Fine selection of up-regulated genes during ovulation by in vivo induction of oocyte maturation and ovulation in zebrafish.
Fine selection of up-regulated genes during ovulation by in vivo induction of oocyte maturation and ovulation in zebrafish.
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DOI：
10.1186/s40851-017-0065-8
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发表时间：
2017
影响因子：
2.7
通讯作者：
Tokumoto T
中科院分区：
文献类型：
--
作者：
Klangnurak W;Tokumoto T
卵母细胞成熟和排卵这两个重要过程是独立诱导的，但作为卵母细胞可受精之前卵子发生的最后一步，它们协同进行。尽管这两个过程是由斑马鱼中相同的成熟诱导类固醇 17α, 20β-二羟基-4-孕烯-3-酮 (17, 20β-DHP) 诱导的，但有人认为每个过程的受体以及信号转导途径是不同的。尽管在了解诱导卵母细胞成熟的分子机制方面已经取得了很大进展，但诱导排卵的机制仍在研究中。在本研究中，使用允许在活体斑马鱼中诱导卵母细胞成熟和排卵的体内诱导技术（体内测定）来选择高度上调的基因（与排卵相关的基因）。使用体内测定，可以获得仅诱导卵母细胞成熟的卵巢组织。这首次使得区分成熟诱导基因和排卵诱导基因成为可能。使用斑马鱼序列的全基因组微阵列，比较乙醇（EtOH）处理组（非激活组）、己烯雌酚（DES）或睾酮（Tes）处理组（成熟诱导组）和17, 20β-DHP处理组（成熟和排卵诱导组）之间的基因表达水平。选择排卵特异性上调基因。通过定量聚合酶链反应（qPCR）测量所选基因的mRNA表达水平。在确定的 34 个基因中，选择了三个显示排卵特异性增加的基因作为可能与排卵相关的候选基因。 qPCR 证实了三个候选物 slc37a4a、zgc:65811 和 zgc:92184 的排卵特异性上调。我们的体内测定提供了一种精确选择与排卵相关基因的新方法。
Two essential processes, oocyte maturation and ovulation, are independently induced, but proceed cooperatively as the final step in oogenesis before oocytes become fertilizable. Although these two processes are induced by the same maturation-inducing steroid, 17α, 20β-dihydroxy-4-pregnen-3-one (17, 20β-DHP), in zebrafish, it has been suggested that the receptor, and thus the signal transduction pathway is different for each process. Although much progress has been made in understanding the molecular mechanisms underlying the induction of oocyte maturation, the mechanisms for inducing ovulation remain under investigation. In the present study, in vivo induction techniques that permit the induction of oocyte maturation and ovulation in living zebrafish (in vivo assays) were used to select highly up-regulated genes (genes associated with ovulation). Using an in vivo assay, ovarian tissues that induced only oocyte maturation could be obtained. This made it possible for the first time to distinguish maturation-inducing genes from ovulation-inducing genes. Using a genome-wide microarray of zebrafish sequences, the gene expression levels were compared among an ethanol (EtOH)-treated group (non-activated group), a diethylstilbestrol (DES)- or testosterone (Tes)-treated group (maturation-induced group), and a 17, 20β-DHP-treated group (maturation- and ovulation-induced group). Ovulation-specific up-regulated genes were selected. The mRNA expression levels of the selected genes were measured by quantitative polymerase chain reaction (qPCR). Among 34 genes identified, three that showed ovulation-specific increases were selected as candidates potentially associated with ovulation. The ovulation-specific up-regulation of three candidates, slc37a4a, zgc:65811 and zgc:92184 was confirmed by qPCR. Our in vivo assay provides a new approach to precisely select genes associated with ovulation.