斑马鱼Z_OTU蛋白在卵子发生和胚胎发育早期的表达_英文_

斑马鱼Z_OTU蛋白在卵子发生和胚胎发育早期的达_英文_ 动 物 学 研 究 2011，Aug. 32(4): 386?390 CN 53-1040/Q ISSN 0254-5853 DOI:10.3724/SP.J.1141.2011.04386 Zoological Research Identification of Z-OTU protein during zebrafish oogenesis and early embryogenesis 1,323,*1,*YUE Ming-Jin, MO Sai-Jun, SONG Ping, GONG Yan-Zhang (1. College of Animal Science, Huazhong Agricultural University, Wuhan 430070, China; 2. College of Basic Medical, Zhengzhou University, Zhengzhou 450001, China; 3. Laboratory of Molecular Genetics and Developmental Biology, Wuhan University, Wuhan 430072, China) Abstract: Zebrafish (Danio rerio) Z-OTU, containing OTU and TUDOR domains, was predicted to be a member of OTU-related protease, a family of the deubiquitylating enzymes (DUBs). A previous report from our laboratory clearly describes the expression patterns of z-otu mRNA. Here, we characterized the Z-OTU protein during zebrafish oogenesis and early embryogenesis. After prokaryotic expression, the recombinant protein of the OTU domain and GST was purified and injected into rabbits to obtain the polyclonal antibody-anti-Z-OTU, which was used for immunohistochemistry in zebrafish ovaries and embryos. Interestingly, obvious differences existed between the expression patterns of z-otu mRNA and its protein during oogenesis and early embryogenesis. In stage I oocytes, z-otu mRNA was detected in cytoplasm while its protein existed in the germinal vesicle. In addition, its protein was distributed during entire oogenesis, while mRNA was not detected in oocytes at stage IV or mature oocytes. The z-otu mRNA disappeared after midblastula transition (MBT) and its protein gradually decreased after this stage. We inferred that Z- OTU protein, like other OTU-related protease with DUB activity, was required for germinal vesicle breakdown of oocytes during meiosis, germinal vesicle migration, and embryo cleavage maintenance. Key words: Danio rerio; OTU-related protease; DUB; Z-OTU protein; Oogenesis; Embryogenesis 斑马鱼 Z-OTU 蛋白在卵子发生和胚胎发育早期的表达 1,323,*1,* 岳明金 , 莫赛军 , 宋平 , 龚炎长 (1. 华中农业大学 动物科技学院, 湖北 武汉 430070; 2. 郑州大学 基础医学院, 河南 郑州 450001; 3. 武汉大学 生命科学学院分子遗传与发育实验室, 湖北 武汉 430072) 摘要:斑马鱼 z-otu 基因编码的蛋白可能具有 DUBs 活性, 它包含 OTU 和 TUDOR 结构域, 属于 OTU 相关蛋 白酶家族的成员。该研究将原核表达的融合蛋白(OTU 结构域和 GST)纯化后免疫新西兰兔, 获得多克隆抗体 anti- Z-OTU, 并利用该抗体对 Z-OTU 蛋白质在斑马鱼卵子发生和早期胚胎发育过程中的表达进行了分析。根据原位 和整体免疫组织化学检测结果并结合以前的研究结论, 分析并比较了 z-otu 基因的 mRNA 和蛋白质的分布, 发现在 卵子发生和早期胚胎发育过程中, z-otu 基因的 mRNA 和蛋白质表达模式存在明显差异:mRNA 仅在卵子发生早 期表达, 卵母细胞受精后才重新开始表达, 而其蛋白在卵子发生过程中均表达; 在卵子发生过程中, mRNA 分布于 细胞质中, 而蛋白质先分布于细胞核中, 然后向细胞质迁移, 接着又向卵母细胞生发泡(germinal vesicle, GV)集中。 推测 Z-OTU 蛋白类似于其他具有去泛素化酶活性的 OTU 相关蛋白酶, 对于卵母细胞减数分裂过程中生发泡破 裂、生发泡迁移及维持胚胎的分裂是必需的。 关键词:斑马鱼; OTU 相关蛋白酶; 去泛素化酶; Z-OTU; 卵子发生; 胚胎发育 中图分类号:Q593.4; Q959.468; Q954.432 文献标志码:A 文章编号:0254-5853-(2011)04-0386-05 Received date: 2010-11-08; Accepted date: 2011-04-27 Foundation items: This work was supported by grants from the ICGEB (International Center for Genetic Engineering and Biotechnology) (CRP/CHN02- 01) (SONG Ping); the National Basic Research Program of China (2004CB117400) (SONG Ping); the National Natural Science Foundation of China (30150005; 30270675) (SONG Ping) * Corresponding authors (通信作者), E-mail: pingsongps@yahoo.com.cn;gong7546@public.wh.hb.cn 收稿日期:2010- 11-08;接受日期:2011-04-27 第一作者简介:岳明金，男，硕士研究生，主要从事动物遗传育种与繁殖研究;E-mail:ymjmsj0618@yahoo.com.cn The ubiquitin-proteasome pathway (UPP) is GG-3') and OR (5'-ATCCTCAACTCCCAGAACACG- important during multiple steps of oocyte meiosis, 3') (Fig.1) with an EcoRI or an XhoI restriction site, fertilization, and early embryonic mitosis. Although respectively. The fragments (otu1) were cloned into pGEX-6p-1 vector and expressed in the prokaryotic numerous mammalian UPP studies have been conducted system. The recombinant protein of the OTU domain and (Ben-Yehoshua Josefsberg et al, 2001; Huo et al, 2004; GST was purified and injected into rabbits to obtain the Sun et al, 2004), understanding of roles and regulatory polyclonal antibody–anti-Z-OTU. The anti-Z-OTU titer mechanisms of UPP in zebrafish remains unclear. in the serum and the antibody specificity were then Deubiquitylating enzymes (DUBs) are important for checked by western blot analysis (Yang et al, 2010). The generating free ubiquitin at various steps of the UPP, for immune and pre-immune sera (obtained before processing inactive ubiquitin precursors, proofreading stimulation) were stored in aliquots at –20?C until use. ubiquitin–protein conjugates, removing ubiquitin from cellular adducts, and keeping 26S proteasome free of inhibitory ubiquitin chains (Wing, 2003). The OTU-related proteases are members of DUBs and belong to an OTU-like super-family that contains the ovarian tumor domain (OTU). This conserved motif encodes for a presumed catalytic core domain containing conserved Cys, His, and Asp residues (Makarova et al, Fig. 1 Locations of primers for amplifying otu1 and OTU 2000). Several OTU-related proteases have been identified. domain alignments of Z-OTU, VCIP135 (Uchiyama For example, Cezanne (Evans et al, 2003), the first et al, 2002), Cezanne (Evans et al, 2003), A20 demonstration of proteolytic activity in OTU proteins, (Opipari et al, 1992) and duo-2 (Sieburth et al, 2005) can cleave ubiquitin monomers from linear or branched OF: otu1 forward primer; OR: otu1 reverse primer; CYS BOX: synthetic ubiquitin chains and ubiquitinated proteins. Cysteine region; HIS BOX: Histidine region. Balakirev et al (2003) observed that the OTU domain of otubains contained an active cysteine protease site. While 1.2 Immunohistochemistry A20, another OTU-related protease containing an N- Ovaries from adult zebrafish were collected and terminal OTU domain, can cleave ubiquitin monomers embedded in paraffin, then 4 µm sections were cut and from branched polyubiquitin chains linked through treated with 0.3% HOin 50 mM PBS for 30 min after 22 Lys48 or Lys63 and bound covalently to a thiol-group dewaxation and rinsed extensively with PBS. These reactive ubiquitin-derived probe, with mutation of sections were then treated with normal non-immune conserved cysteine residue in the catalytic site (Cys103) serum. All antibodies were diluted with 50 mM PBS able to abolish these activities (Opipari et al, 1992; containing 0.05% Triton X-100 and 5% normal goat Evans et al, 2004). serum. Sections were incubated with anti-Z-OTU (1:200) The Z-OTU protein analyzed in this study belongs overnight at 4?C, rinsed with PBS and incubated in to the OTU-like super-family and may exhibit activity of Biotin-conjugated second antibody for 15 minutes at DUBs for containing an OTU domain (Mo et al, 2005). room temperature, rinsed again with PBS, incubated in Our previous work also determined that the zebrafish streptavidin-peroxidase for 15 minutes at room ovary-specific gene was expressed at early stages of temperature, rinsed with PBS, and incubated with DAB oogenesis and embryogenesis (Mo et al, 2005). In this study, solution (Maixin Company, Fujian, China) for 5 minutes. we investigated the distribution of the Z-OTU protein at Following staining and rinsing with PBS, the sections different stages of zebrafish oogenesis, fertilization and were stained with hematoxylin and mounted onto glass early embryogenesis using immunohistochemistry slides with cover slips and viewed with an Olympus and whole mount immunohistochemistry. vanox microscope. As a negative control, the first antibody was replaced in diluted preimmune serum. 1 Materials and Methods 1.3 Whole-mount immunohistochemistry 1.1 Preparation of polyclonal anti-Z-OTU antibody The protocol of whole-mount immunohistochemistry was performed as per Braat et al (2000) with a little DNA fragments of the OTU domain were amplified using primers of OF (5'-AGCATGGACGAGTACCT modification: Embryos were fixed in 4% paraformaldehyde 388 Zoological Research Vol. 32 overnight at 4?C, washed four times for 15 min each in PBT (1% Tween-20 in PBS) and blocked overnight in block buffer (PBT containing 10% heat inactivated sheep serum) at 4?C. Anti-Z-OTU antibodies were diluted in block buffer (1:200) and preabsorbed overnight at 4?C against aceton powder of adult fish from which the gonads had been removed. After discarding the block buffer and adding the antibody, the embryos were incubated overnight at 4?C. After washing in PBT (ten times for 10 min each at room temperature), the embryos were incubated in anti-rabbit IgG-FITC antibody produced in sheep (sigma) diluted in block buffer (1:60) overnight at 4?C. After washing in PBT, the embryos were analyzed on a fluorescence microscope (Olympus). 2 Results Fig. 3 Immunohistochemical staining of Z-OTU To test Z-OTU protein expression during oogenesis, we protein in zebrafish oocyte Ovarian sections were treated with rabbit anti-Z-OTU antibody (A - C) (×40, generated anti-Z-OTU antibodies for immunohistochemistry ×40, and ×100, respectively) or pre-immune antiserum (D and E) (×40). F to examine the localization of Z-OTU in oocytes and (×40): H & E stain. IA: the prefollicle stage of the primary growth phase; IB: embryos. To determine Z-OTU antibody specificity, the follicle stage of the primary growth phase; II: the cortical alveolus stage; western blot analysis showed that Z-OTU antiserum III: the vitellogenesis stage; IV: oocyte maturation stage. Black arrowheads recognized zebrafish Z-OTU (Fig. 2). show germinal vesicle (GV); Red arrows indicate the regions of positive signal of Z-OTU; Blue arrows indicate the cortical alveolus. In zebrafish, egg activation and fertilization can initiate cytoplasmic streaming towards the animal pole, which is thought to result in the movement of determinants to the blastoderm (Jesuthasan and Strahle, 1997; Oppenheimer, 1936). To study the role of Z-OTU Fig. 2 Results of Z-OTU antibody specificity test during this process, we examined its expression in and western blotting analyses mature oocytes prior to and after egg activation. The The antiserum of Z-OTU recognized a signal in zebrafish ovaries (lane 1) results showed that Z-OTU was distributed uniformly and two-cell embryos (Lane 2); the control used was pre-immune sera (Lane 3through the oocyte cytoplasm in inactivated mature 3). The molecular weight of Z-OTU was 96.5 X 10. Equal loading was observed with β-actin antibody. oocytes (Fig. 4A). Upon activation with egg water, however, a dynamic redistribution of Z-OTU was Our data revealed that Z-OTU protein existed in detected (Fig. 4B - E): at 3 min after fertilization, Z-OTU oocytes throughout oogenesis in adult zebrafish ovaries. aggregated in clusters throughout the yolk (arrow in Fig. In stage I oocytes, the positive signal of Z-OTU was 4B), and a positive signal was also detected in the localized in the germinal vesicle (GV) (Fig. 3A); in stage emerging blastoderm (arrowhead in Fig. 4B); at 10 min II and stage III oocytes, the Z-OTU protein was present post-activation, Z-OTU was detected in the yolk cell as in the perinuclear region, cytoplasm, and cell membrane, and the signal was very obvious (red arrow in Fig. 3B, C). aggregates and in the blastoderm (arrowhead in Fig. 4C); Interestingly, most Z-OTU protein was concentrated in at 20 min post-activation, the aggregates in the yolk the perinuclear cytoplasm of the vegetative pole of the became larger (arrow in Fig. 4D) and were closer to the GV at stage IV oocytes (red arrow in Fig. 3C), while blastoderm (arrowhead in Fig. 4D); and at 30 min post- little signal uniformly existed in other cytoplasm and cell activation, Z-OTU was detected exclusively in the membranes. Our results also showed that anti-Z-OTU blastoderm and excluded from the yolk cell (Fig. 4E). staining was absent in the cortical alveolus (blue arrow in Our results also indicated that the positive signal of Z- Fig.3B). The pre-immune rabbit serum did not generate a OTU was highlighted from 1-cell to 1000-cell stage positive signal (Fig. 3D, E). embryos (Fig. 4E-4G). With further embryogenesis, the signal decreased significantly (arrowhead in Fig. 4H, 4I, 2000) and Gibel carp (Xu et al, 2005) may have different 4K). The preimmune rabbit serum did not generate a expression patterns for different biological functions. positive signal (data not shown). Whether z-otu mRNA and its protein have different roles in zebrafish oogenesis and embryogenesis still requires further investigation. Vertebrate oocytes are arrested at the diplotene stage of the first meiotic prophase, also termed the germinal vesicle (GV) stage. Previous research has confirmed that the reinitiation of first meiotic prophase requires the inactivation of the M-phase promoting factor (MPF), a complex of the cyclin-dependent kinase containing p34cdc2 and cyclin B (Murray and Kirschner, 1989; Dekel, 1996). Additionally, cyclin B1 degradation is dependent upon UPP (Tokumoto et al, 1997; Huo et al, 2004). We found that Z-OTU was localized in the GV of stage I oocytes and the signal was gradually enhanced from stage IA to stage IB where chromosomes Fig. 4 Immunofluorescent detection of Z-OTU protein at early decondensed and acquired a lampbrush appearance, a stages of zebrafish embryogenesis Embryos were characteristic of the diplotene stage of first meiotic incubated with rabbit anti-Z-OTU antibody prophase (Pelegri, 2003). Before GVBD (between A: unfertilized embryo; B: fertilized embryo at 3 minutes; C: fertilized embryo at 10 minutes; D: fertilized embryo at 20 minutes; E: fertilized zebrafish stage III and stage IV oocytes), the Z-OTU embryo at 30 minutes; F: 2-cell stage embryo; G: 1000-cell stage embryo; H: protein was distributed in the cytoplasm of the oocytes 30,-epiboly stage embryo; I: 5-somites stage embryo; J: Phase contrast (Fig. 3B). Upon meiosis reinitiation, Z-OTU was photomicrograph of I; K: 13-somites stage embryo; L: Phase contrast accumulated at the perinuclear cytoplasm of oocytes and photomicrograph of K. ×40, applies to A-L. migrated with GV (Fig. 3C). After fertilization, Z-OTU 3 Discussion protein was uniformly distributed in the determinants and blastomere. This expression pattern of Z-OTU was We previously characterized the expression pattern similar to that of ubiquitin protein in mouse oogenesis of z-otu mRNA (Mo et al, 2005). In this study, we and embryogenesis (Huo et al, 2004), where the determined the locations of Z-OTU protein. According to ubiquitin protein was accumulated in the GV of GV- our results, z-otu mRNA and its protein exhibited an stage oocytes and distributed diffusely in whole oocyte overlapping pattern in temporal expression. As z-otu shortly after GVBD. With the development of the mRNA, Z-OTU protein was also expressed at the early stages of oogenesis and embryogenesis, but there existed oocytes, the ubiquitin protein again concentrated around three obvious differences. First, in stage I oocytes, z-otu the condensed chromatin and the localization of ubiquitin mRNA was highly expressed in cytoplasm, while its protein was diffused in the whole oocyte after meiotic protein had lower expression level in GV. Second, in resumption of the oocytes. During early embryonic stage IV oocytes, only Z-OTU protein was detected in mitosis in mice, the ubiquitin protein mainly the cytoplasm and formed an aggregate undergoing accumulated in the nucleus and some staining of the dynamic change with GV migration. This indicated that ubiquitin protein was detected in the cytoplasm (Huo et z-otu was not transcribed during this phase of oogenesis, al, 2004). These results suggest Z-OTU might be but its protein still persisted. Lastly, z-otu mRNA involved in zebrafish oocyte meiosis, fertilization and disappeared after midblastula transition(MBT) while Z- early embryonic mitosis. It also indicates that Z-OTU OTU protein gradually decreased. This demonstrated that might have DUB activity and regulate UPP. Z-OTU protein was still required for the development of embryos after zygotic transcription was activated. 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