Klangnurak Wanlada, Tokumoto Toshinobu
Integrated Bioscience Section, Graduate School of Science and Technology, National University Corporation Shizuoka University, Ohya 836, Suruga-ku, Shizuoka 422-8529 Japan.
Department of Biology, Faculty of Science, National University Corporation Shizuoka University, Shizuoka, 422-8529 Japan.
Zoological Lett. 2017 Feb 28;3:2. doi: 10.1186/s40851-017-0065-8. eCollection 2017.
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, induction techniques that permit the induction of oocyte maturation and ovulation in living zebrafish ( assays) were used to select highly up-regulated genes (genes associated with ovulation). Using an 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 assay provides a new approach to precisely select genes associated with ovulation.
卵母细胞成熟和排卵这两个基本过程是独立诱导的，但在卵母细胞具备受精能力之前，作为卵子发生的最后一步协同进行。尽管在斑马鱼中这两个过程是由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，但有研究表明，每个过程的受体以及信号转导途径是不同的。虽然在理解卵母细胞成熟诱导的分子机制方面已经取得了很大进展，但排卵诱导机制仍在研究中。在本研究中，使用能够在活体斑马鱼中诱导卵母细胞成熟和排卵的诱导技术（试验）来筛选高度上调的基因（与排卵相关的基因）。通过试验，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，比较了乙醇（EtOH）处理组（未激活组）、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。筛选出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。
在鉴定出的34个基因中，选择了3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。通过qPCR证实了3个候选基因slc37a4a、zgc:65811和zgc:92184的排卵特异性上调。
我们的试验为精确筛选与排卵相关的基因提供了一种新方法。
