Expression and segregation of nucleoplasmin during development in Xenopus

The spatial segregation of informational molecules in the unfertilized egg and embryo has been hypothesized to be a necessary phenomenon for the normal progression of development leading to the determination of cellular phenotypes. This study describes the selection of a monoclonal antibody (Mab: 2G6) that identifies an antigen (Ag: 2G6) which is localized in the germinal vesicle of oocytes and has a discrete pattern of inheritance during embryogenesis. The antigen displayed biochemical and physical characteristics very similar to nucleoplasmin, which is the histone-binding and nucleosome-assembly protein previously described. Immunoblot analysis with purified oocyte nucleoplasmin confirmed this relationship. Indirect immunofluorescence was used to study the temporal expression and spatial distribution of nucleoplasmin. From early cleavage stages through gastrulation, it is preferentially localized in nuclei of blastomeres at the animal pole. By tadpole stages, it was detected only in nuclei of postmitotic cells of the central nervous system and in nuclei of striated muscle. It was not detected in adult tissues. Western blot analysis during embryogenesis revealed at least five immunologically related polypeptides that displayed distinct patterns of expression during development. The different species observed most likely represent different levels of phosphorylation of nucleoplasmin. The more acidic forms, known to be more active in nucleosome assembly, were present during cleavage stages. Analysis of labelled oocyte proteins by two-dimensional immunoblots and autoradiography revealed that synthesis of nucleoplasmin was first detected in stage-2 oocytes, reached 60% maximum levels at stage 3, peaked at stage 4 and was undetectable in stage-6 oocytes. The amount of nucleoplasmin message present does not follow a similar pattern during oogenesis. These results suggest that the message undergoes pronounced changes in translational efficiency during oogenesis. A comparative immunoblot analysis using proteins from a variety of adult tissues revealed that, whereas the polyclonal antisera against amphibian vitellogenic oocyte nucleoplasmin recognized several different, tissue-specific polypeptides, two different monoclonal antibodies (Mab: b7-1D1, Mab: 2G6) failed to recognize any of the adult tissues tested. We conclude that nucleoplasmin is a family of closely related proteins with distinct embryonic and adult members.     

Embryonic poly(A)-binding protein is differently expressed and interacts with the messenger RNAs in the mouse oocytes and early embryos

The embryonic poly(A)-binding protein (EPAB) functions in the translational regulation of the maternal messenger RNAs (mRNAs) required during oocyte maturation, fertilization, and early embryo development. Since there is no antibody specific to mammalian EPAB protein, all studies related to the Epab gene could be performed at the mRNA levels except for the investigations in the Xenopus. In this study, we have produced an EPAB-specific antibody. When we examined its expressional distribution in the mouse gonadal and somatic tissues, the EPAB protein was found to be expressed only in the mouse ovary and testis tissues, but it is undetectable level in the somatic tissues including stomach, liver, heart, small intestine, and kidney. Additionally, the spatial and temporal expression patterns of the EPAB and poly(A)-binding protein cytoplasmic 1 (PABPC1) proteins were analyzed in the mouse germinal vesicle (GV) and metaphase II (MII) oocytes, one-cell, and two-cell embryos. While EPAB expression gradually decreased from GV oocytes to two-cell embryos, the PABPC1 protein level progressively increased from GV oocytes to one-cell embryos and remarkably declined in the two-cell embryos ( P < 0.05). We have also described herein that the EPAB protein interacted with Epab, Pabpc1, Ccnb1, Gdf9, and Bmp15 mRNAs dependent upon the developmental stages of the mouse oocytes and early embryos. As a result, we have first produced an EPAB-specific antibody and characterized its expression patterns and interacting mRNAs in the mouse oocytes and early embryos. The findings suggest that EPAB in cooperation with PABPC1 implicate in the translational control of maternal mRNAs during oogenesis and early embryo development.     

DNMT1, DNMT3A and DNMT3B proteins are differently expressed in mouse oocytes and early embryos

DNA methylation is one of the epigenetic mechanisms and plays important roles during oogenesis and early embryo development in mammals. DNA methylation is basically known as adding a methyl group to the fifth carbon atom of cytosine residues within cytosine–phosphate–guanine (CpG) and non-CpG dinucleotide sites. This mechanism is composed of two main processes: de novo methylation and maintenance methylation, both of which are catalyzed by specific DNA methyltransferase (DNMT) enzymes. To date, six different DNMTs have been characterized in mammals defined as DNMT1, DNMT2, DNMT3A, DNMT3B, DNMT3C, and DNMT3L. While DNMT1 primarily functions in maintenance methylation, both DNMT3A and DNMT3B are essentially responsible for de novo methylation. As is known, either maintenance or de novo methylation processes appears during oocyte and early embryo development terms. The aim of the present study is to investigate spatial and temporal expression levels and subcellular localizations of the DNMT1, DNMT3A, and DNMT3B proteins in the mouse germinal vesicle (GV) and metaphase II (MII) oocytes, and early embryos from 1-cell to blastocyst stages. We found that there are remarkable differences in the expressional levels and subcellular localizations of the DNMT1, DNMT3A and DNMT3B proteins in the GV and MII oocytes, and 1-cell, 2-cell, 4-cell, 8-cell, morula, and blastocyst stage embryos. The fluctuations in the expression of DNMT proteins in the analyzed oocytes and early embryos are largely compatible with DNA methylation changes and genomic imprintestablishment appearing during oogenesis and early embryo development. To understand precisemolecular biological meaning of differently expressing DNMTs in the early developmental periods, further studies are required.     

Changes in zinc,copper and metallothionein contents during oocyte growth and early development of the teleost Danio rerio (zebrafish)

In the present report, we investigated zinc, copper and metallothionein (MT) contents in zebrafish oocytes and embryos. Our results demonstrate that the metal content increases during oocytes maturation. Zinc increases from 30 ng/oocyte (stage-1 oocytes) to 100 ng/oocyte (stage-3 oocytes); copper varied from 1 ng/oocyte (stage-1 oocytes) to 3.5 ng/oocyte (stage-3 oocytes). During embryogenesis, zinc and copper contents dramatically increase after fertilisation around the 512-cells stage, then slowly decrease until the mid-gastrula stage. During oocyte growth, the changes in the MT level are proportional to metal content, whereas during embryogenesis the pattern of MT accumulation does not parallel that of the two metals. Indeed, the maternal pool of MT decreases steadily during the early stages of the development until the gastrula stage. We have examined the effect of cadmium on the expression of MT during zebrafish development. After cadmium exposure, MT content increases in embryos at the blastula stage, whereas no induction occurs in embryos at the gastrula stage. However, pre-treatment of embryos at the gastrula stage with 5-aza-2'-deoxycytidine induces MT synthesis following exposure to cadmium. These observations show that changes in metal levels are not correlated to MT content in the embryo, whereas DNA methylation is one of the factors regulating MT expression.     

Identification and isolation of a BTB-POZ-containing gene expressed in oocytes and early embryos of the zebrafish Danio rerio.

In this report, we describe the cloning of a cDNA from the zebrafish Danio rerio encoding a protein containing a BTB-POZ domain closely resembling the BTBD1 and BTBD2 proteins previously identified in mammals. However, unlike other BTB-POZ-containing genes, expression of this gene in adults is most abundant in oocytes, where the RNA can be detected at all stages of oogenesis examined. The presence of the RNA persists through early cleavage, but is decreased significantly by gastrulation. Although the function of this gene has yet to be determined, its resemblance to the BTB-POZ family of genes coupled with its expression pattern suggests that it may have an important function in oogenesis and (or) early zebrafish development.     

扬子鳄FoxO1基因CDS序列的克隆及其在初孵鳄性腺组织的时空表达规律

研究以初孵扬子鳄(Alligator sinensis)性腺组织为实验材料,开展Forkhead box家族蛋白-1(FoxO1)基因CDS区克隆和序列特征分析。利用免疫组织化学检测其在胃、肠和肺脏等组织中的表达谱,结合免疫荧光(IF)、RT-PCR和Western Blot探讨其在不同出壳后日龄(17日龄、63日龄和96日龄)雌性扬子鳄性腺组织中的时空表达规律,探讨其在雌性扬子鳄性腺发育过程中的生物学功能。研究成功克隆获得FoxO1基因长度为1941 bp的完整编码区及646个预测编码氨基酸序列。与其他物种间进行同源性比对和构建进化树分析发现,扬子鳄FoxO1基因与鸟类的亲缘关系相较中华鳖(Pelodiscus sinensis)和原矛头蝮(Protobothrops mucrosquamatus)等爬行动物更近。在其他脊椎动物中,哺乳动物、硬骨鱼和两栖物种也各自聚类称为子群,表明FoxO1兼具功能保守性和种间特异性;免疫组织化学结果表明FoxO1在初孵鳄性腺复合体、肺、胃和肠中均有表达,免疫荧光结果表明17日龄性腺组织中的FoxO1主要表达于肾上腺和中肾区但在皮质部呈微弱表达,在6...     

Zebrafish CTH1, a C3H zinc finger protein, is expressed in ovarian oocytes and embryos

 The Zfcth1 gene is, as the previously cloned carp cth1 gene, related to the mammalian TIS 11 family of primary response genes and encodes a protein with two putative CCCH zinc fingers. This report describes the RNA expression of this gene during oogenesis and early embryogenesis up to gastrulation in the zebrafish (Danio rerio). Maternal cth1 message is present in the ovary of 1-month-old fish and of adult fish in oocytes at all stages of maturation. In the youngest oocytes the message is localized in the cytoplasm all around the nucleus, in larger oocytes the message becomes restricted to the future animal pole of the embryo, and in mature oocytes the expression is sharply localized in the cortical layer under the micropyle. After ovulation the cth1 messenger spreads over the cytoplasmic cap and is distributed over the blastomeres during subsequent cleavages. In subsequent stages maternal expression of cth1 gradually disappears. From early epiboly stages onward embryonic cth1 expression is localized to the germ ring and the hypoblast cells in the central part of the embryonic shield. In the shield, cth1 expression largely overlaps with the area of gooscoid expression in the first involuting cells. In stages after 70% of epiboly cth1 expression diminishes and soon can no longer be detected in the embryo. Next to a developmental role in cell fate determination we propose a function for cth1 during oocyte maturation. Received: 19 October 1998 / Accepted: 16 February 1999     

Spatiotemporal localization of germ plasm RNAs during zebrafish oogenesis

In zebrafish, primordial germ cells (PGCs) are determined by a specialized maternal cytoplasm, the germ plasm, which forms at the distal ends of the cleavage furrows in 4-cell embryos. The germ plasm includes maternal mRNAs from the germline-specific genes such as vasa and nanos1, and vegetally localized dazl RNA is also incorporated into the germ plasm. However, little is known about the distributions and assembly mechanisms of germ plasm components, especially during oogenesis. Here we report that the germ plasm RNAs vasa, nanos1, and dazl co-localize with the mitochondrial cloud (MC) and are transported to the vegetal cortex during early oogenesis. We found that a mitochondrial cloud localization element (MCLE) previously identified in the 3' untranslated region (3'UTR) of Xenopus Xcat2 gene can direct RNA localization to the vegetal cortex via the MC in zebrafish oocytes. In addition, the RNA-binding protein Hermes is a component of the MC in zebrafish oocytes, as is the case in Xenopus. Moreover, we provide evidence that the dazl 3'UTR possesses at least three types of cis-acting elements that direct multiple steps in the localization process: MC localization, anchorage at the vegetal cortex, and localization at the cleavage furrows. Taken together, the data show that the MC functions as a conserved feature that participates in transport of the germ plasm RNAs in Xenopus and zebrafish oocytes. Furthermore, we propose that the germ plasm components are assembled in a stepwise and spatiotemporally-regulated manner during oogenesis and early embryogenesis in zebrafish.     

Histone acetyltransferases and histone deacetyl transferases play crucial role during oogenesis and early embryo development

Dynamic epigenetic regulation is critical for proper oogenesis and early embryo development. During oogenesis, fully grown germinal vesicle oocytes develop to mature Metaphase II oocytes which are ready for fertilization. Fertilized oocyte proliferates mitotically until blastocyst formation and the process is called early embryo development. Throughout oogenesis and early embryo development, spatio-temporal gene expression takes place, and this dynamic gene expression is controlled with the aid of epigenetics. Epigenetic means that gene expression can be altered without changing DNA itself. Epigenome is regulated through DNA methylation and histone modifications. While DNA methylation generally ends up with repression of gene expression, histone modifications can result in expression or repression depending on type of modification, type of histone protein and its specific residue. One of the modifications is histone acetylation which generally ends up with gene expression. Histone acetylation occurs through the addition of acetyl group onto amino terminal of the core histone proteins by histone acetyltransferases (HATs). Contrarily, histone deacetylation is associated with repression of gene expression, and it is catalyzed by histone deacetylases (HDACs). This review article focuses on what is known about alterations in the expression of HATs and HDACs and emphasizes importance of HATs and HDACs during oogenesis and early embryo development.     

Biochemical and molecular characterization of galectins from zebrafish (Danio rerio): notochord-specific expression of a prototype galectin during early embryogenesis

Galectins are a family of beta-galactoside-binding lectins that on synthesis are either translocated into the nucleus or released to the extracellular space. Their developmentally regulated expression, extracellular location, and affinity for extracellular components (such as laminin and fibronectin) suggest a role in embryonic development, but so far this has not been unequivocally established. Zebrafish constitute an ideal model for developmental studies because of their external fertilization, transparent embryos, rapid growth, and availability of a large collection of mutants. As a first step in addressing the biological roles in zebrafish embryogenesis, we identified and characterized members of the three galectin types: three protogalectins (Drgal1-L1, Drgal1-L2, Drgal1-L3), one chimera galectin (Drgal3), and one tandem-repeat galectin (Drgal9-L1). Like mammalian prototype galectin-1, Drgal1-L2 preferentially binds to N-acetyllactosamine. Genomic structure of Drgal1-L2 revealed four exons, with the exon-intron boundaries conserved with the mammalian galectin-1. Interestingly, this gene also encodes an alternatively spliced form of Drgal1-L2 that lacks eight amino acids near the carbohydrate-binding domain. Zebrafish galectins exhibited distinct patterns of temporal expression during embryo development. Drgal1-L2 is expressed postbud stage, and its expression is strikingly specific to the notochord. In contrast, Drgal1-L1 is expressed maternally in the oocytes. Drgal1-L3, Drgal3, and Drgal9-L1 are expressed both maternally and zygotically, ubiquitously in the adult tissues. The distinct temporal and spatial patterns of expression of members of the zebrafish galectin repertoire suggest that each may play distinct biological roles during early embryogenesis.     

Gene expression during mammalian oogenesis and early embryogenesis: quantification of three messenger RNAs abundant in fully grown mouse oocytes

Ribonuclease protection assays have been used to quantitatively assess changes in steady-state levels of specific mRNAs during oogenesis and early embryogenesis in mice. The mRNAs encode ZP3 (a glycoprotein that serves as a sperm receptor), LDH-B (heart-type lactate dehydrogenase), and MOM-1 (a protein of unknown function). MOM-1 and LDH-B are expressed in a variety of adult mouse tissues and midgestation embryos, whereas ZP3 expression is restricted completely to oocytes. All three mRNAs are expressed by growing mouse oocytes and accumulate to unusually high levels in fully grown oocytes as compared to somatic cells; 240,000, 200,000 and 74,000 copies mRNA per fully grown oocyte for ZP3, LDH-B and MOM-1, respectively. Steady-state levels of LDH-B and MOM-1 mRNA undergo a modest decline (approximately 20-40%) during ovulation when fully grown oocytes become unfertilized eggs and, in general, mirror the reported change in poly(A)+RNA levels during this period of development. On the other hand, the level of ZP3 mRNA declines dramatically (approximately 98%) during ovulation, from approximately 240,000 copies per oocyte to approximately 5000 copies per unfertilized egg, and ZP3 mRNA is undetectable in fertilized eggs (less than 1000 copies per fertilized egg). MOM-1 mRNA is expressed at relatively low levels in morulae (approximately 2000 copies per embryo) and blastocysts (approximately 5000 copies per embryo), whereas ZP3 mRNA remains undetectable (less than 1000 copies per embryo) at these stages of preimplantation development. These findings are discussed in the context of overall gene expression during oocyte growth, meiotic maturation and early embryogenesis in mice.     

Le 斑马鱼nanos1基因在配子发生中的原位杂交研究

利用组织原位杂交技术,以地高辛标记的反义RNA为探针,检测了斑马鱼(Danio rerio) nanos1基因在卵子发生及精子发生中的表达分布特点,初步探讨了该基因在斑马鱼配子发生中可能的功能。结果表明：在斑马鱼卵子发生中,nanos1 mRNA均匀分布于卵原细胞和各时期卵母细胞的胞质中;在卵原细胞和Ⅰ、Ⅱ期卵母细胞中,nanos1 mRNA的杂交信号十分强烈,而较晚期卵母细胞中信号明显减弱。在斑马鱼精子发生中, nanos1 mRNA可在精原细胞和初级精母细胞中检测到。nanos1 mRNA的阳性信号在精原细胞中极为强烈,在初级精母细胞中较为微弱,而精子细胞中没有阳性信号。本研究结果初步表明,斑马鱼nanos1基因对生殖干细胞-卵原细胞和精原细胞的维持和正常功能可能起着重要作用。     

Morphological development of the gonads in zebrafish

Gonadogenesis of zebrafish Danio rerio was investigated by means of light microscopy to test the suitability of gonad histology as an endpoint in hazard assessment of endocrine‐active compounds. At age 2 weeks post‐fertilization (pf), primordial germ cells were found in a dorsocaudal position in the body cavity. At 4 weeks pf, the majority of the fish (86%) possessed paired gonads with meiotic germ cells; these gonads represented presumptive ovaries. At week 5 pf, 87% of the fish examined had ovaries with perinucleolar oocytes. Further development of the gonads in female zebrafish up to week 11 pf was characterized by an increase in gonad size as well as in the number and size of perinucleolar oocytes. Starting with week 5, some fish showed alterations of gonad morphology, including a decrease in the number and size of the oocytes, an enhanced basophilia and irregular shape of the oocytes, and finally their degeneration into residual bodies. With the decline in oocyte number, stromal cells became more numerous and they infiltrated the gonadal matrix. In several 7 week‐old zebrafish with altered gonadal morphology, enhanced numbers of gonial cells arranged in cyst‐like groups appeared. These gonads were interpreted as presumptive testes. In one fish out of 32 individuals examined, spermatocytes were detected, in addition to the gonial cells. During the subsequent weeks, the percentage of fish showing early testes with spermatogonia, spermatocytes and spermatids increased and reached 40% at 11 weeks pf. The sequence of gonadal alterations taking place in some of the individuals from week 5 pf onwards was interpreted to reflect the transition of protogynic ovaries into testes. The developmental pattern described identifies zebrafish to be a juvenile hermaphrodite. The results of this study are of relevance for the use of gonadal histopathology as endpoint in endocrine disruption testing, particularly in order to avoid false diagnoses of ‘intersex gonads’ in zebrafish.     

银鲫种系细胞标记分子Vasa: cDNA克隆及其抗体制备

种系细胞始自胚胎发育早期,是动物生殖及生殖工程的基础。为研究鱼类的种系细胞提供标记分子,我们克隆并鉴定了银鲫的vasacDNA即Cagvasa。CagvasacDNA全长2771碱基(nt),编码的蛋白为银鲫Vasa即CagVasa,全长701个氨基酸(aa)。CagVasa蛋白与已知Vasa蛋白的结构特征一致:在N端有14个RGG重复序列,在C端Vasa所特有的8个功能域俱全。银鲫Vasa与鲤鱼、斑马鱼、陆生脊椎动物和果蝇的Vasa蛋白分别有95%,89%,61%-66%和50%的同源性。卵巢切片的RNA原位杂交揭示,Cagvasa限于种系细胞,且表达水平呈现出低-高-低的动态变化:即两头低(卵原细胞跟Ⅳ期成熟卵子),中间高(Ⅱ-Ⅲ期卵子)。为分析鱼类种系细胞提供手段,我们用310aa的N端序列产生细菌的重组蛋白来免疫大白兔,获得了抗Vasa的多克隆抗体αVasa。Western免疫印迹表明,αVasa特异性地识别一个鱼类性腺的蛋白,该蛋白的分子量为75kD,仅见于银鲫的性腺和卵子。卵巢切片的组织免疫荧光共聚焦显微分析表明,抗体αVasa只对种系细胞染色:卵原细胞着色最深,卵母细胞和早期的卵子都浓染,成熟卵则浅染。类似情况亦见之于精子发生早期阶段的雄性种系细胞。卵巢和精巢的体细胞则不着色。因此,Cagvasa编码的当是Vasa同源蛋白,为银鲫种系细胞的第一个标记分子。我们的研究表明,抗体αVasa染色灵敏度高,特异性好,当是鉴别银鲫及其它鲤科鱼类的种系细胞的有效手段