实验海洋生物学重点实验室知识库

生殖细胞移植（Germ cell transplantation，GCT）是一项将供体生殖干细胞移植到不育受体中，从而使受体产生供体后代的技术，在保护濒危鱼类、种质遗传改良等方面具有广阔的应用前景，越来越受到研究者的重视。然而，在经济鱼类GCT研究中，难以获得高纯度的供体生殖干细胞，以及对外源干细胞在受体内增殖、分化及发育的基础认知不足等问题常常导致移植效率低甚至移植失败。大菱鲆（Scophthalmus maximus）和牙鲆（Paralichthys olivaceus）同属鲽形目，都是我国沿海重要的海洋经济养殖鱼种。本研究以大菱鲆和牙鲆为研究对象，探索了大菱鲆精原干细胞（Spermatogonial stem cell，SSC）的体外培养条件，并通过单细胞测序（Single cell RNA-seq，scRNA-seq）对大菱鲆精巢内调控SSC自我更新和分化的重要信号通路和潜在的分子标记开展了系统分析。同时还研究了白消安对牙鲆精巢生殖细胞的消除作用，并确定了使用白消安制备成年牙鲆不育受体的最佳条件。研究结果如下：

1.大菱鲆SSC体外培养

将percoll密度梯度离心纯化后的大菱鲆SSC接种到基础培养基、FSH培养基、GDNF培养基和FSH&GDNF培养基中培养一周，发现只有添加GDNF的后两种培养基能保留更多的SSC。实时荧光定量的结果进一步证明GDNF存在时培养基中的细胞vasa表达量高于其他培养基。同时，FSH和GDNF还能促进体细胞更快地增殖。将经过纯化的大菱鲆SSC接种到基础培养基和鱼血清培养基后，通过VASA/ EdU免疫荧光染色发现第42天时两种培养基中均检测到双阳性的细胞，说明此时生殖细胞仍在增殖。然而，基础培养基无法维持SSC长期存活，鱼血清培养基中生殖细胞则可持续存活超5个月，但数量极少，仅能通过VASA抗体免疫荧光染色证明其存在。

2. scRNA-seq分析揭示大菱鲆SSC自我更新与分化的关键调控网络和各时期生殖细胞基因表达特征

通过scRNA-seq对大菱鲆幼鱼精巢中15469个细胞进行分析，鉴定出9个体细胞群和1个生殖细胞群。通过CellChat分析了不同细胞群之间的细胞通讯作用，共富集到17条信号通路。结果显示GDNF通路在大菱鲆精巢内具有自分泌和旁分泌作用途径，负责调控SSC自我更新；RA通路和FGF信号通路都与SSC分化相关，而且RA通路内关键基因的表达情况比哺乳动物更加复杂，不仅具有多种体细胞来源，还具有生殖细胞表达。PTN通路则主要控制精原细胞分化，同样具有自分泌途径。对生殖细胞群细分显示，该类群细胞可进一步分为2个SSC细胞群、3个不同发育时期的精原细胞群和1个精母细胞群，并通过拟时序分析揭示了不同发育状态SPG的分化轨迹和基因转录表达动态特征。此外，还发现nanos2、gfrα1和id4等基因标记SSC的功能在哺乳动物和大菱鲆上是保守的，首次发现dnmt3bb.1和grb7为大菱鲆SSC的标记基因。

3.白消安制备成年牙鲆不育受体

设置四个白消安浓度，包括40mg/kg、60mg/kg、80mg/kg和120mg/kg，对即将进入繁殖期的成年雄性牙鲆进行腹腔注射。组织学的结果显示，不经注射的对照组牙鲆进入繁殖盛期，精巢中充满精子，而同时期注射过白消安的各实验组牙鲆精巢显示出不同程度的退化，其中60mg/kg实验组牙鲆精巢体积明显缩小，精巢内小叶腔消失，无精子残留，只有精原细胞存在，说明该浓度对繁殖期成年雄性牙鲆精巢生殖细胞具有最好的消除效果。VASA抗体免疫荧光染色和实时荧光定量结果显示60mg/kg处理的牙鲆精巢中精原细胞最多，vasa表达量最高，证明该浓度处理得到的牙鲆精巢适宜精原细胞存活，能够作为GCT的不育受体。

Germ cell transplantation (GCT) involves transplanting germline stem cells from donors into sterile recipients, enabling the recipients to produce offsprings with the donor's genetic characteristics. This technology holds significant promise for conserving endangered fish species and genetic improvement of germplasm. Consequently, it has garnered increasing attention from researchers due to its potential for broad developmental applications. However, in economic fish GCT, it is difficult to obtain donor germline stem cells with high purity, and the lack of basic understanding of the proliferation, differentiation and development of donor cells in recipients often lead to low efficiency of GCT and even failure. Turbot (Scophthalmus maximus) and Japanese flounder (Paralichthys olivaceus), both belonging to Pleuronectiformes, are important marine economic fish species in coastal areas of China. In this study, the suitable conditions for in vitro culture of turbot spermatogonial stem cells (SSC) were explored, and the important signaling pathways and potential molecular markers regulating SSC self-renewal and differentiation of turbot were analyzed by single-cell RNA sequencing (scRNA-seq). Meanwhile, the elimination effect of busulfan on the germ cells of Japanese flounder was studied, and the optimal concentration of busulfan to prepare the sterile recipients of adult Japanese flounder was found. The results were as follows:

1.In vitro culture of turbot SSC

The turbot SSC were purified by percoll density gradient centrifugation and introduced into basic medium, FSH medium, GDNF medium, and FSH& GDNF medium for one week, and it was found that only the latter two media with GDNF could retain more SSC. The results of real-time quantification PCR (qPCR) additionally demonstrated that the vasa expression of cells in the medium supplemented with GDNF exceeded that observed in other media. Meanwhile, FSH and GDNF were found to promote the proliferation of somatic cells. Following inoculation of purified SSC into basic medium and fish serum medium, VASA/EdU immunofluorescence staining (IF) revealed the presence of double-positive cells in both media on day 42, indicating the proliferation of germ cells at this stage. However, while the basic medium failed to sustain the long-term survival of SSC, germ cells in the fish serum medium persisted for over 5 months. Nonetheless, their numbers were diminished, and their presence could only be confirmed by VASA antibody IF.

2. The scRNA-seq analysis revealed the key regulatory networks of SSC self-renewal and differentiation and gene expression characteristics of turbot germ cells at different periods

A total of 15,469 cells from juvenile turbot testes were analyzed by scRNA-seq, obtaining nine somatic cell populations and one germ cell population. Cell communication between different cell populations was analyzed through CellChat, and 17 signaling pathways were enriched. The results showed that in turbot GDNF pathway operated through both autocrine and paracine mechanisms, regulating the SSC self-renewal; RA pathway and FGF signaling pathway were associated wih turbot SSC differentiation, and the expression patterns of key genes in RA pathway were more complicated compared to mammals, with not noly multiple somatic cells origins, but also germ cells expression. PTN was related to spermatogonia (SPG) differentiation and it also had an autocrine pathway. Subsequently, the germ cell population was re-clustered and obtained two SSC clusters, 3 SPG clusters at different developmental stages and 1 spermatocyte cluster. Pseudo-time series analysis was used to reveal the differentiation trajectories and dynamic characteristics of gene transcription and expression of SPG in different developmental states. Besides, it was found that the functions of SSC marker genes such as nanos2, gfrα1 and id4 were conserved in mammals and turbot. Furthermore, dnmt3bb.1 and grb7 were identified as novel SSC marker genes in turbot for the first time.

3.Preparation of sterile recipients in adult Japanese flounder using busulfan

Four distinct injection concentrations (40mg/kg, 60mg/kg, 80mg/kg, and 120mg/kg) were established for the intraperitoneal injection of adult male Japanese flounders approaching the breeding season. The histological examination revealed that the control group, which received no injection, progressed into the fertile stage with testes containing abundant sperm. In contrast, the testes from experimental groups injected with busulfan exhibited varying degrees of degradation. Notably, the testes of 60mg/kg experimental group displayed a significant reduction, with the disappearance of lobular cavities and complete absence of sperm. Only SPG were observed in the testes. These results suggested that 60mg/kg of busulfan had the best effect on eliminating germ cells in adult male Japanese flounders during the breeding period. Additionally, the results of VASA antibody IF and qPCR revealed that the group treated with 60mg/kg had most SPG and the highest expression of vasa in testes. This evidence supported that the testes of Japanese flounders injected with 60mg/kg busulfan were suitable for the survival of SPG. These Japanese flounders could be used as sterile  recipients for GCT.

目 录

第1章 引言

1.1 鱼类GCT研究背景

1.2 鱼类精原干细胞体外培养

1.3 SSC增殖分化调控研究

1.3.1 SSC自我更新的分子调控研究

1.3.2 SSC分化的分子调控研究

1.4 鱼类GCT不育受体研究

1.5 研究目的及意义

第2章 大菱鲆SSC分离纯化及体外培养研究

2.1 研究背景

2.2 材料与方法

2.2.1 实验材料制备

2.2.2 细胞悬液制备

2.2.3 细胞纯化与体外培养

2.2.4 精巢与细胞RNA提取

2.2.5 半定量PCR分析

2.2.6 qPCR分析

2.2.7 细胞滴片的制备

2.2.8 石蜡包埋组织切片和HE染色

2.2.9 免疫荧光染色和EdU染色

2.3 实验结果

2.3.1 大菱鲆幼鱼和成鱼生殖细胞类型差异

2.3.2 不同细胞因子对短期体外培养大菱鲆SSC的影响

2.3.3 大菱鲆SSC体外长期培养

2.4 讨论

第3章 单细胞测序揭示大菱鲆SSC自我更新与分化的关键调控通路以及各时期生殖细胞基因表达特征

3.1 研究背景

3.2 材料与方法

3.2.1 样品制备和单细胞测序

3.2.2 基因组对比和基因表达定量

3.2.3 细胞聚类分析

3.2.4 细胞注释及拟时间轨迹分析

3.2.5 通过CellChat分析不同细胞类型之间的关系

3.2.6 HE染色组织学切片

3.3 结果

3.3.1 大菱鲆幼鱼精巢的单细胞转录组分析

3.3.2 大菱鲆精巢细胞间通信网络

3.3.3 关键信号通路的分析

3.3.4 生殖细胞再分群分析

3.3.5 生殖细胞相关标记基因表达分析

3.4 讨论

3.4.1 调控大菱鲆SSC自我更新与分化的关键信号通路分析

3.4.2 不同类型生殖细胞基因表达特征分析及关键标记基因的探索

第4章 白消安制备成年牙鲆不育受体

4.1 研究背景

4.2 材料与方法

4.2.1 实验材料

4.2.2 实验方法

4.2.3 组织学染色观察

4.2.4 免疫荧光染色与qPCR鉴定

4.2.5 数据分析

4.3 结果

4.3.1 白消安处理对牙鲆体重和GSI影响

4.3.2 不同浓度白消安对牙鲆精巢生殖细胞的影响

4.3.3 不同浓度白消安对牙鲆生殖细胞消除效果的鉴定

4.4 讨论

第5章 结论与展望

5.1 研究结论

5.2 展望

参考文献

致谢

作者简历及攻读学位期间发表的学术论文与其他相关学术成果

图目录

图1-1 鱼类GCT流程示意图

图1-2 成年小鼠未分化精原细胞调控示意图（Makela&Hobbs, 2019）

图1-3 GDNF在调节SSC自我更新中的作用示意图（Wei et al., 2022）

图1-4 鱼类GCT类型

图2-1 大菱鲆幼鱼和成鱼精巢差异

图2-2 不同细胞因子对体外培养大菱鲆SSC的影响

图2-3 大菱鲆SSC体外长期培养

图3-1 大菱鲆幼鱼精巢单细胞转录组分析

图3-2 大菱鲆精巢内细胞间信号通讯网络

图3-3 生殖细胞和体细胞之间的关键信号通路

图3-4 大菱鲆幼鱼生殖细胞再分群及拟时间分析

图3-5 大菱鲆不同类型生殖细胞标记基因表达情况

图4-1 不同浓度白消安对牙鲆体重和GSI影响

图4-2 不同浓度白消安对牙鲆生殖细胞的影响

图4-3 不同浓度白消安对牙鲆精原细胞的影响

图4-4 不同白消安浓度处理下牙鲆精巢vasa和amh的基因表达

表目录

表2-1 大菱鲆半定量实验所用引物名称、序列及Tm值

表2-2 大菱鲆实时荧光定量实验所用引物名称、序列及Tm值

表3-1 过滤前后各个样本中细胞数据量统计表

表3-2 生殖细胞再分群后各细胞群表达量Top 30的DEGs

表4-1 牙鲆实时荧光定量实验所用引物名称、序列及Tm值