Comparative investigation on spindle behavior and MPF activity changes during oocyte maturation between gynogenetic and amphimictic crucian carp

The spindle behavior and MPF activity changes in the progression of oocyte maturation were investigated and compared with cytological observation and kinase assay between gynogenetic silver crucian carp and amphimictic colored crucian carp.MPF activity was measured by using histone H1 as phosphorylation substrate.There were two similar oscillatory MPF kinase activity changes during oocyte maturation in two kinds of fishes with different reproductive modes,but there existed some subtle difference between them.The subtle difference was that the first peak of MPF kinase activity was kept to a longerlasting time in the gynogenetic silver crucian carp than in the amphimictic colored crucian carp.It was suggested that the difference may be related to the spindle behavior changes,such as tripolar spindle formation and spindle rearrangement in the gynogenetic crucian carp.     

Establishment of the diploid gynogenetic hybrid clonal line of red crucian carp × common carp

This study investigated the gynogenetic cytobiological behavior of the third gynogenetic generation (G3), which was generated from the diploid eggs produced by the second gynogenetic generation (G2) of red crucian carp × common carp, and determined the chromosomal numbers of G3, G2×scatter scale carp and G2×allotetraploid hybrids of red crucian carp × common carp. The results showed that the diploid eggs of G2 with 100 chromosomes, activated by UV-irradiated sperm from scatter scale carp and without the treatment for doubling the chromosomes, could develop into G3 with 100 chromosomes. Similar to the first and second gynogenetic generations (G1 and G2), G3 was also diploid (2n=100) and presented the hybrid traits. The triploids (3n=150) and tetraploids (4n=200) were produced by crossing G2 with scatter scale carp and crossing G2 with allotetraploids, respectively. The extrusion of the second polar body in the eggs of G2 ruled out the possibility that the retention of the second polar body led to the formation of the diploid eggs. In addition, we discussed the mechanism of the formation of the diploid eggs generated by G2. The establishment of the diploid gynogenesis clonal line (G1, G2 and G3) provided the evidence that the diploid eggs were able to develop into a new diploid hybrid clonal line by gynogenesis. By producing the diploid eggs as a unique reproductive way, the diploid gyno- genetic progeny of allotetraploid hybrids of red crucian carp × common carp had important signifi- cances in both biological evolution and production application.     

Formation and biological characterization of three new types of improved crucian carp

The improved tetraploids (G1×AT) were obtained by distant crossing and gynogenesis and the high-body individuals accounted for 2% among G1×AT. After mating with each other, the high-body in- dividuals produced three kinds of bisexual fertile diploid fishes: high-body red crucian carp, high-body fork-like-tails goldfish and gray common carp. The high-body red crucian carp mating with each other formed three types of improved crucian carp (ICC) including improved red crucian carp (IRCC), im- proved color crucian carp (ICCC) and improved gray crucian carp (IGCC). The phenotypes, chromo- some numbers, gonadal structure and fertility of the three kinds of ICC and their offspring were observed. All the three kinds of ICC possessed some improved phenotypes such as higher body, smaller head and shorter tail. The ratios of the body height to body length of IRCC, ICCC and IGCC were 0.54, 0.51 and 0.54, respectively. All of them were obviously higher than that of red crucian carp 0.41 (P<0.01). Three kinds of ICC had the same chromosome number as red crucian carp with 100 chromosomes. All the ICC possessed normal gonads producing mature eggs or sperm, which was important for the production of an improved diploid population. Compared with red crucian carp, all the ICC had stronger fertility such as higher gametes production, higher fertilization rate and higher hatchery rate. Three types of improved diploid fish population were generated from the three kinds of ICC by self-crossing, respectively. The ICC can serve as ornamental fish and edible fish. They are also ideal parents to produce triploids by mating with tetraploids. The new ICC plays an important role in biological evolution and fish genetic breeding.     

Screen for stage-specific expression genes between tail bud stage and heartbeat beginning stage in embryogenesis of gynogenetic silver crucian carp

A systemic study was initiated to identify stage-specific expression genes in fish embryogenesis by using suppression subtractive hybridization (SSH) technique. In this study, we presented a preliminary result on screen for stage-specific expression genes between tail bud stage (TBS) and heartbeat beginning stage (HBS) in gynogenetic silver crucian carp (Carassius auratus gibelio). Two SSH plasmid libraries specific for TBS embryos and HBS embryos were constructed, and stage-specific expression genes were screened between the two stages. 1963 TBS positive clones and 2466 HBS positive clones were sampled to PCR amplification, and 1373 TBS and 1809 HBS PCR positive clones were selected to carry out dot blots. 169 TBS dot blot positive clones and 272 HBS dot blot positive clones were sequenced. Searching GenBank by using these nucleotide sequences indicated that most of the TBS dot blot positive clones could not be found homologous sequences in the database, while known genes were mainly detected from HBS d     

雌核发育银鲫和两性融合发育鱼（红鲫和彩鲫）卵壳结构及其组成的比较研究

作者以两性融合发育鱼（红鲫、彩鲫）为对照,对雌核发育银鲫卵壳的表面结构及其可溶性蛋白的组成进行了研究：（１）雌核发育银鲫卵壳表面比较平滑,没有明显的嵴和沟;（２）雌核发育银鲫卵壳可溶性蛋白的含量略高于两性融合发育鱼（红鲫、彩鲫）,并且在ＩＩ区段存在一条分子量为１０万Ｄａｌｔｏｎ的特异蛋白区带。并对银鲫卵壳结构及组成的特殊性与银鲫卵初级控制作用间的关系进行了初步探讨。     

雌核发育银鲫和两性融合发育鱼（红鲫和彩鲫）卵壳结构及其组成的比…

作者以两性融合发育鱼为对照,对雌核发育银鲫卵壳的表面结构及其可溶性蛋白的组成进行了研究：（１）雌核发育银鲫卵壳表面比较平滑,没有明显的嵴和沟;（２）雌核发育银鲫卵壳可溶性蛋白的含量略高于两性融合发育鱼,并且在Ⅱ区段存在一条分子量为１０Ｄａｌｔｏｎ的特异蛋白区带。并对银鲫卵壳结构及组成的特殊性和银鲫卵初级控制作用间的关系进行了初步探讨。     

Evidence for the paternal mitochondrial DNA in the crucian carp-like fish lineage with hybrid origin

In terms of taxonomic status, common carp(Cyprinus carpio, Cyprininae) and crucian carp(Carassius auratus, Cyprininae) are different species; however, in this study, a newborn homodiploid crucian carp-like fish(2n=100)(2nNCRC) lineage(F1–F3) was established from the interspecific hybridization of female common carp(2n=100)×male blunt snout bream(Megalobrama amblycephala, Cultrinae, 2n=48). The phenotypes and genotypes of 2 n NCRC differed from those of its parents but were closely related to those of the existing diploid crucian carp. We further sequenced the whole mitochondrial(mt) genomes of the 2n NCRC lineage from F1 to F3. The paternal mt DNA fragments were stably embedded in the mt-genomes of F_1–F_3 generations of 2n NCRC to form chimeric DNA fragments. Along with this chimeric process, numerous base sites of F1–F3 generations of 2 n NCRC underwent mutations. Most of these mutation sites were consistent with the existing diploid crucian carp. Moreover, the mt DNA organization and nucleotide composition of 2n NCRC were more similar to those of the existing diploid crucian carp than those of the parents. The inheritable chimeric DNA fragments and mutant loci in the mt-genomes of different generations of 2nNCRC provided important evidence of the mt DNA change process in the newborn lineage derived from hybridization of different species. Our findings demonstrated for the first time that the paternal mt DNA were transmitted into the mt-genomes of homodiploid lineage, which provided new insights into the existence of paternal mt DNA in the mt DNA inheritance.     

Comparative studies on histological and ultra-structure of the pituitary of different ploidy level fishes

The histological and ultra-structure of the pituitary in diploid red crucian carp(Carassius auratus red var.),triploid crucian carp and allotetraploid hybrids within and after the breeding season were comparatively studied.The result showed that there were six endocrine cell types in the pituitary of these three kinds of fishes,and there was an obvious difference in cell size among different ploidy level fishes.As for the same type of pituitary cells,the cell size was increased gradually with the in- creasing ploidy level.In the breeding season,the allotetraploid hybrids had higher proportion of go- nadotropin cells(GTH)than triploids,and the triploids had higher proportion of GTH than diploids.The results were related to the earlier sexual maturity of allotetraploid hybrids and sterility of triploid cru- cian carp.On the other hand,among the three kinds of fishes,the proportion of somatotropin(STH) cells in triploids crucian carp was the highest,whereas that in allotetraploid hybrids was the lowest. The results might be connected with the faster growth rate of triploids and slower growth rate of al- lotetraploid hybrids.In addition,in GTH cells of meso-adenohypophysis after the breeding season, there were many endocrine particles in triploids,while those endocrine particles were released from the cells in allotetraploids and diploids.This result showed that the sterility of triploid crucian carp might be related to the hormone which was not released from the GTH cells.In a word,the present study indicated that the differences in the structure of pituitary among different ploidy level fishes contributed to their difference in the growth rate and gonadal development.     

Genome evolution trend of common carp （Cyprinus carpio L.） as revealed by the analysis of microsatellite loci in a gynogentic family

Genome evolution arises from two main ways of duplication and reduction. Fish specific genome duplication （FSGD） may have occurred before the radiation of the teleosts. Common carp （Cyprinus carpio L.） has been considered to be a tetraploid species, because of its chromosome numbers （2n=100） and its high DNA content. Using 69 microsatellite primer pairs, the variations were studied to better understand the genome evolution （genome duplication and diploidization） of common carp from a gynogenetic family. About 48% of primer pairs were estimated to amplify duplicates based on the number of PCR amplification per individual. Segregation patterns in the family suggested a partially duplicated genome structure and disomic inheritance. This indicates that the common carp is tetraploid and polyploidy occurred by allotetraploidy. Two primer pairs （HLJ021 and HLJ332） were estimated to amplify reduction based on the number of PCR amplification per individual. One allele in HLJ002 locus and HLJ332 locus was clearly lost in the gynogenetic family and the same as in six wild populations. Segregation patterns in the family suggested a partially diplodization genome structure. A hypothesis transition （dynamic） and equilibrium （static） were proposed to explain the common carp genome evolution between genome duplication and diploidization.     

Genetic Evidence for Gonochoristic Reproduction in Gynogenetic Silver Crucian Carp (Carassius auratus gibelio Bloch) as Revealed by RAPD Assays

Sex evolution has been a debating focus in evolutionary genetics. In lower vertebrates of reptiles, amphibians, and fish, a species or a bioform reproduces either sexually or asexually but never both. A few species were found to consist of all females in fish. These all-female species can propagate by asexual reproduction modes, such as gynogenesis and hybridogenesis. However, the coexistence of sexuality and asexuality in a single species was recently noted only in a cyprinid fish silver crucian carp, Carassius auratus gibelio. This fish had been demonstrated to be capable of gynogenesis stimulated by sperm from other related species. Surprisingly, natural populations of this fish consist of a minor but significant portion (approx. 20%) of males. As different clones with specific phenotypic and genetic characteristics have been found, and RAPD markers specific to each clone have recently been identified, this fish offers many advantages for analyzing whether or not genetic recombination occurs between different clones. In this study, artificial propagation was performed in clone F and clone D. Ovulated eggs from clone F were divided into two parts and respectively inseminated with sperm from a clone D male and from a red common carp (Cyprinus carpio) male. The control clone D individuals were selected from gynogenetic offspring of clone D activated by sperm of red common carp. The phenotype and sex ratio in the experimental groups were also observed. Using RAPD molecular markers, which allow for reliable discrimination and genetic analysis of different clones, we have revealed direct molecular evidence for gonochoristic reproduction in the gynogenetic silver crucian carp and confirmed a previous hypothesis that the silver crucian carp might reproduce both gynogenetically and gonochoristically. Therefore, we conclude that the silver crucian carp possesses two reproductive modes, i.e., gynogenetic and gonochoristic reproduction. The response mechanism of two reproductive development modes may be the first discovery in vertebrates. Additionally, we discuss the evolutionary implication between gynogenetic and gonochoristic reproduction modes and the contribution of the minor proportion of males to genetic flexibility in the gynogenetic silver crucian carp. Received: 5 January 2000 / Accepted: 3 August 2000     

银鲫与彩鲫卵母细胞cDNA文库构建及周期蛋白A1的cDNA克隆

分别取行天然雌核发育繁殖的银鲫和两性生殖的彩鲫的卵母细胞为材料,提取总RNA,分离mRNA,进而反转录合成cDNA并定向插入λgtll Sfi-Not克隆载体,经体外包装构建了银鲫与彩鲫卵母细胞的表达型cDNA文库。测试结果表明库容量分别达到3.1×106(银鲫)和1.6×106(彩鲫)。进一步人工合成Cyclin A1保守引物,采用PCR扩增文库的方法,克隆了银鲫(1616bp)与彩鲫(1626bp)的Cyclin A1全长cDNA。序列分析结果表明:两种鱼编码区长度均为1173bp,起始于一个包含在脊椎动物起始密码子ANNATG基元内ATG的单一开放读码框,编码391个氨基酸;5'-端非编码区长度也同为70bp,3-'端非编码区长度略有不同,银鲫为373bp,而彩鲫则为383bp;二者3'-端均带有AATAAA的Poly(A)加尾信号以及24bp(银鲫)和27bp(彩鲫)的Poly(A)尾巴。比较银鲫、彩鲫和金鱼与人、爪蟾Cyclin A1氨基酸序列同源性的结果表明,Cyclin A1在人、爪蟾与鱼类之间具有较高同源性;而在银鲫、彩鲫和金鱼之间,Cyclin A1仅在周期蛋白框外存在5个氨基酸的差异,且这些差异均是由个别碱基的变异造成的。       

Genetic Diversity among Different Clones of the Gynogenetic Silver Crucian Carp, Carassius auratus gibelio, Revealed by Transferrin and Isozyme Markers

Genetic diversity among four clones (A, D, E, F) of gynogenetic silver crucian carp was studied using transferrin and isozymes in the blood as markers. Of the five proteins investigated, three (transferrin, esterase and superoxide dismutase) indicated polymorphism and eight polymorphic loci were detected. These loci were probably encoded by codominant alleles and their inheritance patterns were analyzed. Intraclonal homogeneity and interclonal heterogeneity were observed in these clones, which allowed us to infer the clonal nature and evolutionary relationship between them. Clonal diversity in this population of silver crucian carp in China was also compared with data reported from gynogenetic crucian carp in Germany.     

Differential screening and characterization analysis of the egg envelope glycoprotein ZP3 cDNAs between gynogenetic and gonochoristic crucian carp

INTRODUCTIONFertilization in animals is the trigger that ini-tiates development, and results in a series of well-choreographed interactions between molecules lo-cated on the surfaces of egg and sperm[1, 2]. Somecell surface proteins, such as zolla pellucida gly-coprotein ZPI, ZPZ and ZP3, acrosomal proteinbindin, acrosin and lysin, and sperm plasma mem-brane protein a- and p--fertilin, have been identi-fied to mediate the interaction process[2-8]. Al-though studies on the regulative fa…     

A Set of Polymorphic Trinucleotide and Tetranucleotide Microsatellite Markers for Silver Crucian Carp (Carassius auratus gibelio) and Cross-Amplification in Crucian Carp

Silver crucian carp (Carassius auratus gibelio Bloch), as a gynogenetic fish, is a promising model for the study of the evolutionary genetics of vertebrates. We have developed 59 polymorphic trinucleotide and tetranucleotide markers for the silver crucian carp through the biotin capture method and radioactive-labeling hybridization. The number of alleles ranged from 2 to 12 in the population, and the average proportion of heterozygotes (including tri- and diallelic) at polymorphic loci was 76.8%. In addition, these loci were successfully applied to a close relative, the crucian carp (Carassius auratus), by cross-amplification, as shown by the range of alleles (2–19), observed heterozygosity (0.1765–0.9706), expected heterozygosity (0.2392–0.9421), and polymorphism information content (0.2186–0.9236).     

人工诱导雌核发育日本白鲫

分别用遗传失活的散鳞镜鲤(Cyprinus carpio L.)、团头鲂(Megalobrama amblycephala)精子诱导日本白鲫(Carassius cuvieri)进行雌核发育.未经冷休克处理,用UV照射过的镜鲤的精子(其最佳UV照射剂量为4 200 mJ/cm2)与日本白鲫卵子受精获得(32.4±3.3)%雌核发育单倍体和(0.7±0.3)%杂交二倍体,而照射过的团头鲂精子(其最佳UV照射剂量为3 600mJ/cm2)与日本白鲫卵子受精得到了(33.8±1.4)%雌核发育单倍体和(0.5-±0.3)%杂交二倍体.日本白鲫卵子分别经灭活的镜鲤、团头鲂的精子激活(精子经各自最佳UV剂量处理),施以冷休克处理(受精后2min,0-4℃,40min),其孵化率分别为(27.8±2.1)%和(29.4±3.3)%,发育到摄食期,其正常的雌核发育二倍体鱼苗分别为(15.7±3.4)%和(23.6±4.1)%,在镜鲤实验组中,其正常的雌核发育二倍体鱼苗占孵化鱼苗总数的56%,这比团头鲂实验组中正常雌核发育二倍体鱼苗的比率(达到80%)低,结果表明诱导日本白鲫雌核发育,与母本遗传关系远的团头鲂精子较镜鲤的更有效.从染色体数目、外形特征和性腺结构等方面证实雌核发育后代的生物学特性.雌核发育日本白鲫全为雌性,这表明其雌性是同配XX型.雌核发育日本白鲫在生产上将有着潜在的价值,如比普通日本白鲫长得更快,抗病能力强等.所有的雌核发育日本白鲫的染色体数目都是100,而所有的团头鲂与日本白鲫的杂交后代都是三倍体(3n=124),新三倍体鱼在鱼类养殖和理论研究中将存在潜在的价值.     

雌核发育银鲫和两性生殖彩鲫精子蛋白组份的比较研究

对雌核发育银鲫和两性融合彩鲫精子蛋白组份进行了比较分析。通过分级抽提得到精子的不同组份精浆、精头的膜、鞭毛和脱膜精头等,然后经不同的凝胶电泳系统,比较分析了银鲫精子和其两性亲缘种彩鲫精子相应组份可溶性蛋白成份的差异。研究表明,经分级抽提的银鲫精子和彩鲫精子的各个组份都含有其特定的蛋白谱带。精浆蛋白在两种鱼之间和两种鱼的不同个体之间都存在一定差异。精头膜、鞭毛和脱膜精头的可溶性蛋白在同种鱼不同个体间高度一致,但在两种鱼之间表现出差异。两种鱼精头膜的可溶性蛋白在SDS－PAGE电泳图谱上基本一致,而在非变性聚丙烯酰胺凝胶电泳图谱上则具有各自的特征性谱带。鞭毛可溶性蛋白的SDS－PAGE分析在雌核发育银鲫中揭示出一条特异的蛋白带。脱膜精头的可溶性蛋白在SDS－PAGE电泳图谱上差异明显,存在几条特征性蛋白带,并经Acid-Urea PAGE系统分析,证实这些特征性蛋白为碱性蛋白。这些发现为进一步鉴定雌核发育银鲫雄鱼精子的特异性蛋白和揭示其分子机制打下了基础。     

Induction of Gynogenesis in Japanese Crucian Carp (Carassius cuvieri)

Diploid gynogenesis was induced in Japanese crucian carp (Carassius cuvieri) eggs using UV-irradiated genetically inactive spermatozoa from mirror carp (Cyprinus carpio L.) or blunt snout bream (Megalobrama amblycephala), with or without cold shock. The optimal radiation dosage was 4200 mJ/cm² and 3600 mJ/cm² for mirror carp and blunt snout bream sperm, respectively. At this dosage and without cold shock, the yields were (32.4±3.3)% vs. (33.8±1.4)% gynogenetic haploids and (0.7±0.3)% vs. (0.5±0.3)% hybrid diploids, respectively. At the optimal UV dosage but with cold shock (2 min after fertilization, 0-4°C for 40 min), the hatching rates were (27.8±2.1)% and (29.4±3.3)%, respectively. From hatching to feeding, (15.7±3.4)% and (23.6±4.1)% normal gynogenetic diploids were recorded, respectively. Survival of normal gynogenetic diploids was 56% out of the hatched fry when using irradiated spermatozoa of mirror carp, which was lower than that (up to 80%) when using irradiated spermatozoa of blunt snout bream. This indicated that the sperm of blunt snout bream, with distant genetic relation to the maternal Japanese crucian carp, was more effective than that of mirror carp to induce diploid gynogenesis. The nature of the gynogenetic progeny was identified with external appearance, chromosome number and gonad structure. The presence of only females in gynogenetic progeny probably suggested XX genotype in the female Japanese crucian carp. The gynogenetic diploids have potential values such as faster growth and stronger disease resistance than the normal Japanese crucian carp. All gynogenetic progeny possessed 100 chromosomes whereas all J x B crosses were triploid with 124 chromosomes. The formation of the new triploid hybrids in J x B crosses may be useful in aquaculture.     

彭泽鲫雌核发育的细胞学研究

通过对彭泽鲫♀×彭泽鲫♂、彭泽鲫♀×尖鳍鲤♂“杂交”后代的细胞学观察发现 ,同源与异源精子在彭泽鲫卵中的发育情况基本一致。精子入卵后一般呈固缩状态 ,不解凝 ,不形成雄性原核 ,但都能看到未解凝精核与雌性原核接触 (未融合 )的现象。刚产出的成熟卵子没有看到极体 ,但受精 1 0分钟有极体排出 ,是卵子发育排出的惟一极体。根据观察结果 ,彭泽鲫在细胞学上表现出典型的雌核发育行为特征 ,是以雌核发育方式繁殖的种群