Generation of clonal zebrafish line by androgenesis without egg irradiation

Abstract

Generation of clonal zebrafish will facilitate large-scale genetic screening and help us to overcome other biological and biotechnological challenges due to their isogenecity. However, protocols for the development of clonal lines have not been optimized. Here, we sought to develop a novel method for generation of clonal zebrafish by androgenesis induced by cold shock. Androgenetic zebrafish doubled haploids (DHs) were induced by cold shock of just-fertilized eggs, and the eggs were then heat shocked to double the chromosome set. The yield rate of putative DHs relative to the total number of eggs used was 1.10% ± 0.19%. Microsatellite genotyping of the putative DHs using 30 loci that covered all 25 linkage groups detected no heterozygous loci, confirming the homozygosity of the DHs. Thus, a clonal line was established from sperm of a DH through a second cycle of cold-shock androgenesis and heat-shock chromosome doubling, followed by genetic verification of the isogenic rate confirming the presence of identical DNA fingerprints by using amplified fragment length polymorphism markers. In addition, our data provided important insights into the cytological mechanisms of cold-shock–induced androgenesis.

Viable diploid progeny induced from sperm of Chinese tetraploid pond loach by cold-shock androgenesis

“…Our cytological and histological observation confirmed that the cold‐shock treatment could eliminate maternal genetic materials (egg nucleus and second polar body nucleus) and a remaining male pronucleus could initiate androgenetic development after decondensation of sperm nucleus as already reported in Japanese pond loach (Morishima et al., ) and zebrafish (Hou et al., ). Such complete elimination of maternal genetic materials should induce all‐paternal inheritance, while normal release of the second polar body should produce triploid progeny due to the syngamy between haploid egg nucleus and diploid sperm nucleus.…”

“…() developed a new method of androgenesis with cold‐shock treatment of just fertilized eggs. Using such a cold‐shock androgenesis, production of androgenetic doubled haploid and/or clonal progeny has been reported in pond loach (Hou, Saito, Fujimoto, Yamaha, & Arai, ), zebrafish (Hou, Fujimoto, Saito, Yamaha, & Arai, ) and Japanese flounder (Hou et al., ). Induction of viable androgenetic diploid progeny was also achieved by the conventional UV‐irradiation‐androgenesis (Yasui, Fujimoto, & Arai, ) as well as the cold‐shock induced androgenesis (Hou, Fujimoto, Yamaha, & Arai, ), using diploid sperm of neo‐tetraploid males, which had been produced by inhibition of the second polar body after fertilization of eggs of wildtype diploid with diploid sperm of natural tetraploid found in Japanese fish market.…”

“…Recently, Morishima et al (2011) developed a new method of androgenesis with cold-shock treatment of just fertilized eggs. Using such a cold-shock androgenesis, production of androgenetic doubled haploid and/or clonal progeny has been reported in pond loach (Hou, Saito, Fujimoto, Yamaha, & Arai, 2014), zebrafish (Hou, Fujimoto, Saito, Yamaha, & Arai, 2015) and Japanese flounder (Hou et al, 2016).…”

Effects of homozygosity on sex determination in zebrafish Danio rerio

“…Previous reports of androgenesis (Corley‐Smith et al ., ; Hou et al ., ) and gynogenesis (Hörstgen‐Schwark, ; Streisinger et al ., ) in D. rerio have also yielded all‐male, or male‐biased progeny groups. This is consistent with our hypothesis of recessive–over‐dominant male determining alleles that potentially act by interfering with early germ‐cell development.…”

Developmental competence of eggs produced by rainbow trout Doubled Haploids (DHs) and generation of the clonal lines

“…However, clonal lines have been generated in a limited number of fish species due to the low survival of DHs and disturbances during their gonadal development and oogenesis (Hou et al, 2016;Hou, Fujimoto, Saito, Yamaha, & Arai, 2015;Kato et al, 2002;Kobayashi, Ide, Hiasa, Fushiki, & Ueno, 1994;Komen, Bongers, Richter, Van Muiswinkel, & Huisman, 1991;Naruse, Ijiri, Shima, & Egami, 1985;Sarder, Penman, Myers, & McAndrew, 1999;Yamamoto, 1999;Young, Wheeler, Fields, & Thorgaard, 1996;Zhang et al, 2015). DH females may be sterile or produce poor quality eggs exhibiting decreased ability to be fertilized and impaired competences for the proper development of embryos (Migaud et al, 2013).…”