The Expression Of Z-OTU Protein During Zebrafish Oogenesis And Early Embryogenesis

Zebrafish (Danio rerio) is an important model for research of vertebrate ondevelopmental biology and genetics, and more and more researchers pay attention to theregulation mechanism of zebrafish germ cell oogenesis and early embryogenesis.Recently, a few study showed that ubiquitin-proteasome pathway (UPP) is involved innumerous important biological processes, including mammalian oogenesis and earlyembryonic development, such as germinal vesicle breakdown (GVBD) of oocyte, theextrusion of polar body, the prevention and breakthrough of meiotic progression to MПstage and so on. The UPP is an efficient pathway for protein degradation in the vertebratecell and involved in numerous important biological processes, including cell cycles,embryonic development, growth control, apoptosis and neurodegeneration and so on. Theabnormalities of the UPP may cause many diseases. Deubiqitinating enzymes (DUBs) isone of the important component element of UPP and play important roles in regulatingthe UPP. At present, there was no any report about DUBs in zebrafish.Our research showed that the translation product of zebrafish z-otu gene maybe havethe activity of DUB, because the predicted protein contains one typical otu domain and isone member of otu-like cysteine proteinase family. The z-otu was a gene speciallyexpressed in zebrafish ovary and in the early stages of oogenesis and embryogenesis. Inearly oocyte (the oogonium or oocyte that has just entered into meiotic phase and stageⅠ-Ⅲoocyte),the z-otu mRNA was expressed and its expression level was graduallydecreased; no significant signal was observed in stageⅣand stageⅤoocyte; z-otumRNA was also primarily expressed in embryo from zygote period to early- blastulastage while it was disappeared from late-blastula stage to 5dpf (not include 5dpf)。Theanalysis of bioinformation indicated that the Z-OTU was a nuclear protein whichcontaining a transmembrane region located in the region of 491-514 amino acids, and itsN-terminal was existed outside of the nuclear membrane; Z-OTU has no signal peptideand may be a nonsecreted protein.In order to further investigate the function of Z-OTU protein, the specific primerswere designed to amplify the DNA sequence (otu1) of otu domain using PCR. Then theotul fragment was inserted into pGEX-6p-1 vector and the ligated products weretransformed into the BL21 of E.coli. The coding region of constructed recombinantplasmid pGEX-6p-1-otu1 was verified by PCR amplification, digestion of restrictionendonuclease and DNA sequencing, then we obtained the correct recombinant plasmid forprocaryotic expression. After induction by IPTG and checked with SDS-PAGE, we obtained a new about 42kD protein band and its size was same to the expected proteinband. The protein gained by prokaryotic expression was purified and then was injected inrabbits to obtain the polyclonal antibody—anti-Z-OTU, which was used forimmunohistochemistry in zebrafish ovary and embryo.The immunohistochemistral results of zebrafish ovary showed that Z-OTU proteinwas located in germinal vesicle (GV) in the primary growth phase (stageⅠ), while it wasnot detected in cytoplasm; in the stageⅡand the stageⅢoocyte, Z-OTU proteinmigrated to the cytoplasm and mainly distributed in the perinuclear, cytoplasm andcellular membrane, but it did not present in egg yolk and cortical alveolus; at last, Z-OTUprotein mainly concentrated at near vegetative pole of GV, and migrated with GV to thefuture animal pole during the following development.The results of whole mount fluorescence immunohistochemistry indicated that thedistribution of Z-OTU protein was changed in zebrafish embryo of early developmentalstages. After fertilization, Z-OTU protein was located in cytoplasm and flowed withcytoplasm; with the further development of embryo, Z-OTU was uniformly distributed inevery cell during cleavage period, but was mainly located in region where forming somiteafter midblastula transition (MBT). The expression level of Z-OTU protein was alsovaried in zebrafish embryo of early developmental stage. The positive signal of Z-OTUprotein in 30% epiboly stage obviously weak than that in 1000 cell stage; The expressionlevel of Z-OTU protein was gradually decreased during the segmentation period, but itwas not present after 13-somite stage. The expression patterns of Z-OTU in zebrafishoocyte and embryo of early developmental stages suggested that Z-OTU protein isrequired in the meiosis of oocyte, fertilization and early stages of embryogenesis.We analyzed and compared the expression patterns of mRNA and protein of z-otugene, the result suggested that there existed some difference in spatiotemporal expressionbetween the two: z-otu mRNA only expressed during the early developmental stage ofzebrafish embryo, and it appear again after fertilization, while the protein of Z-OTUmaintained expression during the whole stages of zebrafish oogenesis; During the processof oogenesis, the z-otu mRNA distributed in cytoplasm, but Z-OTU protein first existed innuclear, then migrated to cytoplasm, at last it concentrate at the periphery of GV。...