Abstract
BackgroundAndrogenesis (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, Misgurnus anguillicaudatus (a teleost fish).ResultsWhen 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.ConclusionIn 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.
Highlights
Androgenesis is generally induced by means of irradiating the eggs to inactivate the maternal genome, followed by fertilization with normal sperm
Normal wild-type with melanophores appeared in control (Figure 2A), while cold-shock treatments resulted in the significant occurrence of the abnormal albino phenotype (Figure 2B), besides the dominant normal wildtype (Figure 2C)
And paternally derived alleles were segregated in the diploid progeny from the control, while only the paternally derived allele was detected in orange haploid progeny. These results indicate that abnormal orange progeny were androgenetic haploid developing from cold-shocked eggs
Summary
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 experiments to produce triploid salmonids, haploid embryos were cytogenetically recognized in heat-shocked groups [24,25] and pressure-shocked groups [25] Such an incidence of haploid-like progeny was reported in experiments to induce triploidy by inhibiting the second polar body release with cold-shock treatments shortly after fertilization in stickleback [26,27], common carp [28] and loach [29]. The origin of such unusual haploid-like progeny has not been well examined in these fish species, Ueda and Aoki [30] cytogenetically confirmed one androgenetic haploid embryo out of ten diploid hybrid embryos developing from cold shock (0°C for 60 min) at 2 min after fertilization of eggs from Rhodeus ocellatus ocellatus (Rosy bitterling) with sperm from Acheilognathus rhombea (Kanehira bitterling). Further studies are required since their results are from a single trial and a small sample size
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