Molecular and Immunologic Characterization of Gynogenetic Channel Catfish (Ictalurus punctatus)

Second-generation gynogenetic channel catfish were characterized by molecular and immunologic assays to determine if they were isogenic at major histocompatibility complex loci. Southern blot analyses, using channel catfish MHC class II B and class I A gene probes, revealed identical banding patterns among second-generation gynogenetic fish. In contrast, banding patterns from outbred fish differed not only from gynogenetic animals, but also among themselves. Nucleotide sequence analysis of the MHC class II β₁ domain, which encompasses the peptide binding region, from four randomly selected gynogenetic fish showed a single DNA sequence. In contrast, analysis of the same region from three outbred fish showed sequences that differed not only among themselves, but also from those of gynogenetic animals. In cytotoxic assays, peripheral blood leukocytes from outbred fish lysed both gynogenetic and allogeneic targets, whereas those from gynogenetic fish lysed only allogeneic targets. Taken together, these results suggest that this group of second-generation gynogenetic channel catfish is isogenic at MHC loci and may provide an excellent system with which to study cell-mediated immunity in teleosts. Received September 11, 1998; accepted January 14, 1999     

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.     

Hypoxia tolerance of hybrids of carp <Emphasis Type="Italic">Cyprinus carpio</Emphasis> and golden carp <Emphasis Type="Italic">Carassius auratus</Emphasis>

Hypoxia tolerance of diploid gynogenetic and triploid backcross hybrids between golden carp Carassius auratus and carp Cyprinus carpio was investigated in comparison with carp. The more that heredity of golden carp was in their genotype, the better the hybrids survived under hypoxia conditions.     

Investigation on carp, Cyprinus carpio L. gynogenesis

A reliable method for gynogenetic mass-production has been developed in carp ( Cyprinus carpio L.). The effect of different cold shock treatments applied after fertilization were examined. The yield of viable diploid gynogenetic animals could be significantly increased when the cold shock was applied 5 min or 15 min after fertilization. A negative correlation has been found between the effectiveness of these two treatments, so both were used on separate batches of eggs. By means of gynogenesis, the crossover frequency and map distance of the transferrin locus have been determined. Successful pilot experiments were made on grass carp ( Ctenopharyngodon idella Val.) and sheatfish ( Silurus glanis L.) with respect to gynogenetic mass production.     

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     

Impact of MHC class II polymorphism on blood counts of CD4+ T lymphocytes in macaque

While the number of peripheral blood T lymphocytes and of their two main subsets (CD4+CD8− and CD4−CD8+) varies little in a given healthy individual, substantial variation is observed between individuals. It was proposed that these counts could be influenced by MHC polymorphisms because of the well-established role of MHC molecules in thymic T lymphocyte maturation and presentation of antigenic peptides to peripheral T lymphocytes. To test this hypothesis, we have chosen the crab-eating macaque (Macaca fascicularis), an animal model phylogenetically close to man. We selected the Philippine macaque population because of a restriction of the MHC polymorphism in this islander population. Peripheral blood lymphocytes were counted with an automated analyzer and T lymphocyte subsets were assessed by immunolabeling and flow cytometry. The MHC polymorphism was investigated in 200 unrelated subjects using 14 microsatellites markers distributed across the MHC and the DRB locus that was genotyped by denaturing gradient gel electrophoresis and sequencing. All markers were in Hardy–Weinberg equilibrium. Allelic associations were tested with the UNPHASED software. We revealed a significant influence of the MHC class II region on CD4+ T lymphocyte blood count with the largest effect associated with a two-locus haplotypes combining the DRACA allele 274 and the DRB haplotype #8a (p < 8 × 10⁻⁷). Our data should stimulate a similar association study of the CD4+ T cell counts in humans.     

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.     

Water polluted by 17α‐methyltestosterone provides successful male sex inversion of common carp (Cyprinus carpio L.) from gynogenetic offspring

This study investigated the effects of oral treatment and water pollution by 17α‐methyltestosterone (MT) on the masculinization of common carp, Cyprinus carpio, gynogenetic offspring. The oral administration of MT results in detectable levels of the steroid in the water of recirculating systems. Both modes of MT administration (oral/water pollution) caused a high level of masculinization (61.5–100%) of gynogenetic offspring of common carp.     

The haemoflagellate Trypanoplasma borreli induces the production of nitric oxide,which is associated with modulation of carp (Cyprinus carpio L.) leucocyte functions

In an attempt to characterise the role of nitric oxide (NO) in immune responses of carp, carp leucocytes obtained during an acute T. borreli infection were examined, for their capacity to generate NO. In a second set of experiments the impact NO on viability of the parasite and on the modulation of functional carp leucocyte responses were tested in vitro. Both in carp head-kidneys and in the peripheral blood, the fractions of lymphoblasts among separated leucocytes were increased. However, the relative proportions of granulocytes among head-kidney leucocytes (HKL) significantly decreased during infection, whereas granulocytes appeared among peripheral blood leucocytes (PBL). The cellular dynamics of HKL and PBL of infected carp were paralleled by an enhanced spontaneous NO release in vitro. NO production was further increased after addition of viable parasites to these cultures. The hypothesis that NO had a possible role in granulocyte activation and lymphocyte proliferation in carp was supported by the reduction of mitogen-induced proliferative responses of PBL from healthy carp in the presence of NO donor substances. The negative effects of NO on lymphocyte proliferation were contrasted by enhancing effects on granulocyte functions: the inhibition of NO generation in T. borreli-stimulated HKL cultures by the l-arginine analogue L-NMMA reduced the viability of granulocytes and their phagocytic activity. Even massive amounts of nitric oxide produced by donor substances (up to 600 micromol l(-1) NO(-)(2)) caused no reduction in the numbers of viable T. borreli flagellates in vitro. Thus, in carp, T. borreli seems to induce high amounts of NO in vivo which are apparently not harmful for the parasite but which may interfere with co-ordinated interactions of activated cells aiming at the defence of the parasite.     

Response of common carp (Cyprinus carpio L.) leucocytes to hormone-induced stress

The influence of cortisone on leucocytes composition in the blood of common carp (Cyprinus carpio L.) is studied. Following the hormone injection, the relative number of leucocytes decreased and the number of neutrophils and blast-form cells increased in the leucocyte spectrum of experimental fish.     

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。     

Immunochemical ANALYSIS OF GLYCOSYLATED AND nonglycosylated dla class I antigens

Dog peripheral blood lymphocytes, when cultured with ³⁵S-methionine in the presence of tunicamycin, synthesize DLA molecules consisting of ₂-microglobulin and a heavy chain approximately 3000 daltons lower in apparent mol. wt. than observed in control cases. This difference in mol. wt. is consistent with the fact that a single N-linked carbohydrate side chain is present on the heavy chain of DLA class I antigens. There is no evidence of polymorphism in the DLA light chain ( ₂m). Both glycosylated and nonglycosylated forms of the heavy chain, however, show microheterogeneity, which can be related to tissue-type. Analysis by two-dimensional electrophoresis shows that the biochemical heterogeneity in the DLA heavy chain is less than expected from DLA serology, and less than found in HLA class I antigens. The data are consistent with the fact that the products of only a single DLA class I locus are detected.Abbreviations used in this paper ₂m beta-2-microglobulin - 2D two dimensional - DLA dog MHC - HLA human MHC - Ia I-region associated - MHC major histocompatibility complex - PBL peripheral blood lymphocytes - PHA-M phytohaemagglutinin-muco - pI isoelectric point - RLA rabbit MHC - SDS-PAGE sodium dodecyl sulfate-polyacrylamide gel electrophoresis     

Evolution of a new nonclassical MHC class I locus in two Old World primate species

 HLA-G is a nonclassical major histocompatibility complex (MHC) class I molecule that is expressed only in the human placenta, suggesting that it plays an important role at the fetal-maternal interface. In rhesus monkeys, which have similar placentation to humans, the HLA-G orthologue is a pseudogene. However, rhesus monkeys express a novel placental MHC class I molecule, Mamu-AG, which has HLA-G-like characteristics. Phylogenetic analysis of AG alleles in two Old World primate species, the baboon and the rhesus macaque, revealed limited diversity characteristic of a nonclassical MHC class I locus. Gene trees constructed using classical and nonclassical primate MHC class I alleles demonstrated that the AG locus was most closely related to the classical A locus. Interestingly, gene tree analyses suggested that the AG alleles were most closely related to a subset of A alleles which are the products of an ancestral interlocus recombination event between the A and B loci. Calculation of the rates of synonymous and nonsynonymous substitution at the AG locus revealed that positive selection was not acting on the codons encoding the peptide binding region. In exon 4, however, the rate of nonsynonymous substitution was significantly lower than the rate of synonymous substitution, suggesting that negative selection was acting on these codons. Received: 22 April 1998 / Revised: 15 July 1998     

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。       

Histocompatibility analyses in tetraploids induced from clonal triploid crucian carp and in gynogenetic diploid goldfish

Histocompatibility analyses in goldfish were performed using the tetraploid goldfish-crucian carp hybrid and the first generation of gynogenetic diploid (GD₁) goldfish. Tetraploids were obtained by crossing clonal triploid crucian carp with goldfish. GD₁ goldfish were produced by the suppression of the second meiotic division. Tetraploid scale grafts on triploid clone members evoked an acute rejection in 4–6 days, whereas the reverse transplants were accepted or rejected chronically. Reciprocal grafting between tetraploids showed subacute rejection in 10–12 days, although some fish showed chronic rejection in 20–30 days. On the other hand, scale grafts reciprocally exchanged among triploids were intact even 3 months after grafting, although some of them showed a unidirectional rejection pattern. Furthermore, allograft rejection among gynogens occurred between 5 and 20 days, whereas all the scale allografts between members of control siblings were rejected within 9 days. In addition, neither accelerated acute rejection nor acceptance of allografts was observed in grafts exchanged among GD₁ goldfish. These results suggest that single doses of histocompatibility alleles are effective in eliciting acute rejection, and each of the fourth haploid set of chromosomes originating from paternal goldfish might share the same histocompatibility antigens to a large extent. This experiment also indicates that the genecentromere recombination rate is quite high with respect to the histocompatibility loci in this species.     

Rainbow trout offspring with different resistance to viral haemorrhagic septicaemia

To study immunological and immunogenetical parameters related to resistance against viral haemorrhagic septicaemia (VHS), attempts to make gynogenetic strains of rainbow trout selected for high and low resistance to VHS were initiated in 1988. The first gynogenetic generation of inbreeding resulted in the more resistant offspring E8 and the low resistance offspring K3; the K3 offspring having the same high mortality as the susceptible reference strain of outbred trout in infection trials. A second gynogenetic generation derived from the E8 strain resulted in some low resistance offspring, and two gynogenetic families in which all, or nearly all, fish survived challenge with VHS virus. In this study, an attempt to associate the distribution of different MHC class II genotypes with low and high resistance gynogenetic offspring was performed. Two different MHC haplotypes could be distinguished, and in both low and high resistance families all three genotypes were found, which could be explained by the fact that the mother fish carried the heterozygous genotype. Although no significant differences in MHC II genotypes were found between the high and low resistance offspring, a significantly different distribution of haplotypes in the low resistance offspring was observed, that could not be explained by a one- or two-locus model.     

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.     

Segmental exchange between MHC class I genes in a higher primate: recombination in the gorilla between the ancestor of a human non-functional gene and an A locus gene

Classical human major histocompatibility complex (MHC) class I molecules are the products of highly diverse gene loci. It has been suggested that segmental exchange may play a role in the generation of diversity at the antigen recognition site of MHC class I molecules. Here we present the cloning, sequencing and expression of two gorilla A locus cDNAs. One of these cDNAs shows remarkable similarity to the non-functional HLA-AR locus gene (5.4-LBF) only in exon 2. The remainder of the cDNA, however, is most closely related to other classical higher primate A locus genes. This suggests that a segmental exchange may have occurred between the ancestor of the non-functional HLA-AR gene and a classical gorilla A locus gene. Furthermore, the recombination event resulting in Gogo-A3 has affected its antigen recognition site. These data, therefore, demonstrate that segmental exchange can generate diversity at the antigen recognition sites of primate MHC class I molecules and suggest that non-functional genes can contribute to the generation of diversity of classical MHC class I genes.The nucleotide sequence data reported in this paper have been submitted to the GenBank nucleotide sequence data base and have been assigned the accession numbers X54375 (Gogo-A3) and X54376 (Gogo-A4). Address correspondence and offprint requests to: D. I. Watkins.     

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。