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

The Mechanism for Primordial Germ-Cell Migration Is Conserved between Japanese Eel and Zebrafish

Primordial germ cells (PGCs) are segregated and specified from somatic cells during early development. These cells arise elsewhere and have to migrate across the embryo to reach developing gonadal precursors. Several molecules associated with PGC migration (i.e. dead-end, nanos1, and cxcr4) are highly conserved across phylum boundaries. However, since cell migration is a complicated process that is regulated spatially and temporally by multiple adaptors and signal effectors, the process is unlikely to be explained by these known genes only. Indeed, it has been shown that there are variations in PGC migration pattern during development among teleost species. However, it is still unclear whether the actual mechanism of PGC migration is conserved among species. In this study, we studied the migration of PGCs in Japanese eel (Anguilla japonica) embryos and tested the migration mechanism between Japanese eel and zebrafish (Danio rerio) for conservation, by transplanting eel PGCs into zebrafish embryos. The experiments showed that eel PGCs can migrate toward the gonadal region of zebrafish embryos along with endogenous PGCs, even though the migration patterns, behaviors, and settlements of PGCs are somewhat different between these species. Our results demonstrate that the migration mechanism of PGCs during embryonic development is highly conserved between these two distantly related species (belonging to different teleost orders)

Histological Differentiation of Primordial Germ Cells in Zebrafish

In this study, primordial germ cells (PGCs) in zebrafish were described histologically using eosinophilic granules as a marker. PGC-like cells (PL-cells) with eosinophilic granules were identified initially at the sphere stage (4-hr postfertilization), and were observed until the bud stage, the earliest stage to which PGCs with proper morphology could be traced. The morphology and distribution of eosinophilic granules in PL-cells changed during epiboly. Mitoses of PL-cells were observed only from the shield to bud stage, after eosinophilic granules aggregated to the perinuclear region in PGCs. These shifts of eosinophilic granules corresponded histologically to those of germ plasm described in Xenopus. These results suggest that eosinophilic granules represent germ plasm in fish and that PL-cells with these granules correspond to the PGCs or presumptive PGCs (pPGCs)

Improvement in group identification of dojo loach, Misgurnus anguillicaudatus, using PCR-restriction fragment length polymorphism

In most Japanese populations of dojo loach (Misgurnus anguillicaudatus), gonochoristic diploids of genetically diversified groups (A and B, further subdivided into B1 and B2) are present, whereas unisexual clonal lineages inhabit certain localities in the Hokkaido and Ishikawa Prefectures in Japan. Through a series of genetic studies including DNA markers, the clonal loaches were deemed to originate from a hybridization event(s) between the A and B1 groups. However, combined analyses with other DNA markers are needed to identify each genetic group. In this study, we improved the PCR-restriction fragment length polymorphism (RFLP) analysis of the recombination activating gene 1 (RAG1) gene using digestion with two restriction enzymes, PvuII and StuI. The improved RAG1-RFLP analysis showed different fragment patterns for each group: two fragments (245 and 198 bp) for group A, three fragments (198, 147, and 98 bp) for group B1, and a single fragment (443 bp) for group B2. The clonal loaches exhibited four fragments (245, 198, 147, and 98 bp) derived from both groups A and B1. Moreover, the DNA markers were able to detect two different hybrid genotypes (A x B2 and B1 x B2). Thus, the improved RAG1-RFLP markers allowed for quick and accurate group identification of the dojo loaches

Production of androgenetic diploid loach by cold-shock of eggs fertilized with diploid sperm

Diploid androgenotes were produced without egg irradiation in the loach, Misgurnus anguillicaudatus. Eggs of wild-type diploid females were fertilized with diploid sperm of a neo-tetraploid male and then cold-shock treated at 3 °C (range, ±0.5 °C) for 30 minutes just after fertilization to eliminate the female nucleus. After hatching, ploidy status of the hatched larvae was analyzed by flow cytometry, which revealed putative diploid androgenotes as well as larvae possessing other ploidies. Five independent microsatellite DNA markers were genotyped to confirm all-male inheritance of the resultant diploid larvae. The mean ± SD yield rate of diploid androgenetic larvae to total eggs used was 12.29 ± 3.25% in the cold-shock group and 22.23 ± 13.42% in the UV-irradiated group (P > 0.05). No diploid androgenetic larvae were detected in the intact control group. To our knowledge, this is the first report demonstrating successful induction of diploid androgenotes without egg irradiation in fish

Developmental biotechnology for aquaculture, with special reference to surrogate production in teleost fishes

This review introduces surrogate production as a new technique for fish-seed production in aquaculture. Surrogate production in fish is a technique used to obtain the gametes of a certain genotype through the gonad of another genotype. It is achieved by inducing germ-line chimerism between different species during early development. Primordial germ cells (PGCs) are the key material of this technique to induce germ-line chimera. In several species, it has been reported that PGCs differentiated from the blastomeres inherited some maternally supplied mRNA located in the terminal regions of the early cleavage furrows. PGCs from donor species (or strains) are isolated and transplanted into host species to induce the germ-line chimera. Four methods for inducing germ-line chimera are described: blastomere transplantation, blastoderm-graft transplantation, transplantation of PGC from the genital ridge, and transplantation visualised PGC with GFP fluorescence. Several problems preventing the successful induction of germ-line chimera in various fish species are discussed. Surrogate production, however, opens the possibility of efficient fish-seed production and effective breeding and transfer of biodiversity to an aquaculture strain. Conservation and efficient utilisation of genetic resources will be achieved through surrogate production combined with the cryopreservation of PGCs

Intersubfamilial Hybridization of Two Danio and Six Related Cyprinid Fishes

Production of sterile individuals is the key technique for surrogate propagation in teleosts. Sterile hybrids may be the ideal surrogate host when they do not generate their own germ cells in their gonads. Here, we attempted hybridization experiments between zebrafish (Danio rerio) and six closely related species (Danio albolineatus, Aphycypris chinensis, Hemigrammocypris rasborella, Opsariichthys platypus, Nipponocypris temminckii, N. sieboldii) and one remotely related species (Tanichthys albonubes). Intersubfamilial hybridizations in the family Cyprinidae resulted in the occurrence of inviable abnormal larvae with the two parental genomes, except for the T. albonubes x A. chinensis hybridization, in which normal larvae survived. Allotetraploidy and spontaneous gynogenetic diploidy were infrequently detected in T. albonubes x A. chinensis and D. albolineatus x A. chinensis, respectivel

Androgenetic doubled haploids induced without irradiation of eggs in loach (Misgurnus anguillicaudatus)

Androgenetic doubled haploids (DHs) were induced in the loach Misgurnus anguillicaudatus (Cobitidae) without irradiation of the eggs. The eggs of wild-type females were activated with the intact sperm of an orange-phenotype male, and treated (within 10 s of activation) at 3 ± 0.5 °C for 30 min, to eliminate the female nucleus. The eggs were then incubated in a water bath at 20 ± 0.5 °C for 35 min. Finally, diploidy was restored (65 min after activation) by heat-shock treatment at 42 ± 0.5 °C for 2 min. Under these conditions, the yield rate (mean ± SD) of putative DHs relative to the total number of eggs used was 10.43 ± 1.69%, which was significantly higher (P < 0.05) than the yield rates obtained under the remaining heat-shock initiation conditions (55 min, 60 min, and 70 min after activation). We analyzed the ploidy status of the putative DH by using flow cytometry. All-male inheritance was confirmed by the expression of the recessive orange body color trait and microsatellite genotypes. We detected no maternally derived alleles or heterozygous genotypes at any of the 28 loci (covering 27 linkage groups) of loach, indicating the exclusively paternal inheritance and homozygosity of the obtained androgenetic DHs