Conditionally induced apoptosis and ablation of transgenic zebrafish oocytes
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摘要:目的
构建针对斑马鱼Danio rerio卵细胞的可诱导消融品系，并了解其卵巢组织在条件诱导下的去除规律和特性，为相关领域的应用提供材料基础。
方法利用Tol2转座系统建立卵细胞特异表达硝基还原酶(Nitroreductase，NTR)的转基因斑马鱼品系；通过定期采样，连续观察、解剖、组织切片等方法研究甲硝唑(Metronidazole，Mtz)对成熟雌鱼外观及卵巢结构的影响；冰冻切片结合TUNEL检测，了解条件性诱导对卵巢细胞凋亡的影响。
结果获得囊胚细胞具特异荧光标记的转基因鱼，并形成稳定遗传品系。经Mtz诱导，该品系成熟雌鱼卵巢结构逐步退化、萎缩，卵母细胞渐渐消融，提示NTR在卵细胞特异表达；检测发现Mtz可导致卵细胞的凋亡，推测卵巢的组织变化为细胞凋亡所致；撤销Mtz胁迫卵巢可再生并恢复生育功能，说明卵巢的消融过程是可逆的。
结论获得的转基因斑马鱼可通过条件诱导实现卵巢消融与再生。
Abstract:ObjectiveTo develop a transgenic zebrafish, Danio rerio, line in which oocyte ablation can be conditionally induced, study the characteristics of oocyte ablation, and provide materials for future reproduction studies.
MethodThe Tol2 transposon system was used to generate a transgenic zebrafish line which expressed nitroreductase (NTR) enzyme under control of the oocyte-specific gene promoter. The effects of metronidazole (Mtz) on appearance of mature females and internal ovary structure were evaluated by regular sampling, continuous observation, dissection and tissue sectioning. Frozen sections stained by TUNEL were used to detect the induced oocyte apoptosis.
ResultA transgenic fish line with stable inheritance was obtained and its blastula cells showed specific mCherry fluorescence. Induced by Mtz, the transgenic mature female ovary gradually degenerated and atrophied, and the oocytes progressively ablated. The results indicated NTR expression in the oocytes. TUNEL detection confirmed that Mtz could induce oocyte apoptosis, leading to histological changes in the ovary. Furthermore, the ovarian ablation induced by Mtz was reversible. When the Mtz stress was withdrawn, the oocytes regenerated and the ovary restored its function.
ConclusionThe transgenic zebrafish obtained in this study can implement ovarian ablation and regeneration through conditional induction.
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图 1 转基因鱼质粒构建及荧光观察
A：转基因表达元件；B：Tol2转基因表达载体；C、D分别为转基因F1代囊胚期自然光与荧光下图片; E、F分别为转基因F1代性成熟卵巢自然光与荧光下图片；标尺=50 μm。
Figure 1. Transgenic fish plasmid construction and fluorescence observation
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图 2 转基因斑马鱼卵巢消融与再生的外观及解剖图
A：Tg(zpc:nfsB-mCherry)鱼处理前；B、C、D分别为Tg(zpc:nfsB-mCherry)鱼浸泡5 mmol·L-1 Mtz 4、8、12 d；E、F分别为Tg(zpc:nfsB-mCherry)鱼撤销Mtz恢复10、20 d；G：野生型鱼浸泡5 mmol·L-1 Mtz 12 d。
Figure 2. External appearance and internal ovarian morphology of Tg(zpc:nfsB-mCherry) female during the process of ovarian ablation and regeneration
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图 3 转基因斑马鱼卵巢消融的石蜡切片H&E染色
Ⅱ、Ⅲ、Ⅳ分别代表第Ⅱ、Ⅲ、Ⅳ时相卵母细胞; A为Tg(zpc:nfsB-mCherry)鱼Mtz处理前；B、C、D分别为Tg(zpc:nfsB-mCherry)鱼浸泡5 mmol·L-1 Mtz 4、8、12 d；标尺=100 μm。
Figure 3. Images of Tg(zpc:nfsB-mCherry) female ovarian paraffin sections with H&E staining during ovarian ablation
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图 4 转基因斑马鱼Mtz (5 mmol·L-1)处理1 d卵巢的TUNEL检测
A、B分别为野生型鱼卵巢自然光、荧光图片；C、D分别为Tg(zpc:nfsB-mCherry)鱼卵巢自然光、荧光图片；标尺=100 μm。
Figure 4. TUNEL detection of Tg(zpc:nfsB-mCherry) female ovarian after 1 d exposure to Mtz (5 mmol·L-1)
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