中华绒螯蟹多倍体的诱导研究 : Ⅱ.热休克诱导中华绒螯蟹三倍体胚胎和四倍体 状幼体

采用热休克方法对中华绒螯蟹进行了三倍体、四倍体的人工诱导研究。结果表明,在卵子受精后（发育水温２０±１℃）１０～２５分钟,用３８～４１℃的高温处理１～２分钟,三倍体的平均诱导率为１０％～５０％,而对受精后３０分钟的卵子进行诱导,或在４０℃下处理超过２５分钟和处理温度高于４２℃诱导１５分钟的各试验组,均未检测到有三倍体胚胎。依据诱导三倍体的适宜诱导温度和处理时间,将受精后８１／２～９５／６小时的卵子用４０℃热休克处理１５分钟,均获得了一定比例的四倍体胚胎,其中四倍体最高诱导率为５２９６％。检查卵受精后８５／６小时经热休克处理所孵得的氵蚤状幼体,其四倍体的出现频率约为１９７％。讨论了热休克处理促使第一极体保留生产三倍体和抑制第一次卵裂诱导四倍体的条件和技术关键,以及诱导参数与胚胎发育、胚胎存活的相互关系等问题。     

静水压休克诱导水晶彩鲫三倍体和四倍体的细胞学机理初探

本文分析了静水压休克诱导水晶彩鲫三倍体和四倍体的细胞学机理,促使第二极体保留的压力敏感期较短,在卵子第二次成熟分裂中后期的某段时间,也就是受精后４－５ｍｉｎ时;而在这之前,为卵子启动期,施加压力会破坏卵子的激动和修整过程,造成发育受阻;其后,为压力不应期,此时第二次成熟分裂态势也趋明朗,压力对保留第二极体失去作用。本文还对促使第二极体保留生产鱼类三倍体和抑制第一次卵裂诱导鱼类四倍体的条件等问题进行     

三倍体九孔鲍生物学特性的初步探讨

采用细胞松弛素B(Cytochalasin B)抑制九孔鲍受精卵第二极体的释放获得三倍体材料,进行三倍体与普通二倍体的生长对比试验,试验分为仔鲍和成鲍两个养殖阶段。观察了三倍体性比和性腺发育情况。结果表明,三倍体九孔鲍比普通二倍体九孔鲍具有显著的生长优势。投放的担轮幼虫经5个月培育,三倍体平均壳长可达2.25cm,壳长绝对生长量是普通二倍体的1.20倍;平均壳长3.25cm的个体经6个月培育后壳长为5.43cm,绝对增长量是普通二倍体的1.80倍。三倍体成鲍性比和二倍体没有显著差异,接近1:1;三倍体性腺发育程度较低,雌、雄性腺大小和体积比例均明显小于同期培育的二倍体成鲍。     

6-DMAP抑制栉孔扇贝（♀）×虾夷扇贝（♂）受精卵第一极体释放后染色体的分离状况

三倍体贝类因其具有生长快、个体大、肉质佳和成活率高等优良的经济性状而存水产养殖业中具有重要的应用价值。获得100％三倍体贝类的最佳途径是通过四倍体贝类与二倍体贝类杂交来获得。目前已经发展出多种诱导四倍体的方法,包括抑制受精卵第一极体的释放、抑制受精卯的卵裂、细胞融合和利用人工雌核发育等方法。人工诱导四倍体技术已经在多种鱼类上获得成功。     

白鲫（♀）×红鲫（♂）异源四倍体鱼的倍性操作及其生殖力的研究

采用热休克调控技术诱导出103尾第一代白鲫（♀）×红鲫（）异源四倍体鱼,并对其生殖力进行了研究。1或2龄的四倍体雄鱼能产生精子,而雌鱼不能产生正常的卵。将异源四倍体雄鱼与二倍体白鲫雌鱼交配产生倍间三倍体鱼,染色体检查证明是整三倍体（3N＝150）,但其受精率很低（11．4—51．3％,平均32．4％）．网箱养殖结果表明,倍间三倍体白鲫的生长速度比白鲫快30％以上,雌雄均不育．并用冷休克处理回收异源四倍体4N（）×白鲫2N（♀）受精卵的第二极体产生了新的四倍体鱼．文中还对第一代异源四倍体鱼的批量生产、生殖能力以及异源三倍体鱼的生产应用进行了讨论．     

6-DMAP抑制栉孔扇贝（♀）×虾夷扇贝（♂）受精卵第一极体释放后染色体的分离状况

三倍体贝类因其具有生长快、个体大、肉质佳和成活率高等优良的经济性状而在水产养殖业中具有重要的应用价值[1].获得100%三倍体贝类的最佳途径是通过四倍体贝类与二倍体贝类杂交来获得.目前已经发展出多种诱导四倍体的方法,包括抑制受精卵第一极体的释放、抑制受精卵的卵裂、细胞融合和利用人工雌核发育等方法.人工诱导四倍体技术已经在多种鱼类上获得成功[2].迄今,国内外学者已对多种贝类[3]进行了四倍体育种研究,都获得了一定比例的四倍体胚胎或幼虫,甚至是稚贝,但是除了太平洋牡蛎Crassostrea gigas(Thunberg)[4]外大部分由于存活率低最后都没有获得两性能育且形成群体的四倍体贝类.     

罗氏沼虾四倍体诱导的研究

对罗氏沼虾卵子极体排出时间和第一次卵裂时间进行了观察,并在此基础上,采用热休克和细胞松弛素 B(C,B)两种诱导方法成功诱导出了罗氏沼虾四倍体。石蜡切片显示,当罗氏沼虾卵子完全成熟时,第一极体已经 排出。第二极体排出的时间约在受精后55min。经过雌性原核和雄性原核的联会,第一次卵裂的时间约在受精后 210-230min。设计了多种处理条件,其中热休克诱导罗氏沼虾四倍体的最好条件是:受精后210min,用40℃水温处 理胚胎1．5min,在这种条件下,最高可以得到35．86％的四倍体率;而C．B诱导罗氏沼虾四倍体的最好条件是:受精 后230min,用1．0mg/L的C．B处理胚胎10min,此时最高四倍体诱导率达33．78％。     

由鲤(Cyprinus carpio)和鲫(Carassius auratus)染色体组叠加构建的 两个单性人工多倍体克隆

具有天然雌核发育的多倍体杂种鱼可防止杂种优势的分离并保持其后代的杂种优势. 由于假设诱发的多倍体鱼类的生殖模式是天然雌核发育的, 我们进行了鲤鲫杂种的多倍体诱发, 目的是描述经染色体组叠加由有性鲤鲫二倍体转化为异源三倍体及异源四倍体克隆谱系. 鲤鲫杂种产生未减数而具有两亲本染色体组杂种卵子, 未减数的雌核可与入卵的雄核融合叠加形成三倍体合子. 鲤鲫异源三倍体胚胎发育正常, 部分异源三倍体雌性个体可产生未减数的、仍保留母本的三套染色体的成熟卵子. 绝大部分鲤鲫异源人工三倍体个体的成熟卵子的雌核不与入卵的雄核融合, 具有天然雌核发育特性. 异源三倍体卵子在入卵精子的激动下由雌核发育产生全雌后代, 并形成一个单性克隆系, 后代保留异源三倍体母本的形态特征, 并靠雌核发育的生殖方式形成异源三倍体克隆系. 极少数异源三倍体个体的成熟卵子的雌核可与入卵的雄核融合, 再通过染色体组叠加形成鲤鲫异源四倍体. 所有异源四倍体的雌性产生未减数的、含有4个染色体组的成熟卵子. 异源四倍体的成熟卵子保持雌核发育特性, 在近类的精子诱发下产生单性后代, 形成一个异源四倍体单性克隆.     

乌鳢三倍体诱导及其生长

采用热休克抑制第二极体排放的方法诱导乌鳢(Channa argus)三倍体,以探索人工诱导乌鳢三倍体的理想热休克条件。采用DNA含量测定法和红细胞核大小鉴定法对获得的鱼苗倍性进行鉴定,同时对普通二倍体乌鳢群体与三倍体群体的生长差异进行了比较研究。结果表明,(1)热休克法适宜诱导条件为,(28±0.5)℃培育水温授精后4 min,在水温42℃条件下持续处理3 min,三倍体诱导率最高,达到87.69%;(2)三倍体与二倍体DNA含量差异极显著(P0.01),其比值为1.50︰1.00;(3)三倍体和二倍体在红细胞核长径、红细胞核体积、红细胞核面积等6项指标上存在极显著差异(P0.01);与二倍体相比,三倍体红细胞体积和核体积分别是其1.69倍和1.60倍;(4)在4月龄与8月龄,三倍体的体长和体重比二倍体稍高,但两者差异不显著(P0.05)。本实验结果为进一步开展乌鳢倍性育种奠定了基础。     

二倍体鲫鲤F2产生不同倍性卵子的证据

在检测到鲫鲤F2产生3种不同大小(直径分别为0.13 cm,0.17cm和0.2 cm)类型的卵子基础上,进行了F2(♀)×红鲫(♂)及F2(♀)×四倍体鲫鲤(♂)的交配实验.通过染色体计数和流式细胞仪分析,在F2(♀)×红鲫(♂)后代中获得了四倍体、三倍体、二倍体鱼;在F2(♀)×四倍体鲫鲤(♂)后代中获得了四倍体和三倍体鱼.这两个交配组合后代中出现的不同倍性的鱼类为证明鲫鲤F2能产生三倍体、二倍体和单倍体卵子提供了进一步证据.F2(♀)×红鲫(♂)中雄性四倍体鱼的存在说明在四倍体后代中存在基因型为XXXY的个体.对上述两个交配组合后代的四倍体鱼和三倍体鱼的性腺结构观察表明四倍体鱼是可育的,而三倍体鱼是不育的.作者认为鲫鲤F2能够产生二倍体和三倍体卵子与核内复制机制和生殖细胞的融合有关.     

A Tripolar Spindle Formed at Meiosis I Assures the Retention of the Original Ploidy in the Gynogenetic Triploid Crucian Carp, Ginbuna Carassius auratus langsdorfii

A triploid crucian carp, ginbuna ( Carassius auratus langsdorfii ), reproduces by gynogenesis, in which sperm of diploid ginbuna or of other species triggers the development of the triploid eggs, but a male genome makes no contribution to the zygotic genome. Gynogenesis is maintained by two mechanisms: exclusion of male genome during fertilization and retention of somatic ploidy levels during oogenesis. We examined the mechanisms responsible for producing unreduced eggs. Microfluorometry with a DNA staining dye showed that DNA content in the ginbuna oocytes was not reduced in half during meiosis I. Cytological observations revealed that a tripolar spindle was formed at meiosis I and the first polar body was not extruded, whereas an ordinary bipolar spindle was formed and the second polar body was extruded at meiosis II. Activity of histone H1 kinase (as an indicator of maturation-promoting factor) decreased transiently between meiosis I and II, strongly suggesting a "normal" meiotic cycle progression in the ginbuna oocytes. These results have indicated that in the gynogenetic ginbuna the somatic ploidy levels are maintained by inhibiting the first polar body extrusion via the formation of the tripolar spindle at meiosis I.     

A Complementary Method for Production of Tetraploid Crassostrea gigas Using Crosses Between Diploids and Tetraploids with Cytochalasin B Treatments

We present a new method to produce tetraploid Crassostrea gigas by cytochalasin B inhibition of polar body 2 expulsion in diploid females crossed with tetraploid males. This offers a means of direct introgression of genetic characters from selected diploid to tetraploid lines, avoiding a triploid step. Offspring larval ploidy shifted over time and depended on size, with tetraploids more frequent among the smaller larvae and triploids among the large. Viable tetraploids were found at 4 and 6 months, indicating the technique was successful. The possibility that gynogenesis occurred was tested by microsatellite analysis to confirm the presence of paternally inherited alleles. These were present in all animals of the 2n × 4n + CB (female first) cross. However, a 4n × 2n + CB cross produced triploids, including some gynogens. Our method illustrates for the first time that diploid C. gigas eggs, if selected for large size, can give viable tetraploid offspring.     

Triploid and Tetraploid Zhikong Scallop, <Emphasis Type="BoldItalic">Chlamys farreri</Emphasis> Jones et Preston,Produced by Inhibiting Polar Body I

: Triploid scallops are valuable for aquaculture because of their enlarged adductor muscle, and tetraploids are important for the commercial production of triploids. We tested tetraploid induction in the zhikong scallop by inhibiting polar body I in newly fertilized eggs. The ploidy of resultant embryos was determined by chromosome counting at 2- to 4-cell stage and by flow cytometry thereafter. Embryos from the control groups were mostly diploids (79%), along with some aneuploids. Embryos from the treated groups were 13% diploids, 18% triploids, 26% tetraploids, 13% pentaploids, and 36% aneuploids. Tetraploids, pentaploids, and most aneuploids suffered heavy mortality during the first week and became undetectable among the larvae at day 14. Five tetraploids (2%) were found among a sample of 267 spat from one of the replicates, and none was detected at day 450. The adductor muscle of triploid scallops was 44% heavier (P < .01) than that of diploids, confirming the value of the triploid technology in this species.     

Delayed meiosis and polar body release in eggs of triploid Pacific oysters, Crassostrea gigas, in relation to tetraploid production

The dynamics of polar body release are important for creating polyploid shellfish. For producing triploids, these dynamics concern meiosis in diploid eggs and are well understood. For creating tetraploids, eggs from triploids are employed and the dynamics, variation, and environmental influences upon polar body release are less studied. We investigated the effects of several agents on the timing of 50% first polar body (PB1) release in eggs of triploids. PB1 release is generally slower in triploid eggs than diploid ones at 26 degrees C. Lowering the temperature (from 26 to 19 degrees C) had a marked effect on timing of 50% PB1 in both diploid and triploid eggs. While lower temperature merely slowed development in diploid eggs, it nearly halted it in triploid eggs. At any temperature, the variability in 50% PB1 release was much higher in triploid eggs than diploid ones; this variation occurred both within eggs from individual females and among eggs from different females. The amount of time eggs remain in seawater between the time they are stripped and fertilized (or time of hydration) also affected rate of meiosis. In triploid eggs, the average time necessary for the expulsion of 50% PB1 was 23 min post-fertilization (PF) for 75 min of hydration versus 29 min PF for 35 min. However, increasing the time of hydration had no effect on the variability in the timing among females. Serotonin also had no effect on the dynamics of polar body release in triploids. Variability among triploid females in timing of meiosis cannot be improved with any treatments we tried. Consequently we recommend that treatments of triploid eggs to produce tetraploids incorporate a single female at a time.     

Reproduction and the origin of polyploids in hybrid salamanders of the genus Ambystoma

Eggs and larvae produced by diploid, triploid, and tetraploid females collected from breeding ponds on Pelee Island in Lake Erie were studied to examine the reproductive mechanism. No instance of parthenogenesis was found as all examined females required sperm to produce viable progeny. Diploid females produced diploid and triploid larvae, triploid females produced triploid and tetraploid larvae, and tetraploid females produced triploid and tetraploid larvae. The majority of the eggs produced by hybrid females do not develop or do not complete embryogenesis. Electrophoretic examination of females and their offspring demonstrate that the male genome is being incorporated in reduced as well as unreduced eggs produced by all three ploidy classes of females. The elevation of ploidy among Pelee Island Ambystoma is attributed to sperm incorporation in unreduced eggs. Triploid as well as tetraploid individuals are constantly being produced. A critical examination of the literature on parthenogenetic or gynogenetic modes of reproduction in North America Ambystoma hybrids shows no conclusive evidence supporting these modes and it is suggested that the reproductive mechanism found among Pelee Island female hybrids may be more generally applied to other hybrid Ambystoma populations.     

Male meiosis in polyploid silkworms,Bombyx mori L. (Lepidoptera : Bombycidae)

Tetraploid and hexaploid silkworms, Bombyx mori L. (Lepidoptera Bombycidae) were induced by applying a cold shock to diploid and triploid eggs at the first cleavage stage. Male meiosis in the primary spermatocytes of these silkworms having a different ploidy was observed. In the polyploid cells, chromosome bridges, 2 for the tetraploid and 3 for the hexaploid, occurred between the newly formed daughter nuclei at telophase. Observation in the living spermatocytes showed that tetraploid cells needed a longer time than the diploid ones to complete the first meiotic division. The delay in the cell division may be responsible for the high sterility of the tetraploid males.     

Microsatellite-centromere distances and microsatellite diversity in different ploidy classes of Chinese shrimp (Fenneropenaeus Chinensis)

This is the first report of microsatellite-centromere mapping in this commercial species Fenneropenaeus Chinensis, and will be important for providing fixed points in the linkage groups of genetic maps. Triploid Chinese shrimp was induced by heat shock. The fertilized eggs were treated either by retention of the first polar body or the second polar body to produce Meiosis I (MI) or Meiosis II (MII) triploid. The triploidy status in each Chinese shrimp could be confirmed by nine polymorphic microsatellite loci, in which the parents with different alleles and the female parents were each heterozygous. The nine loci were mapped in relation to their centromeres in three MII triploid families, which were induced by retention of the second polar bodies after fertilization with sperm. Microsatellite-centromere (M-C) distances ranged from 9.6 cM to 37 cM under the assumption of complete interference. Information on the positions of centromeres in relation to the microsatellite loci will represent a contribution towards assembly of genetic maps in F. chinensis. Twelve polymorphic microsatellites were used to assess the heterozygosity and allelic diversity in different ploidy classes. As expected, triploids were significantly more polymorphic than diploids. The diploids had an average heterozygosity and allelic diversity value of 0.86, whereas the triploids heterozygosity averaged 0.93 and had allelic diversity value of 1.29. However, MI triploids were not significantly more polymorphic than MII in the microsatellite loci.     

The formation of the polyploid hybrids from different subfamily fish crossings and its evolutionary significance

This study provides genetic evidences at the chromosome, DNA content, DNA fragment and sequence, and morphological levels to support the successful establishment of the polyploid hybrids of red crucian carp x blunt snout bream, which belonged to a different subfamily of fish (Cyprininae subfamily and Cultrinae subfamily) in the catalog. We successfully obtained the sterile triploid hybrids and bisexual fertile tetraploid hybrids of red crucian carp (RCC) (female symbol) x blunt snout bream (BSB) (male symbol) as well as their pentaploid hybrids. The triploid hybrids possessed 124 chromosomes with two sets from RCC and one set from BSB; the tetraploid hybrids had 148 chromosomes with two sets from RCC and two sets from BSB. The females of tetraploid hybrids produced unreduced tetraploid eggs that were fertilized with the haploid sperm of BSB to generate pentaploid hybrids with 172 chromosomes with three sets from BSB and two sets from RCC. The ploidy levels of triploid, tetraploid, and pentaploid hybrids were confirmed by counting chromosomal number, forming chromosomal karyotype, and measuring DNA content and erythrocyte nuclear volume. The similar and different DNA fragments were PCR amplified and sequenced in triploid, tetraploid hybrids, and their parents, indicating their molecular genetic relationship and genetic markers. In addition, this study also presents results about the phenotypes and feeding habits of polyploid hybrids and discusses the formation mechanism of the polyploid hybrids. It is the first report on the formation of the triploid, tetraploid, and pentaploid hybrids by crossing parents with a different chromosome number in vertebrates. The formation of the polyploid hybrids is potentially interesting in both evolution and fish genetic breeding.     

Diploid clone produces unreduced diploid gametes but tetraploid clone generates reduced diploid gametes in the Misgurnus loach

Most individuals of the loach Misgurnus anguillicaudatus reproduce bisexually, but cryptic clonal lineages reproduce by natural gynogenesis of unreduced diploid eggs that are genetically identical to maternal somatic cells. Triploid progeny often occur by the accidental incorporation of a sperm nucleus into diploid eggs. Sex reversal from a genetic female to a physiological male is easily induced in this species by androgen treatment and through environmental influences. Here, we produced clonal tetraploid individuals by two methods: 1) fertilization of diploid eggs from a clonal diploid female with diploid sperm of a hormonally sex-reversed clonal diploid male and 2) artificial inhibition of the release of the second polar body in eggs of clonal diploid females just after initiation of gynogenetic development. There is no genetic difference between the clonal diploid and tetraploid individuals except for the number of chromosome sets or genomes. Clonal tetraploid males never produced unreduced tetraploid sperm, only diploid sperm that were genetically identical to those of a clonal diploid. Likewise, clonal tetraploid females did not form unreduced tetraploid eggs, just diploid eggs. However, the eggs' genotypes were identical to those of the original clone, and almost all the eggs initiated natural gynogenesis. Thus, gametogenesis of the clonal tetraploid loach is controlled by the presence of two chromosome sets to pair, thereby preserving the normal meiotic process, i.e., the formation of bivalents and subsequently two successive divisions.     

Artificial fertilization and induction of triploidy and meiogynogenesis in the European sea bass, Dicentrarchus labrax L.

The European sea bass, Dicentrarchus labrax L., was successfully subjected to chromosome manipulation. Triploidy was induced by cold-shocking eggs at 0–2°C for 20 min starting 5 min after fertilization in order to prevent the extrusion of the second polar body. Meiogynogenesis was also obtained by fertilizing eggs with UV-irradiated sperm (3300 and 6600 erg m⁻²) and subsequently doubling the chromosome set by meiotic block as above. The commercial advantages of culturing triploid and gynogenetic sea bass are discussed.