Cold-shock eliminates female nucleus in fertilized eggs to induce androgenesis in the loach (Misgurnus anguillicaudatus), a teleost fish

<p>Abstract</p> <p>Background</p> <p>Androgenesis (all-male inheritance) is generally induced by means of irradiating the eggs to inactivate the maternal genome, followed by fertilization with normal sperm. In fish, the conventional technique for induced androgenesis has been applied for rapid fixation to traits, recovery of cryopreserved genotypes, sex-control, etc. A new method of androgenesis that eliminates the need to irradiate the egg was proposed using the loach, <it>Misgurnus anguillicaudatus </it>(a teleost fish).</p> <p>Results</p> <p>When the eggs of wild-type females were fertilized with sperm of albino or orange phenotype males and cold-shocked at 0 to 3°C for 60 min duration just after fertilization, generally more than 30% (with a peak of 100%) of the hatched progeny were androgenotes. While a few of them were the normal diploid, most of them turned out to be abnormal haploid. All-male inheritance was verified by the expression of the recessive color trait (albino or orange) and microsatellite genotypes comprising only paternally derived alleles. Nuclear behavior after the cold-shock treatment was traced by microscopic observation of DAPI (4'6-diamidino-2-phenylindole)-stained samples and hematoxylin-eosin stained histological sections, and the extrusion of egg (maternal) nucleus was observed in eggs treated in the optimum timing.</p> <p>Conclusion</p> <p>In this paper, we demonstrate that cold-shock treatment (at 0 and 3°C) of loach eggs for 60 min just after fertilization successfully induces androgenetic haploid development. The most likely mechanism of cold-shock induced androgenesis is an elimination of the egg nucleus together along with the second polar body and subsequent development of a decondensed sperm nucleus or male pronucleus.</p

Functional Sperm of the Yellowtail (Seriola quinqueradiata) Were Produced in the Small-Bodied Surrogate, Jack Mackerel (Trachurus japonicus).

Production of xenogeneic gametes from large-bodied, commercially important marine species in closely related smaller surrogates with short generation times may enable rapid domestication of the targeted species. In this study we aimed to produce gametes of Japanese yellowtail (Seriola quinqueradiata) using jack mackerel (Trachurus japonicus) as a surrogate with a smaller body size and shorter maturation period. Donor spermatogonia were collected from the testes of yellowtail males and transferred into the peritoneal cavity of 10- and 12-day-old jack mackerel larvae. Twenty days later, 59.5% of the recipients survived of which 88.2% had donor-derived germ cells in their gonads. One year later, genomic DNA templates were prepared from the semen of 96 male recipients and subjected to PCR analyses using primers specific for the yellowtail vasa sequence, resulting in the detection of positive signals in semen from two recipients. The milt collected from the recipients was used for fertilization with yellowtail eggs. Of eight hatchlings obtained from the crosses, two were confirmed to be derived from donor yellowtail by DNA markers, although the others were gynogenetic diploids. These findings indicate that it is possible to produce donor-derived sperm in xenogeneic recipients with smaller body size and shorter generation time by transplanting spermatogonia. Thus, the xenogeneic transplantation of spermatogonia might be a potential tool to produce gametes of large-bodied, commercially important fish although the efficiency of the method requires further improvement. This is the first report demonstrating that donor-derived sperm could be produced in xenogeneic recipient via spermatogonial transplantation in carangid fishes

Hybridogenesis in triploid loach

Triploid loaches Misgurnus anguillicaudatus are derived from unreduced diploid gametes produced by an asexual clonal lineage that normally undergoes gynogenetic reproduction. Here, we have investigated the reproductive system of two types of triploids: the first type carried maternally inherited clonal diploid genomes and a paternally inherited haploid genome from the same population; the second type had the same clonal diploid genomes but a haploid genome from another, genetically divergent population. The germinal vesicles of oocytes from triploid females (3n=75) contained only 25 bivalents, i.e. 50 chromosomes. Flow cytometry revealed that the majority of the progeny resulting from fertilization of eggs from triploid females with normal haploid sperm were diploid. This indicates that triploid females mainly produced haploid eggs. Microsatellite analyses of the diploid progeny of triploid females showed that one allele of the clonal genotype was not transmitted to haploid eggs. Moreover, the identity of the eliminated allele differed between the two types of triploids. Our results demonstrate that there is preferential pairing of homologous chromosomes as well as the elimination of unmatched chromosomes in the course of haploid egg formation, i.e. meiotic hybridogenesis. Two distinct genomes in the clone suggest its hybrid origin

Inter-populational difference in microsatellite-centromere map distances in the loach, Misgurnus anguillicaudatus

Microsatellite-centromere recombination rates were estimated at 21 loci in relation to centromere of chromosomes in gynogenetic diploid lines induced from loaches of two different populations in Japan. All the microsatellite loci gave allelic segregation according to the Mendelian manner of inheritance in normal diploid families. Since loaches from Kita population in the southern area of Hokkaido Island and those from Memanbetsu population in the northern area, Hokkaido, Japan, were reported to be genetically diversified by previous genetic studies, map distances were compared between loaches from the two different populations. Three (Mado7, Mac3 and Mac49) of five loci, which could be compared inter-populationally, gave significantly different recombination rates, i.e., map distances. The results support the presence of genetic difference between the two populations

Cross-species amplification of microsatellite markers for the brown sole in the family Pleuronectidae

The amplification of eight microsatellite loci previously developed and characterized in brown sole Pleuronectes herzensteini was attempted in 11 other flatfish species. Two loci Phz6 and Phz12 were amplified in all the species examined. Cross-species amplification was succeeded in eight loci of Kareius bicoloratus and Pleuronectes yokohamae, but in seven loci of Microstomus achne, Pleuronectes punctatissimus and Pleuronectes schrenki. Five to three loci could be amplified in other six species. In the three species selected, K. bicoloratus, P. punctatissimus and P. yokohamae, cross-amplified seven to eight loci exhibited polymorphisms comprising one to 22 alleles. Expected heterozygosity (He) ranged from 0.66 to 0.96 in K. bicoloratus, from 0.62 to 0.96 in P. punctatissimus and from 0.43 to 0.91 in P. yokohamae. Observed heterozygosity (Ho) ranged from 0.65 to 1.00 in K. bicoloratus, from 0.55 to 0.95 in P. punctatissimus and from 0.40 to 0.95 in P. yokohamae. The Phz2, Phz3 and Phz12 loci significantly deviated in certain or all the three species from Hardy-Weinberg equilibrium. The mean values of homology to the flanking-region sequences of brown sole were 93.7% in K. bicoloratus, 91.2% in P. punctatissimus, and 93.9% in P. yokohamae. These results suggest that microsatellite markers for brown sole are applicable for genetic studies in flatfish species including at least these three species

Genetic verification of induced gynogenesis and microsatellite–centromere mapping in the barfin flounder, Verasper moseri

Primer sets were newly developed for 34 polymorphic microsatellite loci in the barfin flounder Verasper moseri. Mendelian inheritance was confirmed by examining the genotypic segregation in 2 normal diploid full-sib families. All the 34 loci showed genotypic segregation according to the Mendelian manner of inheritance; in some cases, null alleles were assumed. The genotypes at 27 loci were also examined in 4 meiotic gynogenetic diploid lines produced by fertilizing eggs with UV-irradiated sperm, followed by inhibition of the second meiotic division by cold shock. The absence of paternal alleles verified the success of gynogenetic development in all 4 meiotic gynogenetic diploid lines; the proportion of heterozygous progeny of a heterozygous mother, i.e., the frequency of second division segregation (y), was used to estimate the map distance of each microsatellite locus in relation to the centromere. Marker-centromere distances were estimated to be in the range of 0 to approximately 50 centiMorgan (cM) under the assumption of complete interference. Using 8 diagnostic loci located at the telomeric region of the chromosome, complete homozygosity was confirmed in 1 mitotic gynogenetic diploid line that was produced by suppressing the first cleavage via hydrostatic pressure shock

Parentage assignment of stocked black sea bream Acanthopagrus schlegelii in Hiroshima Bay using microsatellite DNA markers

The genetic contribution of 51 broodstock, comprising 29 females and 22 males, reared at Hiroshima City Marine Products Promotion Center for the production of stocked black sea bream was monitored during two consecutive years using seven microsatellite DNA loci. The high discrimination ability of these markers was reflected in the polymorphic identification content (PIC=0.831), the exclusion probability (Q∼1), and the low probability of identity index (I=3.635^−10). The total number of breeders contributing to the mating process was estimated at 32 (62.7%) in 2000 and 30 (58.8%) in 2001. On pedigree reconstruction, 69.3% of the offspring were successfully assigned to a single broodstock pair. Loss of alleles accounted for 16.9% during seed production; nevertheless, 90.9% of males and 69.0% of females participated in the mating process. Based on microsatellite genetic tagging, 58.9% of the fish sampled during the two months after release were identified as hatchery stock, presenting no significant differences from wild conspecifics in either fork length or body weight