Induced Androgenesis in Fish: Obtaining Viable Nucleocytoplasmic Hybrids

A self-review of long-standing studies of induced androgenesis in fish. The data are provided on the induction of diploid androgenesis in teleost fish via blocking the first cleavage division and the development of the method of dispermic androgenesis in sturgeon fishes. Androgenetic nucleocytoplasmic hybrids were obtained using the method of dispermic androgenesis and eggs of previously produced true interspecific hybrids. Specific morphological features of androgenetic hybrids were described and dispermic androgenesis was realized using cryopreserved sperm. The mechanism of sex determination in sturgeon fishes is discussed with reference to possible use of androgenesis for restoration of rare and disappearing species.__________Translated from Ontogenez, Vol. 36, No. 4, 2005, pp. 254–264.Original Russian Text Copyright © 2005 by Grunina, Recoubratsky.     

Dispermic androgenesis in sturgeons with the help of cryopreserved sperm: production of androgenetic hybrids between Siberian and Russian sturgeons

Dispermic androgenesis was used to produce, for the first time, an androgenetic progeny of the Siberian sturgeon (Acipenser baerii) and the androgenetic nuclear cytoplasmic hybrids (Siberian sturgeon, A. baerii x Russian sturgeon, A. gueldenstaedtii) using cryopreserved sperm. Microsatellite DNA analysis confirmed exclusively paternal inheritance in the androgenetic progeny of Siberian sturgeon. Heterozygotes for certain microsatellite loci were detected among the androgenetic hybrids, thereby confirming a dispermic nature of androgenesis. According to the data of comparative morphological analysis, the obtained androgenetic hybrid, by the age of 15 months old, was completely identical to the paternal species. Both a female and a male were detected in the androgenetic sturgeon progenies, which is of interest for producing bisexual progenies via androgenesis. The data of this study confirm the feasibility of dispermic androgenesis using cryopreserved sperm to preserve and recover the gene pools of endangered sturgeon species.     

Dispermic androgenesis in sturgeons with the use of cryopreserved sperm: Production of androgenetic siberian sturgeon and androgenetic hybrids between Siberian and Russian sturgeons

Dispermic androgenesis was used to produce, for the first time, an androgenetic progeny of the Siberian sturgeon (Acipenser baerii) and the androgenetic nucleo-cytoplasmic hybrids (Siberian sturgeon, A. baerii × Russian sturgeon, A. gueldenstaedtii) using cryopreserved sperm. Microsatellite DNA analysis confirmed exclusively paternal inheritance in the androgenetic progeny of Siberian sturgeon. Heterozygotes for certain microsatellite loci were detected among the androgenetic hybrids, thereby confirming a dispermic nature of androgenesis. According to the data of comparative morphological analysis, the obtained androgenetic hybrid, by the age of 15 months old, was completely identical to the paternal species. Both a female and a male were detected in the androgenetic sturgeon progenies, which is of interest for producing bisexual progenies via androgenesis. The data of this study confirm the feasibility of dispermic androgenesis using cryopreserved sperm to preserve and recover the gene pools of endangered sturgeon species.     

Induced androgenesis in acipenserids may be obtained by ultraviolet radiation

The results are presented on haploid androgenesis in Siberian sturgeon and sterlet induced by UV irradiation of ovicells. During irradiation, the cells in Ringer solution were rotated around a UV lamp. The efficiency of genetic inactivation of ovicells was estimated by the following parameters: manifestation of Hertwig effect, the fraction of embryos demonstrating haploid syndrome at final developmental stages, by arrest of embryonic development in hybrids Siberian sturgeon × great sturgeon, and by absence of maternal alleles of microsatellite loci in embryos. The dose-effect curve suggests that, during UV irradiation of ovicells of Siberian sturgeon, the complete genetic inactivation is attained at exposition of 120 s, while that in sterlet is 90 or 105 s. The results show a principal possibility of inactivation of ovicells by UV irradiation and use of such cells for producing androgenetic progeny of acipenserids.     

Nucleocytoplasmic Incompatibility in Androgenetic Fish Hybrids Can Be Overcome

Many androgenetic fish hybrids are nonviable due to the disturbed interaction between the foreign nucleus and the cytoplasm. It has been proposed that this incompatibility can be overcome if eggs from interspecific hybrids and sperm from one of the parental species are used for diploid androgenesis. Indeed, the androgenetic progeny obtained as a result of the insemination of the enucleated eggs of goldfish and carp hybrids by the carp spermatozoa proved to be viable and fertile. This approach may be used for reconstitution of the genotypes of rare or disappearing fish species with the help of induced androgenesis, if their native or cryoconserved sperm is available.     

Distant hybridization leads to different ploidy fishes

Distant hybridization makes it possible to transfer the genome of one species to another, which results in changes in phenotypes and genotypes of the progenies. This study shows that distant hybridization or the combination of this method with gynogenesis or androgenesis lead to different ploidy fishes with genetic variation, including fertile tetraploid hybrids, sterile triploid hybrids, fertile diploid hybrids, fertile diploid gynogenetic fish, and their derived progenies. The formations of the different ploidy fishes depend on the genetic relationship between the parents. In this study, several types of distant hybridization, including red crucian carp (Carassius auratus red var.) (2n=100, abbreviated as RCC) (♀)×common carp (Cyprinus carpio L.) (2n=100, abbreviated as CC) (♂), and RCC (2n=100) (♀)×blunt snout bream (Megalobrama amblycephala) (2n=48, abbreviated as BSB) (♂) are described. In the distant hybridization of RCC (♀)×CC (♂), bisexual fertile F3–F18 allotetraploid hybrids (4n=200, abbreviated as 4nAT) were formed. The diploid hybrid eggs and diploid sperm generated by the females and males of 4nAT developed into diploid gynogenetic hybrids and diploid androgenetic hybrids, respectively, by gynogenesis and androgenesis, without treatment for doubling the chromosome. Improved tetraploid hybrids and improved diploid fishes with genetic variation were derived from the gynogenetic hybrid line. The improved diploid fishes included the high-body RCC and high-body goldfish. The formation of the tetraploid hybrids was related to the occurrence of unreduced gametes generated from the diploid hybrids, which involved in premeiotic endoreduplication, endomitosis, or fusion of germ cells. The sterile triploid hybrids (3n=150) were produced on a large scale by crossing the males of tetraploid hybrids with females of diploid fish (2n=100). In another distant hybridization of RCC (♀)×BSB (♂), different ploidy fishes were obtained, including diploid bisexual fertile natural gynogenetic fish (2n=100), sterile triploid hybrids (3n=124), and bisexual fertile tetraploid hybrids (4n=148). Furthermore, two kinds of pentaploid hybrids (5n=172 and 5n=198) were formed. The biological characteristics and the mechanisms of formation of the different ploidy fish were compared and discussed at the cellular and molecular level. The results indicated distant hybridization or the combination of this method with gynogenesis or androgenesis affects the formation of different ploidy fish with genetic variation.     

Nucleocytoplasmic incompatibility in androgenetic fish hybrids can be overcome

Many androgenetic fish hybrids are nonviable due to the disturbed interaction between the foreign nucleus and the cytoplasm. It has been proposed that this incompatibility can be overcome if eggs from interspecific hybrids and sperm from one of the parental species are used for diploid androgenesis. Indeed, the androgenetic progeny obtained as a result of the insemination of the enucleated eggs of goldfish and carp hybrids by the carp spermatozoa proved to be viable and fertile. This approach may be used for reconstitution of the genotypes of rare or disappearing fish species with the help of induced androgenesis, if their native or cryoconserved sperm is available.     

Discrimination of interspecific hybrids in natural populations of Amur sturgeon fish by means of multilocus RAPD-PCR markers

A RAPD-PCR analysis of a sample of Amur sturgeon fish (46 individuals) is performed. An estimate of the genetic state of native populations of Amur sturgeon Acipenser schrenckii (Brandt, 1869) and kaluga fish Huso dauricus (Georgi, 1775) is given. Genetic evidence for the hybrid origin of two phenotypical hybrids is obtained. Estimates of the genetic distances between the species and the hybrids proved to be at the level of interspecific distances. It is shown that an Exact Test for differentiation of the populations is the most effective means of discriminating the two species, while multidimensional scaling (MDS) is the most effective means of discriminating the hybrids. It is concluded that the populations of sturgeon fish in the Amur River have maintained a substantial level of genetic diversity; the presence of hybrids in these populations is judged to be one of the risk factors. Multilocus RAPD-PCR markers are acknowledged as a convenient and reliable tool for conducting genetic monitoring of populations of Amur sturgeon fish for the purpose of preserving the gene pool of the fish.     

Genetic evidence for hybridization of pallid and shovelnose sturgeon

To determine the genetic origin of individual sturgeon that are morphologically intermediate to pallid (Scaphirhynchus albus) and shovelnose (Scaphirhynchus platorhynchus) sturgeon, we combined previously published mitochondrial DNA (mtDNA) and microsatellite data with additional microsatellite data. Two sympatric populations of pallid and shovelnose sturgeon from the upper Missouri River and a sympatric population containing pallid, shovelnose, and putative pallid-shovelnose hybrids from the Atchafalaya River were analyzed using an index of hybridization and a principle components analysis of individual relatedness scores. The addition of new microsatellite data improved our ability to genetically differentiate individual pallid and shovelnose sturgeon collected in both areas. Our methods distinguished morphologically intermediate Atchafalaya River sturgeon, which appear to be genetically intermediate between pallid and shovelnose sturgeon. The results support a hybrid origin for morphologically intermediate individuals, although it is unclear whether they are all first-generation hybrids or if some are the result of subsequent backcrossing with the more common shovelnose sturgeon.     

Identification of interspecific hybrids by amplified fragment length polymorphism: the case of sturgeon

The identification of interspecific hybrids represents an important issue for conservation biology and trade controls. In Italy, the commercial demand for sturgeon is rapidly increasing and interspecific hybrids represent a relevant part of aquacultural production. In this study we tested the suitability of the amplified fragment length polymorphism (AFLP) technique for sturgeon hybrid detection. Multilocus AFLP profiles were analysed by cluster analysis and assignment tests based on observed and simulated samples. Our results show that this approach can easily identify sturgeon hybrids, encouraging its application not only in sturgeon but also in other systematic groups.     

Diet of farm‐escaped sturgeon in the Yangtze River

The diet of farm‐escaped sturgeon accidentally introduced into the Yangtze River ecosystem was investigated from samples collected over 10 days. The fish had consumed adequate quantities of natural food, with prey type related to fish body size. The fish were categorized according to their primary dietary component: demersal fish were most common prey of kaluga Huso dauricus and H. dauricus × Acipenser schrenckii hybrids of total length 120.3–188.7 cm. Shrimp Macrobrachium nipponensis made up the major portion of the diet of H. dauricus × A. schrenckii and Acipenser baerii × A. schrenckii of total length 91.1–106.8 cm. Gammarid amphipods were consumed by A. baerii × A. schrenckii of total length 35.0–81.2 cm. The body length of prey was proportional to the fork length of the sturgeon, Y = 0.19X‐9.46, R² = 0.997. Escaped sturgeon had travelled at least 898 km downstream from the point of escape in the Yangtze River and were feeding on native macrobenthos and demersal fish.     

Use of heterologous sperm for the dispermic induction of androgenesis in barbs

Dispermic activation of genome‐inactivated eggs of the grey tiger barb Puntius tetrazona , facilitated by 2·5% polyethylene glycol (PEG)‐incubated sperm of the golden rosy barb Puntius conchonius , resulted in the generation of interspecific androgenetic clones of the golden rosy barb. A 10 min incubation of the golden rosy barb sperm in increasing concentrations (1·0, 1·5, 2·0, 2·5 and 3·0%) reduced the frequency of motile sperm (to 70%), motility duration (110–50 s) and fertilizability (to 80%) of the sperm; however, the frequency of sperm with double head size increased. At 3% PEG, motility pattern of the sperm completely changed from 'zig‐zag' to 'circular'. Incubation in 2·5% PEG facilitated the dispermic entry and production of diploid androgenetic female and male progenies at the ratio of 0·27 : 0·73. Polymerase chain reaction (PCR) analysis using Tc1 transposon specific primers confirmed the purity of paternal inheritance of P. conchonius through the surrogate eggs of P. tetrazona . Survival and breeding, body colour, diploidy (karyotyping and erythrocyte measurements) and 0·27F : 0·73M sex ratio of the androgenotes provided evidence for the successful induction of dispermic androgenesis. Despite increased heterozygosity and reduced 'damage cost' on restoration of diploidy, survival of dispermic androgenotes induced in heterologous eggs was lower (1·7%) than those reported for androgenetic golden rosy barbs induced using homologous sperm (14·0%) and heterologous eggs (7·0%), i.e . tiger barb eggs.     

Sturgeon production trials in the waste-heat effluents of a pulp and paper plant in Archangelsk, Russia

Sturgeons were cultured at the Solombalsky cellulose and paper plant (CPP) in Arkhangelsk over a 3 year period. The warm-water effluents of the biological treatment unit were utilized to rear the Siberian sturgeon (Acipenser baerii) and its hybrids with the sterlet (Acipenser ruthenus). Another culture trial included the Russian sturgeon (Acipenser gueldenstädtii) and its hybrids with the beluga (Huso huso). The most profitable culture (in terms of growth rates) was with the Siberian sturgeon and its hybrids with the sterlet. After the second year of culture, Siberian sturgeon and its hybrid averaged about 2.3 kg in wet weight while Russian sturgeons gained less (1.5 kg). One- and two-year old sturgeons grown at the Solombalsky CPP met practically all requirements needed for a successful culture.     

Comparative characterization of the ultrastructure of intestinal epithelium of various sturgeon species

The ultrastructure of the mid-intestine of four sturgeon species and their hybrids was studied. Apart from enterocytes and glandular goblet cells, the cells with microvilli, endocrine cells, and immunocompetent elements were revealed. Specific cells responsible for the primary immune response, analogous to mammalian M-cells, were discovered in sturgeon fishes. The ultrastructural characteristics common for the mid-intestinal epithelium of all the sturgeon fishes studied were determined. Species-specific features of intestinal epithelium of the parent species, which were characteristic of the hybrids, were revealed.     

The effect of genotype on the rate of regeneration of plants in a microspore culture of Triticum aestivum L

The effect of genotype on the productivity of in vitro microspore culture of wheat (Triticum aestivum) was studied. Wheat cultivars and hybrids bred in Kazakhstan and used in the programs on breeding new high-productivity and disease-resistant cultivars and forms of plants were tested. The genotypes that are responsive to androgenesis have been found among the cultivars tested: cultivars Kazakhstanskaya 4, Kazakhstanskaya 5, and hybrids obtained from crosses with the participation of these cultivars (K-6, B-5, and B-10). Such genotypes are recommended to be used as model objects in the studies on androgenesis and plant biotechnology. Based on the use of haploid technology, wheat dihaploid lines were created that are currently tested by breeders.     

Fertility of females of sturgeon hybrids obtained from species with different levels of ploidy (Acipenser ruthenus and A. dauricus) and their cloning

Main purpose of this work is the identification of females of artificial sturgeon hybrids capable to produce unreduced oocytes. The importance of this task is due to the ability to receive clonal all-female lines. Experiments were performed on the previously obtained reciprocal hybrids of sterlet, Acipenser ruthenus (S) with ~120 chromosomes and kaluga, Acipenser dauricus (K) with ~260 chromosomes. Karyotypes of backcross hybrids of (S × K) female (obtained by crossing sterlet female with kaluga male) and sterlet male included 180 – 190 chromosomes. This means that (S × K) female produced eggs with ~125 chromosomes and its karyotype consisted of ~250 chromosomes. This number was confirmed by a comparative analysis of erythrocyte size in this female and species with different ploidy. Karyotype with ~250 chromosomes can occur in (S x K) female only as a result of fertilization of a diploid sterlet egg (120 chromosomes) with kaluga haploid sperm (~130 chromosomes). Eggs of hybrid fertile (S × K) female, inseminated with inactivated sperm of Amur sturgeon and sterlet, developed into viable gynogenetic offspring, confirmed by the analysis of five microsatellite loci in this progeny, (S x K) female, and males used for UV-inactivated sperm. These data allow us to propose a method for obtaining fertile females of sturgeon hybrids from species with different ploidy. For this, experimentally obtained diploidized eggs from diploid 120-chromosome species must be fertilized by 250–270-chromosome male. Karyotypes of backcross hybrids of (K × S) female (obtained by crossing kaluga female with sterlet male) and sterlet male included ~250 chromosomes and hybrids of this female with kaluga male had ~320 chromosomes. These results proved an ability of hybrid (K × S) female to produce unreduced eggs, resulting in triploid backcrosses. The absence of reduction during egg development is well known in clonal forms (species) of vertebrates, which are of hybrid origin, and in artificially created fish hybrids. However, this has not been reported previously for sturgeons. Insemination of eggs of (K × S) female with UV-inactivated sperm of sterlet and Amur sturgeon led to offspring generation for which the genetic identity to their mother was proved using microsatellite analysis. That is, clonal inheritance was observed. These results suggest the possibility of developing a technology to produce all-female offspring. Artificial production of clonal lines in hybrid vertebrates can be also considered as experimental reproduction of the first stages of reticular speciation in nature.     

Triple hybrids obtained by artificial hybridization of the Russian sturgeon Acipenser gueldenstaedtii with the hybrid of the starred sturgeon A. stellatus and the great sturgeon A. huso (Acipenseridae): The kind of inheritance of some morphological characters and fertility of the parental hybrid form

Results are described of experimental crossing for obtaining of triple hybrids of the Russian sturgeon Acipenser gueldenstaedtii, the great sturgeon A. huso, and the starred sturgeon A. stellatus and of comparative analysis of some quantitative morphological characters in these hybrids, in the progeny of parental species and of reciprocal hybrids of starred sturgeon and great sturgeon. By most investigated characters, in triple hybrids, the states of the paternal species (Russian sturgeon) are inherited, by some other characters-of great sturgeon-the state of starred sturgeon is not inherited. Therefore, on the basis of external appearance, they may be identified as the hybrid of Russian sturgeon with great sturgeon. The observed segregation by the form of gill rakers into paternal, maternal, or intermediate variants leads to a conclusion of nonmonogenous inheritance of a wide form and a conical form of gill rakers in sturgeons. On the whole, the obtained results clearly demonstrate how, due to effects of matrocliny and partocliny, and different combination of dominant and recessive alleles of the genes determining the diagnostic characters, the hybrid individuals may acquire a different extent of similarity and differences from the parental species. The investigated triple hybrids are an interesting example of mutual leveling of divergent traits in the course of combination of different genomes within limits of one closely related Atlantic group of sturgeons. As a result, a certain averaged habitus is formed which might take place also in case of formation of polyploidy species in the process of evolution.     

Stock structure of pallid sturgeon analyzed with microsatellite loci

Recovery efforts for the endangered pallid sturgeon (Scaphirhynchus albus) include supplementation of wild stocks with hatchery reared progeny. Identifying the extent of genetic stock structure, which has previously been detected in samples from the range extremes, will help to determine whether stock transfers might be harmful. DNA microsatellite genotypes were screened in pallid sturgeon from the upper Missouri River, lower Missouri River, middle Mississippi River and Atchafalaya River and analyzed using a combination of Bayesian model‐based and more traditional F‐statistic based methods to characterize genetic differentiation. Scaphirhynchus specimens were collected by researchers active in the recovery effort and genotypes were screened at 16 microsatellite loci. Because there is considerable genetic and morphological overlap between pallid sturgeon, shovelnose sturgeon, and their hybrids, a combination of morphological and genetic techniques were used to eliminate shovelnose and possible hybrids from the sample. Genetic differentiation was detected among samples (overall θ = 0.050, P = 0.001). Pairwise θ, genetic distances, and Bayesian assignment testing reveal that pallid sturgeon from the upper Missouri River are the most distinct group with pairwise comparisons of pallid sturgeon among all the remaining samples exhibiting lower θ values, higher genetic distances, and self assignment scores. Our results indicate that using local broodstock, when available, should be used for pallid sturgeon propagation. If local broodstock are not available, geographically proximate individuals would limit genetic differences between native and stocked individuals.     

Recording of Processes of Hemoglobin Oxidation and Cryoprotective Effect of Glycerol

It was shown earlier that functional properties of hemoglobin changed during storage. Our goal was to evaluate processes of hemoglobin autooxidation and to develop simple methods for conservation of hemoglobin samples. It is revealed that cryoprotective effect of glycerol rapidly rises with increase of its concentration up to 5% for hemoglobins of all 4 studied species of sturgeon fish, sterlet, Russian sturgeon, starred sturgeon, and great sturgeon. The cryoprotective effect of glycerol is the highest on hemoglobin of the starred sturgeon, the lowest, of the sterlet. The results of this study allow proposing the method for cryoconservation of the sturgeon fish hemoglobin with addition of glycerol (up to 5% ) to the frozen solution of hemoglobin.     

Production and propagation of fully inbred clonal lines in the Nile tilapia (Oreochromis niloticus L.).

Fully inbred clonal lines of fish are likely to be of great value in research on immunology, sex determination, quantitative genetics, and toxicology. In this study on the Nile tilapia (Oreochromis niloticus), gynogenesis or androgenesis were used to produce a first generation of completely inbred fish, from which clonal lines were established using gynogenesis, androgenesis, hormonal sex reversal and intraline crosses. The clonal nature of these lines was verified by using multilocus DNA fingerprinting and the isozyme locus ADA*. Although these lines might be expected to be monosex in nature (all-female XX or all-male YY depending on the clone), one line did contain both sexes of fish. The presence of males in this gynogenetic clonal line and data from progeny testing of these males suggested that this line was homozygous for an allele or combination of alleles at an autosomal locus or loci which caused female to male sex reversal but with limited penetrance. Outbred clonal lines were also produced by crossing between different inbred clones. J. Exp. Zool. 284:675-685, 1999.