| The Chinese mitten crab(Eriocheir sinensis), also known as the fresh water crab, is one of the important aquaculture species in China. Compared with mammals, crabs require more complex environment to induce mating and ovulation, and therefore crab has unique reproductive regulative mechanism. There often appears precocious phenomenon during crab aquaculture process, causing great losses for crab aquaculture. Therefore, to explore the molecular mechanism of gametogenesis of crab not only provides the basis for fully illustrating crab reproductive development, enriching the content of invertebrate development regulation, but also provides important theorical basis for guiding crab aquaculture. The main functions of RNA in gametes are normal regulation of gamete formation, maintenance gamete function and play a role in fertilization and embryo formation. RNA function mainly depends on its structure, particularly the secondary and tertiary structure, which is the critical factor in the exercise of their functions. DEAD-box family, a class of ATP-dependent RNA helicase, can use the energy of ATP hydrolysis to effective repair misfolded RNA structure, ensuring the proper folding of RNA. DEAD-box RNA helicase is involved in almost all processes of RNA metabolism, including transcriptional, pre-mRNA splicing, ribosome biogenesis, RNA export, translation, organelle-specific RNA metabolism, RNA decay, RNA storage and so on. Therefore, this family is closely related with the gametogenesis. In addition, vitlogenesis, the requirement of oocyte maturation, is the key of crustacean ovarian development, directly affect the quantity and quality of offspring larvae and crustacean fecundity.This study is to explore the regulatory mechanism of DEAD-box family and the related genes to gametogenesis and VTG expression.The main contents include:(1) Tissue and gonadal specific expression, and subcellular localization analyses of DDX6 in gametogenesis of E. sinensis; (2) Tissue and gonadal specific expression, and subcellular localization analyses of DDX52 in spermatogenesis of E. sinensis; (3) Regulation of FOXL2 on vitellogenin expression at ovarian mature stage of E. sinensis.The main experimental results are as follows:1. According to the testis and accessory gland transcriptome analysis in Eriocheir sinensis, the full-length cDNAs of DDX6, DDX52, FOXL2, FTZ-F1 and partial sequence of DDX20 (DDX6 full-length cDNA is 1799bp, including 1536bp ORF, encoding 512 amino acids; FOXL2 full-length cDNA is 1336bp, including 1050bp ORF, encoding 349 amino acids; FTZ-F1 ORF is 1512bp, encoding 504 amino acids; DDX52 full-length cDNA is 2225bp, including ORF 1635bp, encoding 544 amino acids; the obtained sequence of DDX20 is 1440bp, encoding 479 amino acids).2. Expression and distribution of DDX6 mRNA and protein were analyzed using qRT-PCR, western blot and IHC. It shown that DDX6 mRNA highly expressed in testis and ovary, and expression level was very low in other tissues. In testicular development process, DDX6 mRNA expression level firstly increased slightly from July and reached the peak in October, then its expression level decreased from October. During ovarian development, the expression level of DDX6 mRNA in primary oocyte was significantly higher than the level in pre-mature stage. DDX6 protein expressed in both testicular and ovarian tissues, furthermore, in the testis, DDX6 protein mainly localized in the cytoplasm of spermatogonia and primary spermatocytes, less in spermatid. In the ovary, DDX6 proteins mainly distribute in the cytoplasm of oocyte.3. Expression and distribution of DDX52 mRNA and protein were analyzed using qRT-PCR, ISH,western blot and IF. It shown that DDX52 mRNA mainly expressed in the ovary, accessory gland and testis, relatively low in hemolymph, heart, muscle, stomach and thoracic ganglia. During testicular development, DDX52 mRNA level reached the peak in August and January. DDX52 mRNA mainly localized in the nucleus of spermatogonia at spermatogonium stage, however, it seems undetectable DDX52 mRNA expression at spermatocyte stage. In the process of spermiogenesis, DDX52 mRNA predominantly diffused in the nucleus of spermatids, slightly in the acrosome cap and acrosome tubule of later spermatids. DDX52 protein mainly expressed in the cytoplasm of spermatogonia at the early spermatogenesis. In the early and middle stage of spermiogenesis, DDX52 protein highly expressed in the nucleus of spermatid, but it mainly expressed in the preacrosome vesicle and less in nucleus of spermatid in the late stage of spermiogenesis.4. The results of qRT-PCR and western blot shown that DDX20, FOXL2 and FTZ-F1 were highly expressed in ovary at both mRNA and protein levels. At ovarian mature stage, the levels of FOXl2, DDX20 and FTZ-F1 mRNA were increased gradually, but the VTG mRNA level was decreased. With the rising of Etoposide concentration, the levels of FOXL2 and DDX20 mRNA and protein in follicular cells were increased, and the interaction of FOXL2 and DDX20 proteins were demonstrated using CO-IP. In addition, with the time accumulation after unilateral eyestalk treatment, levels of FOXl2, DDX20, FTZ-F1 and VTG mRNA were increased, furthermore, the expression pattern of FOXL2, DDX20 and FTZ-F1 mRNAs were same. Finally, it demonstrated that FOXL2 interacted with FTZ-F1 through its forkhead domain using CO-IP.In a word, it researched the related genes of reproductive regulation in Eriocheir sinensis using qRT-PCR, IF, IHC, western blot and Co-IP. It has demonstrated that DDX6 was involved in the testicular and ovarian development, and mainly participated in the oocyte growth process of Eriocheir sinensis. In addition, DDX52 may be involved in the regulation of mitosis and spermiogenesis in spermatogenesis, and may play a critical role in the subsequent sperm-oocyte fusion and early embryo development. At the mature stage of E. sinensis, FOXL2 interacted with DDX20 was involved in the apoptosis of follicular cell surrounding oocyte to inhibit oocyte recruit VTG from hemolymph through follicular cell. On the other hand, FOXL2 interacted with FTZ-F1 was probably involved in the regulation of P450 aromatizing enzyme expression in the steroidogenesis, and finally prevented hepatopancreas releasing VTG, promoted by steroid hormone, by inhibiting the steroidogenesis. |
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