Chromosome Doubling in Early Spermatogonia Produces Diploid Spermatozoa in a Natural Clonal Fish1

Abstract

The natural clonal loach Misgurnus anguillicaudatus (Teleostei: Cobitidae) is diploid (2n ¼ 50) and produces genetically identical unreduced eggs, which develop into diploid individuals without any genetic contribution from sperm. Artificially sexreversed clones created by the administration of 17alpha-methyltestosterone produce clonal diploid sperm. In metaphase spreads from testicular cells of the sex-reversed clones, spermatocytes had twice the normal number of chromosomes (50 bivalents) compared with those of normal diploids (25 bivalents). Thus, the production of unreduced diploid spermatozoa is initiated by premeiotic endomitosis (or endoreduplication), chromosome doubling before meiosis, and is followed by two quasinormal divisions. Larger nuclei in the germ cells were observed in all stages of type B spermatogonia in the testes of the sex-reversed clones. In contrast, besides having larger type A spermatogonia, the sex-reversed clones also had the type A spermatogonia that were the same size as those of normal diploids. It follows that chromosome duplication causing unreduced spermatogenesis occurred in the type A spermatogonia. The presence of tetraploid type A and early type B spermatogonia, identified by labeling with antispermatogoniaspecific antigen 1, was verified using DNA content flow cytometry. These results support the conclusion that chromosome doubling occurs at the type A spermatogonial stage in diploid spermatogenesis in the clonal fish.

Meiotic chromosome configurations in triploid progeny from reciprocal crosses between wild-type diploid and natural tetraploid loach Misgurnus anguillicaudatus in China

“…Similar polyploid cells were also noticed in spermatocytes of salmonids (Nygren et al 1972). Such unusual polyploids presumably due to endomitosis have been observed in the unreduced egg production process of triploid (Zhang et al 1998) and clonal diploid (Itono et al 2006), and in the unreduced sperm formation of sex-reversed clonal diploid (Yoshikawa et al 2009) in the loach. Other possible mechanism responsible for the presence of polyploid spermatocytes may be the meiotic arrest after chromosome replication, followed by formation of spermatozoa or speramatozoon-like cells with unusual proceeding of spermiogenesis.…”

“…Thus, the appearance of trivalents may relate to the presence of three sets of homologous chromosomes in triploid animals. No such trivalents have been seen in the meiosis of wild-type diploid loaches (Zhang et al 1998;Yoshikawa et al 2009;Li et al 2011). However, all the triploids did not exhibit the configuration including trivalents: artificially induced triploids and previous triploids from diploid 9 tetraploid hybridization, they did not exhibit trivalents in the process of meiosis (Zhang and Arai 1999;Zhang et al 2002).…”

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

“…In 254 general, natural or artificially induced tetraploid males generate diploid sperm. Some 255 diploid-triploid mosaic [35] and masculinized clonal loach individuals also spawn 256 diploid sperm [36][37][38]. These diploid sperm can be cryopreserved and used to restore 257 viable diploid progeny through androgenesis, thus enabling the recovery of special 258 fish strains or endangered species [21,30].…”

Restoration of the loach, Misgurnus anguillicaudatus, from cryopreserved diploid sperm and induced androgenesis

“…In the loach, however, other sources of diploid sperm may be obtained from masculinized clonal individuals (Morishima et al, 2002;Yoshikawa et al, 2007Yoshikawa et al, , 2009) and diploid-triploid mosaics (Morishima et al, 2004).…”