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.     

Spontaneous diploidization of maternal chromosome set in ornamental (koi) carp, Cyprinus carpio L.

An unusually high frequency of spontaneous diploidization in a maternal chromosome set (SDM) was discovered in one ornamental carp (koi) female in an experiment on these fish for induced gynogenesis. Spontaneous appearance of diploid embryos in the gynogenetic offspring (intact eggs × irradiated sperm) and appearance of triploids among control fish (intact eggs × intact sperm) obtained from koi female and males of edible carp indicated spontaneous diploidization of maternal chromosomes (SDM). Possible cytological processes causing this phenomenon are discussed.     

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.     

雌核发育银鲫的受精生物学研究——天然雌核发育银鲫繁殖方式的讨论

本文研究了同源雌核发育银鲫精子在4种类型的雌核发育银鲫卵中的发育特征。初步揭示了天然雌核发育银鲫根据精子的来源不同而分别具有二种不同的繁殖方式,对其在维持雌核发育银鲫种群生存,促使克隆分化等方面的独特的生物学意义进行了讨论。     

鲫鱼的人工和天然雌核发育

用照射处理的草鱼或华南鲤的精液授精的鲫鱼卵为单倍体雌核发育;所有单倍体胚胎都是畸形,并在孵化前死亡。如果用照射精液授精的鲫鱼卵在18—19℃的水中放置2分钟,然后在0—3℃的冰水中放置20分钟,再放回室温水中孵化,可以从每百颗受精卵中得到4.5—11.9(平均8.34)尾成活的二倍体仔鱼。根据38尾银鲫(其中33尾捕自黑龙江省方正县双凤水库)的细胞学和胚胎学检查,证实它们是雌核发育繁殖的。       

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.     

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 gynogenetic progeny of allotetrapioid hybrids of red crucian carp × common carp had important significances in both biological evolution and production application.     

异源精子在银鲫雌核发育子代中的生物学效应

黑龙江省方正县双凤水库的两性型银鲫群体是三倍体雌核发育种群。异源精子不仅能刺激银鲫卵雌核发育,而且还能影响雌核发育子代的某些性状,如对于子代的生长、性比、体色和肝脏LDH同工酶等都产生了影响。为区别于原有术语“雌核发育gynogenesis”,我们把这种表现了异源精子生物学效应的雌核发育称之为“异精雌核发育allogynogenesis”,发育的子代称之为“异育银鲫”。异育银鲫已以其明显的生长优势在生产上显示了优良的经济性状。       

Morphology of androgenetic and gynogenetic grass carp, Ctenopharyngodon idella (Valenciennes)

Crosses between female carp x male grass carp resulted in androgenetic grass carp and hybrids. Fertilizing grass carp eggs with carp milt that had been irradiated with ultraviolet light for 15 min at 1.0 mW/cm² produced gynogenetic grass carp. We compared the morphology of experimental progeny with that of the parental species to determine if inheritance was strictly matroclinous in gynogenesis and strictly patroclinous in andro-genesis. Dorsal and anal fin rays, lateral line scales, gill rakers, pharyngeal teeth, and the relative length of the dorsal fin all suggested that gynogenetic grass carp were identical with the maternal species and androgenetic grass carp with the paternal species. Hybrids were intermediate between the parents except that the number of anal rays was identical with that in carp. The number of dorsal rays, the body depth, and the pharyngeal teeth in hybrids were more like those in carp than in grass carp, whereas the number of gill rakers was more similar to that in the paternal species.     

天然雌核发育银鲫卵子控制异源精核发育的受精学机制

作者对两性融合生殖鱼和雌核发育银鲫脱膜卵受精的精核发育进行了观察,并采用鱼类卵子无细胞系对以上两类卵质提取物体外诱导经Ｔｒｉｔｏｎ—Ｘ１００处理的精子及其发育进行了初步研究,结果表明在两性融合生殖型脱膜鱼卵中精核通过解凝最终形成原核,而在雌核发育的银鲫脱膜卵子中部分精核体积虽有一定程度的增加,但始终没有观察到原核的发育;在体外诱导实验中,经Ｔｒｉｔｏｎ—Ｘ１００处理的精子在两类卵质提取物中充分发育,都出现了类似体内原核的状态。该现象提示在银鲫卵质中存在有促使精核形成原核的因子,但在正常受精状态下,由于银鲫卵质促使精核核膜解体的功能的异常,使覆盖精子头部的核膜不能象在两性融合生殖受精卵子中进行崩解,精核进一步的原核发育受到抑制。另外,建立体外诱导系统的重要意义,在于它为研究雌核发育调控的分子学机制提供了一条有效途径。     

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。       

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     

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。     

人工转性异育银鲫精子在两性融合型和雌核发育型卵质中的发育特征及其应用意义

通过甲基睾丸素诱导产生的雄性雌核发育银鲫产生的精子,能与两性融合生殖的鲤鱼卵受精结合,并能正常地转化为雄性原核。但在卵裂开始后,可观察到诸如核物质丢失、多极分裂等异常现象。从而进一步证实了作者过去提出的染色体不相容假设。转性异育银鲫精核在雌核发育银鲫卵中能初步地转化为雄性原核,但其发育程度低于雌核。根据双重控制假设,作者初步认为,转性异育银鲫精子在进入雌核发育型卵的过程中没有受到初级控制的作用。这或许是因为转性雄鱼的雌性遗传基础导致精子的某种特异性因子缺失的缘故。由于雌核发育银鲫卵质中次级控制的存在,使得解凝的精核不能完全转化为雄性原核。     

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.     

Production of F1 and F2 diploid gynogenetic tilapias and analysis of the “Hertwig curve” obtained using ultraviolet irradiated sperm

Summary In this study, a Hertwig effect with a non-typical biphasic curve was obtained using sperm irradiated with increasing intensities of UV. The first phase of the UV curve appeared to be quite different from that normally demonstrated using or x-ray irradiation. This difference is characterised throughout the length of the first phase by (1) low and stable embryo hatching rates of about 3.5%, and (2) exclusive formation of haploid embryos at any irradiation intensity. Additionally, at both phases, the ability of the sperm to induce morula formation was not affected at all, and no aneuploidy nor chromosomal fragments could be seen. Therefore, it was suggested that in this fish the lethal effect of UV irradition on sperm is mainly expressed on early differentiative events during embryogenesis, which lead to a degeneration of the embryos during early stages of their development. The possible mechanism by which haploidy is achieved during the first phase is discussed. Two generations of diploid gynogenetic tilapias were induced by activating Oreochromis aureus eggs with UV-irradiated O. niloticus sperm and by using the heat-shock technique, at optimized conditions, for the prevention of the second polar body extrusion. Species specific dominant genetic markers (serum esterases and tail striation) were used to confirm the exclusive content of the maternal genome in gynogenetic offspring. Very low survival rates (0.36%) were shown in F₁ gynogenetic fish as well as a high incidence of malformations among survivors. In the second gynogenetic generation, both significantly higher survival rates (3.6%) and a considerably reduced incidence of malformations were obtained. We suggest that low frequencies of recombination occur in this species and cause a rapid increase in the inbreeding level. This is followed by the expression of lethal and defective genes that are considerably reduced after second generation selection.     

Growth and feed utilization in two strains of gibel carp, Carassius auratus gibelio: paternal effects in a gynogenetic fish

Gibel carp ( Carassius auratus gibelio Bloch) is a natural gynogenetic fish which requires sperm of the same or related species to activate egg development. The eggs of one gibel carp were divided into two batches. One batch was 'fertilized' with sperm from gibel carp (strain DD), and the other 'fertilized' with sperm from red common carp ( Cyprinus carpio red variety) (strain DR). The juveniles were transferred to the laboratory 36 days post-hatch. Triplicate groups of each strain were fed a formulated diet at either 3% or satiation ration for 8 weeks. At both the restricted and satiation rations, specific growth rate was significantly higher in strain DR than in strain DD. At the 3% ration, there was no significant difference in feeding rate or feed conversion efficiency between the two strains. At the satiation ration, strain DR had a significantly lower feeding rate but higher feed conversion efficiency than strain DD. At the satiation ration, strain DR had a significantly lower intake protein, but higher recovered protein than strain DD. There was no significant difference in faecal protein loss between the two strains. At the 3% ration, strain had no significant effects on intake protein, faecal protein or recovered protein. Neither faecal energy loss nor recovered energy was affected by strain or ration. At both the 3% and satiation ration, final body contents of dry matter and lipid were significantly lower in strain DR than strain DD, while there was no significant difference in protein and energy content between the two strains at either ration level. The results suggested that gibel carp 'fertilized' with sperm of common carp grew faster than those 'fertilized' with sperm of gibel carp through increased feed conversion efficiency and protein retention.     

Breakdown of the sperm nuclear envelope is a prerequisite for male pronucleus formation: direct evidence from the gynogenetic crucian carp Carassius auratus langsdorfii

The gynogenetic fish, Carassius auratus langsdorfii (the ginbuna, a crucian carp), provides an interesting model for the study of the mechanisms controlling male pronucleus formation. When the sperm nucleus of a different subspecies (C. a. cuvieri) is incorporated into the gynogenetic egg, the nuclear envelope of the spermatozoon is not broken down, and the pronucleus fails to develop, although dispersion of the sperm chromatin occurs to some extent within the space limited by the nuclear envelope. When spermatozoa without plasma membranes and nuclear envelopes were microinjected into mature activated eggs, the sperm nuclei underwent chromatin dispersion, nuclear envelope formation, DNA synthesis, and transformation into male pronuclei. These results indicate that the failure of the male pronucleus to form in ginbuna is primarily due to the failure of sperm nuclear envelope breakdown. We conclude that sperm nuclear envelope breakdown is an indispensable step for the development of the male pronucleus.     

Two unisexual artificial polyploid clones constructed by genome addition of common carp (<Emphasis Type="Italic">Cyprinus carp</Emphasis>) and crucian carp (<Emphasis Type="Italic">Carassius auratus</Emphasis>)

A polyploid hybrid fish with natural gynogenesis can prevent segregation and maintain their hybrid vigor in their progenies. Supposing the reproduction mode of induced polyploid fish being natural gynogenesis, allopolyploid hybrid between common carp and crucian carp into allopolyploid was performed. The purpose of this paper is to describe a lineage from sexual diploid carp transforming into allotriploid and allotetraploid unisexual clones by genome addition. The diploid hybrid between common carp and crucian carp reproduces an unreduced nucleus consisting of two parental genomes. This unreduced female pronucleus will fuse with male pronucleus and form allotriploid zygote after penetration of related species sperms. Allotriploid embryos grow normally, and part of female allotriploid can produce unreduced mature ova with three genomes. Mature ova of most allotriploid females are provided with natural gynogenetic trait and their nuclei do not fuse with any entrance sperm. All female offspring are produced by gynogenesis of allotriploid egg under activation of penetrating sperms. These offspring maintain morphological traits of their allotriploid maternal and form an allotetraploid unisexual clone by gynogenetic reproduction mode. However, female nuclei of rare allotriploid female can fuse with penetrating male pronuclei and result in the appearance of allotetraploid individuals by means of genome addition. All allotetraploid females can reproduce unreduced mature eggs containing four genomes. Therefore, mature eggs of allotetraploid maintain gynogenetic trait and allotetraploid unisexual clone is produced under activation of related species sperms.