Diploidized eggs reprogram adult somatic cell nuclei to pluripotency in nuclear transfer in medaka fish (Oryzias latipes)

Abstract

Reprogramming of adult somatic cell nuclei to pluripotency has been unsuccessful in non-mammalian animals, primarily because of chromosomal aberrations in nuclear transplants, which are considered to be caused by asynchrony between the cell cycles of the recipient egg and donor nucleus. In order to normalize the chromosomal status, we used diploidized eggs by retention of second polar body release, instead of enucleated eggs, as recipients in nuclear transfer of primary culture cells from the caudal fin of adult green fluorescent protein gene (GFP) transgenic medaka fish (Oryzias latipes). We found that 2.7% of the reconstructed embryos grew into adults that expressed GFP in various tissues in the same pattern as in the donor fish. Moreover, these fish were diploid, fertile and capable of passing the marker gene to the next generation in Mendelian fashion. We hesitate to call these fish 'clones' because we used non-enucleated eggs as recipients; in effect, they may be chimeras consisting of cells derived from diploid recipient nuclei and donor nuclei. In either case, fish adult somatic cell nuclei were reprogrammed to pluripotency and differentiated into a variety of cell types including germ cells via the use of diploidized recipient eggs.

Electroactivation of zebrafish (Danio rerio) eggs

“…In mammals, the use of non-ionic media in electroactivation processes is common (Chang et al, 1992) although the ionic media are also efficient (Elsheikh et al, 1995;Rickords and White, 1992). In contrast with this, to our knowledge the only information available in fish refers to Bubenshchikova et al (2007) and Wakamatsu (2008). In those cases, an ionic medium was used (a balanced salt solution medium designed for medaka) for the electroactivation of medaka fish eggs.…”

“…These set of techniques are nowadays of marked importance in regenerative medicine and/or assisted reproduction in vertebrate models and even, in humans (ICSI). To our knowledge, the use of egg electroactivation in fish nuclear transplant (NT) has only been carried out on medaka fish (Bubenshchikova et al, 2007;Wakamatsu, 2008). In zebrafish, the NT of somatic nucleus is able to induce embryo development when activation is only promoted by water, usually reaching the MBT stage (Huang et al, 2003;Pérez-Camps et al, 2009;Siripattarapravat et al, 2009b).…”

EMBRYOLOGICAL AND MICROMANIPULATION TECHNIQUES IN ZEBRAFISH (Danio rerio) AND PACIFIC OYSTER (Crassostrea gigas)

“…Such strategy was based on the fact that diploidized eggs have been shown as suitable recipients for nuclear transplant in medaka due to the more stable ploidy status, among other aspects (Bubenshchikova et al, 2007). But, the activation by sperm fertilization prevented to know if the donor nucleus conditioned embryonic and early larval developmental ability.…”

“…Although fish cloning is less developed, several recent works in medaka have been reported, in which both blastomeres (Bubenshchikova et al, 2005) and somatic larval and adult cells (Bubenshchikova et al, 2005, Bubenshchikova et al, 2007 were used as donors, and non enucleated and activated eggs were used as recipients in all cases. In this species, embryonic nuclear transplants using functionally enucleated and non activated eggs have been achieved only with blastomeres as nuclei donors (Wakamatsu et al, 2001).…”

“…To date, due to technical (Nüsslein-Volhard and Dahm, 2002) or biological (Westerfield, 2000) limitations, fish somatic nuclear transplant in these two laboratory species with somatic embryonic (10-15 somites; Huang et al, 2003) or adult cells (Bubenshchikova et al, 2007) has only been attempted using pre-activated eggs as recipients. These limitations have hindered the study of the reprogramming effects of cytoplasmic factors characteristic of the metaphase II status in the oocyte, the effects of the synchrony degree between activation and nuclear transplant, egg aging, or the previous donor cell reprogramming treatments.…”