Katsutoshi Arai

Germ cells are not the primary factor for sexual fate determination in goldfish

The presence of germ cells in the early gonad is important for sexual fate determination and gonadal development in vertebrates. Recent studies in zebrafish and medaka have shown that a lack of germ cells in the early gonad induces sex reversal in favor of a male phenotype. However, it is uncertain whether the gonadal somatic cells or the germ cells are predominant in determining gonadal fate in other vertebrate. Here, we investigated the role of germ cells in gonadal differentiation in goldfish, a gonochoristic species that possesses an XX-XY genetic sex determination system. The primordial germ cells (PGCs) of the fish were eliminated during embryogenesis by injection of a morpholino oligonucleotide against the dead end gene. Fish without germ cells showed two types of gonadal morphology: one with an ovarian cavity; the other with seminiferous tubules. Next, we tested whether function could be restored to these empty gonads by transplantation of a single PGC into each embryo, and also determined the gonadal sex of the resulting germline chimeras. Transplantation of a single GFP-labeled PGC successfully produced a germline chimera in 42.7% of the embryos. Some of the adult germline chimeras had a developed gonad on one side that contained donor derived germ cells, while the contralateral gonad lacked any early germ cell stages. Female germline chimeras possessed a normal ovary and a germ-cell free ovary-like structure on the contralateral side; this structure was similar to those seen in female morphants. Male germline chimeras possessed a testis and a contralateral empty testis that contained some sperm in the tubular lumens. Analysis of aromatase, foxl2 and amh expression in gonads of morphants and germline chimeras suggested that somatic transdifferentiation did not occur. The offspring of fertile germline chimeras all had the donor-derived phenotype, indicating that germline replacement had occurred and that the transplanted PGC had rescued both female and male gonadal function. These findings suggest that the absence of germ cells did not affect the pathway for ovary or testis development and that phenotypic sex in goldfish is determined by somatic cells under genetic sex control rather than an interaction between the germ cells and somatic cells.

Genomic Constitution and Atypical Reproduction in Polyploid and Unisexual Lineages of the Misgurnus Loach, a Teleost Fish

The loach (Misgurnus anguillicaudatus) is an excellent animal model to elucidate biological origin and evolutionary significance of genome duplication and unisexual reproduction because artificially induced and naturally occurring polyploids and parthenogenetic (gynogenetic, androgenetic) animals can be compared. First, we summarize the chromosome manipulation techniques to induce triploids and tetraploids by inhibiting meiotic or mitotic divisions of inseminated eggs, respectively, as well as parthenogenetic animals, obtained after fertilization with genetically inactivated gametes. Then, we review the knowledge on natural polyploid and unisexual lineages found in Misgurnus loaches. A natural diploid-tetraploid complex occurs in wild populations in central China, and these diploid and tetraploid loaches reproduce bisexually. Chinese tetraploids are considered autotetraploid, which may have arisen by doubling of the entire genome of an ancestral diploid, based on cytogenetic results from FISH (fluorescence in situ hybridization) karyotypes and meiotic configurations. In contrast, gynogenetically reproducing clonal diploid lineages have been discovered in a few wild populations in Japan, although most wild-type individuals are bisexually reproducing diploids. Such clonal diploid loaches sometimes produce triploid progeny by accidental incorporation of a sperm nucleus into an unreduced diploid egg, and the resulting triploid generates haploid eggs by meiotic hybridogenesis. Unreduced diploid gametes of clonal loaches are generated by a cytological mechanism, premeiotic endomitosis, which likely occurs in the early (gonium stage) germ cells. Initiation of gynogenetic development is related to a failure of decondensation of the male (sperm) pronucleus in unreduced diploid eggs of a clonal loach. Clonal lineages may have arisen from a past hybrid event between genetically divergent groups, but their exact origins are unknown at present. See also the sister article focusing on plants by Hegarty et al. in this themed issue.

Induction of viable gynogenetic progeny using eggs and UV-irradiated sperm from the Chinese tetraploid loach, Misgurnus anguillicaudatus

When eggs from the Chinese tetraploid loach that had 100 chromosomes were fertilized with UV-irradiated sperm, we obtained viable gynogenetic progeny without any additional treatment for the duplication of maternal chromosomes, which survived beyond first feeding towards adult stage of development. Gynogenetic progeny were determined to be diploid since they possessed 50 chromosomes, along with two chromosomes bearing nucleolar organizing regions (NORs), detected by silver nitrate staining (Ag-NORs), chromomycin-A3 (CMA3)-positive sites and fluorescence in situ hybridization (FISH) signals for rDNA loci. In contrast, when gynogens were induced using eggs from diploid loach fertilized by UV-irradiated sperm, but without chromosome doubling, we found that all resultant progeny were non-viable haploid gynogens with 25 chromosomes, along with one NOR-bearing chromosome detected by Ag-NORs, CMA3 and FISH. These observations demonstrate the true genetic tetraploid nature of the Chinese loach possessing 100 chromosomes, and the potential use of this tetraploid as a source of functional diploid gametes for further ploidy manipulation experiments.

Genetic structure of the Peruvian scallop Argopecten purpuratus inferred from mitochondrial and nuclear DNA variation.

The population genetic structure of the Peruvian scallop Argopecten purpuratus from three different wild populations along the Peruvian coast was analyzed using nine microsatellite loci and a partial region (530bp) of the mitochondrial 16S rRNA gene. A total of 19 polymorphic sites in the 16S rRNA gene defined 18 unique haplotypes. High genetic diversity was presented in all populations. Statistical analysis of mitochondrial DNA revealed no significant genetic structure (ΦST=0.00511, P=0.32149) among the three localities. However, microsatellite analysis showed low (2.86%) but highly significant (P=0.0001) genetic differentiation among populations, most of the variation was found in Independencia Bay population, which is located in the Peruvian National Reserve of Paracas. Neutrality tests based on mitochondrial haplotypes were performed to assess signatures of recent historical demographic events. Overall results from Tajimas D and Fus FS tests indicated significant deviations from neutrality. To our knowledge, this study constitutes the first investigation based on mitochondrial and microsatellite markers on the genetic structure of A. purpuratus.

Technical note: Viability and motility of vitrified/thawed primordial germ cell isolated from common carp (Cyprinus carpio) somite embryos

The feasibility of cryopreserving common carp (Cyprinus carpio) primordial germ cells (PGC) by vitrification of whole embryos at the 22- to 28-somite stage was investigated. Green fluorescent protein (GFP)-labeled PGC were cooled rapidly using liquid nitrogen after exposure to a pretreatment solution containing 1.5 M cryoprotectant (ethylene glycol or dimethyl sulfoxide, 30 or 50 min) and a vitrification solution containing 3 M cryoprotectant and 0.5 M sucrose (5, 10, 20, or 30 min). Embryonic cells that were pretreated for 30 min and vitrified for 20 min with ethylene glycol had the greatest rate of survival of embryonic cells (68.6%; P < 0.01), an optimal highest percentage of viable PGC (73.8 to 74.9%; P < 0.05), and no evidence of ice formation after thawing. The vitrified/thawed PGC were transplanted into blastula-stage embryos from goldfish (Carassius auratus). The PGC maintained their motility and moved to the gonadal ridge of the host embryo. Thus, the combination of vitrification and transplantation to produce germ-line chimeras is a powerful tool for the artificial production of next-generation offspring.