Spermatogonial stem cells (SSCs) were isolated and purified from the cryopreserved whole testes of Japanese flounder, summer flounder and turbot, then labeled with PKH-26, and intraperitoneally transplanted into triploid Japanese flounder larvae within 22 days post hatching, exploring the cell transplantation of genetic relationships from from near to far (intra-species, intra-genus, and inter-family). Fluorescence tracking revealed that donor cells almost all migrated to the primordial genital ridge at 14 days after transplantation (14dpt), and were randomly distributed and incorporated into the developing recipient gonads at 50 dpt; Histology showed that in intra-species and intra-genus transplantations, the recipients differentiated into male and female chimeras, while in in inter-family transplantation, the recipients differentiated into male and intersex chimeras; The In situ hybridization and immunohistochemistry identified the survival and proliferation of turbot germ cells in the triploid Japanese flounder recipient. It also proved that the intersex chimera included not only early oocytes and male germ cells of Japanese flounder, but also the spermatogonia and spermatocyte of turbot. Subsequently, the donor-derived spermatozoa and its function were further confirmed by scanning electron microscopy, PCR, and artificial insemination. Moreover, the intersex chimera could mature and produce functional turbot spermatozoa. Based on the above identification, the transplantation efficiency was turbot-Japanese flounder (33-50%), summer flounder-Japanese flounder (75-95%) and Japanese flounder-Japanese flounder (100%). This firstly realized inter-family transplantation in marine fish species and shortened the maturation time of turbot spermatozoa.

4. Transplantation of spermatogonial stem cells within Scorpaeniformes

   The SSCs were isolated from the cryopreserved whole testis of black rockfish, labeled with PKH-26, and then intraperitoneally transplanted into allogeneic hatched larvae at 5-10 days post hatching. Fluorescence observed that the donor cells migrated to the genital ridge at 20dpt (100%), and incorporated into the recipient gonad at 90dpt (90%). Histology showed that the recipient gonad was normally differentiated, and the morphology was not significantly different from that of the control. One year after transplantation, 29 recipients were identified by microsatellite, and the chimerism rate of intra-species transplantation reached 82.76%. At the same time, the SSCs of other species in Scorpaeniformes, such as Hwanghae Rockfish (Sebastes koreanus) and marbled rockfish (Sebastiscus marmoratus), were transplanted into black rockfish to explore the inter-species transplantation of viviparous fish. Through fluorescence tracking, it was found that the donor cells migrated to the genital ridge of the recipient at 15 dpt, with a migration efficiency of more than 60%.

第1章 引言... 1

1.1　鱼类生殖细胞的起源研究进展... 1

1.1.1　PGCs形成... 1

1.1.2　PGCs迁移... 3

1.1.3　影响PGCs迁移因素... 7

1.2　鱼类生殖干细胞... 8

1.2.1　鱼类生殖干细胞标记... 9

1.2.2　鱼类生殖干细胞分离... 10

1.2.3　鱼类生殖干细胞鉴定... 11

1.2.4　鱼类生殖干细胞冷冻保存... 11

1.3　鱼类生殖细胞移植的研究进展... 11

1.3.1　鱼类生殖细胞移植技术意义... 12

1.3.2　鱼类生殖细胞移植技术发展... 13

1.3.3　鱼类生殖细胞移植技术类型... 16

1.3.4　鱼类生殖细胞移植技术问题与展望... 21

1.4　本研究的目的及意义... 22

第2章 海洋经济鱼类生殖细胞的起源及标记研究... 25

2.1 研究背景... 25

2.2 材料与方法... 27

2.2.1　实验材料... 27

2.2.2　主要试剂和仪器... 27

2.2.3　序列分析... 28

2.2.4　克隆nanos3/vasa 3’UTR序列... 28

2.2.5　胚胎整体原位杂交... 30

2.2.6　构建融合载体及合成嵌合mRNA.. 31

2.2.7　显微注射和荧光观察... 34

2.3　结果... 35

2.3.1　鱼类Vasa蛋白的结构和系统发育分析... 35

2.3.2　许氏平鲉生殖细胞起源... 37

2.3.3　许氏平鲉vasa 3’UTR定位功能验证与分析... 38

2.3.4　硬骨鱼类nanos3 3’UTR的分离与生物信息学分析... 43

2.3.5　通过nanos3 3’UTR介导荧光蛋白体内可视化大菱鲆PGCs. 45

2.3.6　四种鱼类vasa 3’UTR分子克隆和序列分析... 47

2.3.7　四种鱼vasa 3’UTR标记大菱鲆PGCs能力... 49

2.4　讨论... 51

第3章　鲽形目鱼类精原干细胞移植... 57

3.1　研究背景... 57

3.2 材料与方法... 58

3.2.1　实验材料... 58

3.2.2　主要试剂和仪器... 59

3.2.3　精巢整体冷冻保存... 59

3.2.4　性腺组织学... 60

3.2.5　供体细胞的分离、鉴定和标记... 60

3.2.6　移植操作... 61

3.2.7　荧光定期观察供体来源的生殖细胞... 61

3.2.8　切片原位杂交... 62

3.2.9　免疫组化... 62

3.2.10 扫描电镜观察受体产生精子形态... 62

3.2.11 PCR检测供体来源的精子或后代... 62

3.2.12 亲子鉴定... 63

3.3 结果... 64

3.3.1　分离、鉴定和标记供体细胞... 64

3.3.2　评估受体中移植的供体细胞的迁移和定殖率... 66

3.3.3　分析移植后受体的性腺发育和性别分化... 68

3.3.4　检测移植受体中供体细胞的来源和增殖... 72

3.3.5　鉴定成熟受体产生供体来源的精子或后代... 74

3.4 讨论... 78

第4章　鲉形目鱼类精原干细胞移植... 81

4.1 研究背景... 81

4.2 材料与方法... 82

4.2.1　实验材料... 82

4.2.2　主要试剂和仪器... 82

4.2.3　切片原位杂交... 83

4.2.4　许氏平鲉精巢整体冷冻保存... 83

4.2.5　性腺解剖组织学... 83

4.2.6　供体细胞的分离和标记... 83

4.2.7　移植操作... 83

4.2.8　荧光定期观察供体来源的生殖细胞... 83

4.2.9　微卫星鉴定嵌合比率... 84

4.3 结果... 84

4.3.1　许氏平鲉性腺形成和发育... 84

4.3.2　许氏平鲉精原干细胞同种移植... 89

4.3.3　鲉科鱼类精原干细胞种间移植... 95

4.4 讨论... 96

第5章　结论与展望... 99

5.1 研究结论... 99

5.2 展望... 100

参考文献... 101

附　录... 113

作者简历及攻读学位期间发表的学术论文与研究成果... 117