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.     

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.     

Induced Gynogenesis in Grass Carp (Ctenopharyngodon idellus) Using Irradiated Sperm of Allotetraploid Hybrids

Grass carp (Ctenopharyngodon idellus) eggs were activated by UV-irradiated diploid sperm of allotetraploid hybrids derived from red crucian carp (♀)?×?common carp (♂) and then duplicated by cold shock in 4-6°C water for 10-12 min. Different cold shock initiation times resulted in two types of diploid gynogenetic grass carp: meiotic gynogenetic (meiG) and mitotic gynogenetic (mitG). Over a 5-year period, a total of 17,170 meiG and 1,080 mitG fry were produced and 6,862 meiG and 372 mitG grass carp survived. The gynogenetic fish were confirmed by morphological characteristics, chromosome examination, and microsatellite DNA analysis. The morphological traits of the gynogenetic grass carp were similar to those of wild diploid grass carp. Normal gynogenetic fish were identified as diploid with 48 chromosomes by chromosomal metaphases examination, while nonviable abnormal embryos were detected as haploid with 24 chromosomes. Microsatellite DNA analysis indicated that after one generation of gynogenesis, the genetic purity of meiG and mitG grass carp was significantly increased over that of wild grass carp. In addition, both meiG and mitG grass carp groups were 100% female, and 88% of these showed normal ovary development. Thus, the sex determination mechanism in female grass carp was homogamety. The ability to establish pure all-female groups of meiG and mitG grass carp should be a valuable contribution to both fish genetics and grass carp breeding.     

Induction of Tetraploid Gynogenesis in the European Sea Bass (Dicentrarchus labrax L.)

A preliminary study on tetraploid gynogenetic induction in the European sea bass was performed by pressure-blocking the second polar body release and the first cleavage in eggs fertilized with ultraviolet-irradiated sperm. Fertilization of eggs with genetically inactivated sperm produced only haploid development that terminated around hatching. Pressure treatments (8.500 psi for 2 min) applied at 6 and 65 min after fertilization (a.f.) produced variable levels (7–95%) of tetraploid larvae at hatching. A small proportion of mosaics (3.8n/4.2n) was also recorded.     

Artificial Gynogenesis and Sex Determination in Half-Smooth Tongue Sole (Cynoglossus semilaevis)

Half-smooth tongue sole (Cynoglossus semilaevis) is an important cultured marine fish as well as a promising model fish for the study of sex determination mechanisms. In the present study, a protocol for artificial gynogenesis of half-smooth tongue sole was developed in order to identify the sex determination mechanism and to generate all-female stock. The optimal UV-irradiation dose for genetically inactivating sea perch spermatozoa was determined to be ≥30 mJ/cm². The optimal initiation time for cold shock of gynogenetic embryos was determined to be 5 min after fertilization, while the optimal temperature and treatment duration were determined to be 20–25 min at 5°C. Chromosomes from common diploids, gynogenetic haploids, and diploids were analyzed. WW chromosomes were discovered in some of the gynogenetic diploids. The microsatellite marker was applied to analyze gynogenetic diploid fry. Among the 30 gynogenetic diploid fry, 11 fry contained only one allele, while 19 contained two alleles, which had the same genotype as their mother. The female-specific DNA marker was observed in four individuals out of ten gynogenetic diploid fry. Ploidy analysis of 20 putative gynogenetic fry showed them all to be diploid. Thus, a protocol for the induction of artificial gynogenesis has been developed for the first time in half smooth tongue sole, and the sex determination mechanism in the tongue sole was determined to be female heterogametic with the ZW chromosome.     

两个雌核发育白鲢群体同工酶分析及遗传标记的确定

取源于武汉两个不同渔场两尾白鲢的卵子,经紫外照射遗传物质失活的鲤鱼精子刺激雌核发育和热休克诱导第二极体保留的基因组操作技术,获得了两个不同的人工雌核发育白鲢群体。采用聚丙烯酰胺垂直板电泳技术,分析了这两个不同人工雌核发育白鲢群体(分别称为Hy-G1和Hy-G2)内不同个体的肝脏、肌肉组织以及红细胞中乳酸脱氢酶(LDH)、苹果酸脱氢酶(MDH)、酯酶(EST)、超氧化物歧化酶(SOD)等几种同工酶的表达谱式,并与普通繁殖的同龄白鲢进行了比较。结果表明,各个雌核发育白鲢群体内不同个体间的酶谱表现出很大程度的一致性,具较高的纯合度,而两个不同雌核发育群体Hy-G1和Hy-G2之间表现有明显差异。特别是肝脏和肌肉组织迁移率较快的酯酶谱带等以其稳定的差异,建议作为区分这两个人工雌核发育白鲢群体的生化遗传标记。       

草鱼雌核发育后代不同群体的微卫星遗传分析及指纹识别

以紫外线灭活的团头鲂精子激活草鱼卵子,冷休克抑制第二极体排出的方法诱导出长江水系优良F2代草鱼减数雌核发育子代。在后代中不仅存在雌核发育后代,还存在草鲂杂交后代,雌核发育后代的体型与草鱼一致,而草鲂杂交后代的体型介于草鱼与团头鲂之间。Partec CyFlow倍性分析仪测定结果显示:普通草鱼与雌核发育草鱼的相对DNA含量分别为23.01和22.72,二者的DNA含量接近;而高体型子代的相对DNA含量为25.38,介于草鱼与团头鲂（DNA含量28.21）之间,属于草鲂杂交后代。选取17个微卫星标记对草鱼群体、雌核发育草鱼群体和草鲂杂交后代的遗传多样性进行了检测,共检测出59个等位基因,其中43.18个有效等位基因。草鱼对照群体、草鲂杂交后代和雌核发育草鱼群体的平均等位基因依次为3.57、2.86和2.79,平均有效等位基因依次为2.93、2.37和1.96,平均期望杂合度在依次为0.6502、0.5573和0.3775,多态信息含量（PIC）平均值依次为0.5738、0.4649和0.3791。与草鱼对照群体相比,雌核发育草鱼群体的遗传多样性显著下降,表明通过减数雌核发育方法可获得纯合性较高的草鱼个体。构建了草鱼后代不同群体的DNA指纹模式图,筛选到不同群体的9个特异微卫星标记,为草鱼优良群体的选育提供了基础资料。     

银鲫精子在两性融合生殖鱼类卵质中的受精特征

红鲤(♀)×银鲫(♂)的杂种胚胎均不能成活。受精细胞学研究结果表明,雌核发育银鲫的精核在两性融合生殖的鲤鱼卵质中能转化为雄性原核,并和雌性原核融合成合子核。但在卵裂开始后,可观察到某些异常现象,如染色体丢失,多极纺锤体形成,以及第二次卵裂时受精卵的两分裂球的非同步性分裂等。我们初步认为,雌核发育银鲫的染色体和两性融合生殖的鱼类染色体之间存在某种不相容性,表现为彼此间的互相排斥。出现这种现象可能和银鲫染色体组的雌核发育遗传背景有关。     

Induction of meiotic gynogenesis in Atlantic cod,Gadus morhua (L.)

Gynogenesis is one of several chromosome‐manipulating techniques used in fish. In gynogenesis the male does not contribute to the genetic material of the offspring, and the sperm cells act only as stimulators in order for the egg to start development. This technique has several applications, both in aquaculture and in biological research: gynogenetic fish may be used as a step in the production of all‐female populations, the production of isogenetic‐ and inbred lines, revealing of the sex determination mechanism, construction of genetic maps, and testing of environmental vs genetic control of different traits. The aim of this study was to develop a simple protocol for production of gynogenetic cod (Gadus morhua L.) for further use in aquaculture research. Various milt dilutions and UV‐irradiation doses were tested, in order to inactivate the sperm without destroying its ability to induce egg development. This was followed by pressure treatment of the eggs shortly after ‘fertilization’ to suppress the completion of meiosis II, and thereby restoring diploidy. A dose of 9000 erg mm^?2, followed by a 5‐min pressure treatment (58.6 MPa) 180 min‐degrees after fertilization gave 100% gynogenetic larvae. Histologically, sexual differentiated fish were all females, possibly confirming female homogamety in Atlantic cod. No particular signs of reduced growth, survival or enhanced deformity rates were observed after the fish had reached the juvenile phase. Mortality was, however, high during the egg and larval stages. This protocol has made capable the production of gynogenetic cod juveniles in significant amounts using relatively simple means; the next step will be to elaborate on the technique in order to produce mitotic gynogenetic (double haploid) individuals, which are 100% homozygous.     

Occurrence of aneuploidy in radiation gynogenesis of carp, Cyprinus carpio L.

DNA content and chromosome numbers were studied in gynogenetic and normal carp embryos. Gynogenetic embryos showed the haploid chromosome number; the occurrence of spontaneous diploidization was rare. Early cold shock treatment of the gynogenetic zygotes resulted in the retention of the second polar body producing a much higher proportion of normally developing viable larvae. The appearance of large numbers of aneuploid embryos in the cold-treated gynogenetic group is explained by the loss or breakdown of chromosomes that are caused in their turn by the absorbtion of the second polar body through the cold treatment.     

雌核发育银鲫与两性生殖彩鲫卵壳蛋白组分的比较分析及其受精前后的变化

以雌核发育银鲫和两性生殖彩鲫的成熟卵为材料,分离卵壳,经处理得到卵壳可溶性蛋白组分。SDS－PAGE梯度凝胶电泳分析在雌核发育银鲫中揭示出3条较明显的差异蛋白带。同时,采用相同处理方法对受精前后卵壳蛋白组分进行比较分析后发现,这些差异蛋白带在受精后发生了变化,其带纹表现为减弱或消失,表明这些差异蛋白可能与受精过程相关。 Abstracts:Egg chorions were isolated from unfertilized and fertilized eggs of gynogenetic silver crucian carp and gonochoristic color crucian carp by homogenization and further purification techniques.Then,soluble proteins were extracted from the isolated egg chorions,and were analyzed by gradient SDS-PAGE.Three differential protein bands were revealed between the gynogenetic silver crucian carp and gonochoristic color crucian carp.Furthermore,these differential proteins were demonstrated to undergo obvious changes during fertilization.It was suggested that these differential proteins should be related to the special fertilization process in gynogenetic silver crucian carp.     

雌核发育和两性融合发育鱼卵调控精核受精发育的生化特性研究

两性融合生殖的鱼卵受精后,精核能疏松、解凝,形成雄性原核：雌核发育银鲫卵子受精后,精核发育受到抑制,无法形成原核。采用显微注射去膜精核以及细胞学和电镜观察的方法,本文对两类鱼卵受精后精核早期发育的生化性质进行了初步探讨,并着重研究了雌核发育银鲫卵子控制精核发育的生化特征。实验结果显示,两性融合生殖鱼类卵质中,一定量的Ｃａ２＋的存在,二硫键的还原作用对于精核的发育显然是必要的;而在雌核发育银鲫卵中,Ｃａ２＋的功能和二硫键的还原作用与精核发育受到抑制之间并无直接联系。银鲫卵质中似乎显示出异常的磷酸酶脂解活性,导致磷酸化过程无法进行,使精核解凝受到阻碍。另外,两性融合生殖的鱼卵重质层中具有大量诱导精核原核化的有关因子,而银鲫卵质中则缺少该因子（或活性极低）。银鲫卵质中还可能缺乏某些与雄性原核的核膜重组装有关的大分子物质。     

人工雌核发育鲢的遗传多样性及异源遗传物质整入的RAPD分析

运用RAPD技术对连续二代人工雌核发育鲢的遗传多样性及异源遗传物质的整入进行了分析 ,结果表明 :一代雌核发育鲢 ,个体间遗传相似度为 0 94 5— 0 995 6 ,多样性指数为 0 175 ;二代雌核发育鲢 ,个体间遗传相似度为0 96 15— 1 0 0 ,平均为 0 985 2 ,多样性指数为 0 0 6 2。研究揭示经过连续二代人工雌核发育后 ,其遗传多样性明显减少 ,种质进一步纯化。通过对雌核发育鲢二代、亲本鲢和雄鲤的RAPD扩增比较 ,发现雌核发育鲢含有少数与父本相同的特异DNA扩增带 ,而亲本鲢没有 ,在基因水平上表明雌核发育鲢整入了雄鲤的遗传物质     

High efficiency of meiotic gynogenesis in sea lamprey Petromyzon marinus

Induction of androgenesis and gynogenesis by applying a pressure (PS) or heat shock (HS) to double the haploid chromosomal set results in progenies possessing only chromosomes from a single parent. This has never been accomplished in representatives of Agnatha. The objective of this study was to induce gynogenesis and androgenesis in sea lamprey Petromyzon marinus. For gynogenesis experiments, ultraviolet (UV)-irradiated sperm was used to activate sea lamprey eggs and HS or PS were applied to inhibit the second meiotic division and consequently induce diploidy in the embryos. The UV irradiation of immobilized sperm was performed for 1 min at 1,719 J m(-2). HS of 35+/-1 degrees C for 2 min and PS of 9,000 psi for 4 min were applied at different times after egg activation (8, 12, 20, and 24 min or 8, 16, and 24 min for HS or PS, respectively). Regardless of the induction time of the HS, survivals at pre-hatching stage were similar. In contrast, PS applied 8 min after activation appears to increase survival rate of pre-hatched embryos in comparison to 16 and 24 min after activation. In control groups, without shock treatment (no diploidization), there were no survivors. All deformed, gynogenetic embryos were confirmed to be haploids and died prior to burying themselves in the sand. We confirmed by flow cytometry that progenies produced using both shock methods surviving to the next stage, burying in the substrate, were diploid gynogenetic. For the androgenesis experiments, UV-irradiated eggs (1,719 J m(-2) for 1 min) were fertilized with non-treated sperm and HS was applied to restore diploidy of the eggs. Several attempts have been made to optimize the parameters used. HS of 35+/-1 degrees C was applied 110, 140, 170, 200, and 230 min after activation for 2 min. Low yields of androgens were obtained and all animals died within a week after hatching. These techniques will allow to establish meiotic gynogenetic lines of sea lamprey for determining sex differentiation in this species and to analyze its hormonal and environmental regulation.     

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.     

Microsatellite analysis of different ploidy offspring of artificial gynogenesis in Cyprinus carpio

Gynogenesis was induced by using UV-irradiated spermatozoa of blunt snout bream Megalobrama amblycephala to activate eggs of common carp Cyprinus carpio. The maternal genome was then duplicated by cold shock in 0 to 4° C cold water to retain the second polar body. Two kinds of fry, normal fry and abnormal tortuous fry, were hatched. Their DNA content was measured by flow cytometry. The normal fry were identified as diploid, representing the successful gynogenesis in C. carpio whereas the abnormal tortuous fry were haploid. Ten microsatellite loci were used to study the genetic diversity among C. carpio, diploid gynogenetic C. carpio and unduplicated haploid tortuous fry. The results indicated that the genetic homozygosity of gynogenetic C. carpio was significantly higher than that of C. carpio. The genetic homozygosity of the haploid C. carpio was intermediate between that of gynogenetic C. carpio and C. carpio. It might be easier for the allogenetic DNA fragments to be integrated into the haploid genome than into diploid gynogenetic genome.     

两个人工雌核发育红白锦鲤群体的RAPD标记分析

采用PAPD方法,对两个人工雌核发育红白锦鲤群体进行了多态性及分子标记分析。结果表明,同一雌核发育群体具有基本一致的扩增产物,而不同雌核发育群体间的扩增产物则有较大不同;并从30个随机引物的扩增谱带中找到了7个引物（Opo-7,Opo-9,Opo-12,Opo-14,Opj-4,Opj-8和Opj-10）的扩增谱带可以作为两个不同雌核发育群体间的遗传标记。由UPGMA聚类法构建的分支系统树清晰地反映了两个雌核发育群体及其个体间的相互关系。     

鞍带石斑鱼精子诱导斜带石斑鱼雌核生殖

石斑鱼性成熟时间长和雌雄同体的特性延长了其育种的周期, 采用人工雌核生殖策略能够显著提高选育效率。研究筛选了鞍带石斑鱼(Epinephelus lanceolatus)精子人工诱导斜带石斑鱼(Epinephelus coioides)雌核生殖的最佳条件, 并通过形态学、流式细胞术和微卫星分子标记等方法进行鉴定。结果显示, 灭活的异源精子可成功诱导斜带石斑鱼人工雌核生殖, 精子灭活的最佳紫外照射强度为518 mJ/cm2; 卵子受精后6min在4—6℃的条件下进行冷休克处理, 可获得最优的40%的表观受精率(卵子正常分裂比率)和29.7%的孵化率, 而未冷休克的对照组具有83.5%的表观受精率和80%的单倍体率。多重比较结果表明, 在表观受精率方面人工诱导的时间差异(P=0.579)和水温差异(P=0.133)并不显著; 而在孵化率上, 处理时间组间差异极显著(P=0.002), 而在水温组中差异不显著(P=0.396)。胚胎发育结果显示, 人工雌核生殖诱导会导致孵化率降低, 单倍体胚胎出现明显的发育障碍。微卫星分子标记鉴定结果显示, 石斑鱼人工雌核生殖子代的遗传物质大部分来自于母本, 同时也存在异精效应。     

Microsatellite markers in common carp (Cyprinus carpio L.)

Microsatellite markers of the poly (CA) type in common carp ( Cyprinus carpio L.) are described. Clones containing a (CA) repeat were isolated from a common carp genomic library and sequenced. The number of repeats found was high compared to mammals but comparable with other teleost fishes. Classification of the repeats (perfect, imperfect and compound) are compared with the Atlantic cod ( Gadus morhua L.), rainbow trout ( Oncorhynchus mykiss ), and Atlantic salmon ( Salmo salar L.). A total of 41 primer sets were designed and tested for polymorphism on a test panel of eight animals (derived from outbred lines, inbred lines and gynogenetic clones). Thirty-two markers were found to be polymorphic. The heterozygosity in the outbred animals was 60·4%, 51·1% in the inbred animals and 0% in the gynogenetic clones. The average number of alleles among the eight animals was 4·7 per marker. Six markers (18·8%) gave an additional polymorphic amplification product besides the polymorphic amplification product in the expected size range. The possibility that these loci are tetraploid is discussed. The polymorphic loci described for common carp will be valuable as genetic markers for use in population, breeding, and evolutionary studies.