Suzu Sakao

Developmental Stages and Germ Cell Lineage of the Loach (Misgurnus anguillicaudatus)

Abstract The staging of embryonic and larval development, and the germ cell lineage of the loach, Misgurnus anguillicaudatus, are described. Fertilized eggs were obtained by artificial insemination. For the convenience of detailed observation and photography of the external appearance, we use dechorionated embryos. Through a series of operations, these embryos were cultured at 20°C in an incubator. Embryonic and larval development of the loach was divided into five periods: cleavage, blastula, gastrula, segmentation, and hatching. Stages were assigned within each of these periods. Developmental stages were determined and named by morphological features and somite number. The staging series were photographed and tabulated. The germ cell lineage was then elucidated by whole mount in situ hybridization of mRNA expression of the germ-cell-specific marker vasa and histological analysis. Primordial germ cells (PGCs) of the loach derived from the cleavage furrows of 8-cell stage embryos began proliferation in the late blastula period and migrated to the gonadal anlagen through a migration pathway similar to that of the zebrafish. However, it is characteristic of the loach that PGCs migrate a long distance and stay in the posterior part of the yolk-extension region.

Recovery of fertility in male hybrids of a cross between goldfish and common carp by transplantation of PGC (primordial germ cell)-containing graft

In germ-line chimera, gametes originate from both the donor and recipient. In order to increase the proportion of gametes from the donor, the elimination or reduction of primordial germ cells (PGCs) from the recipient is required. In the present study, histological and genetic analyses were performed in the chimeric fish obtained when sterile goldfish × common carp hybrid and fertile goldfish embryos were used as a recipient and donor, respectively. Chimerism was induced by transplantation of the lower part of the goldfish blastoderm into the hybrid blastoderm at the blastula stage. Neither spermatid nor spermatozoa were observed in the testis of the male hybrid. Motile sperm were obtained from 15 chimeric males by human chorionic gonadotropin (HCG) injection. When the sperm of chimeric fish were genetically analyzed, only goldfish-specific repetitive DNA sequences were detected. These results revealed that chimeric fish of the cross between a sterile male hybrid and fertile goldfish produced sperm exclusively derived from the donor goldfish.

Developmental potential of embryonic cells in a nucleocytoplasmic hybrid formed using a goldfish haploid nucleus and loach egg cytoplasm

In teleosts, viable nucleocytoplasmic hybrids, formed by combining a nucleus from one species with the egg cytoplasm of another, have been used as one of the methods for breed improvement in aquaculture, but have been little exploited for developmental biology studies. Here, we used an artificial androgenesis technique to form nucleocytoplasmic hybrids comprising a goldfish haploid nucleus and loach egg cytoplasm. These hybrids were used to investigate interactions between the nucleus and cytoplasm during embryonic development. Additionally, the developmental characteristics of embryonic cells of nucleocytoplasmic hybrids were examined in chimeras produced by transplantation of blastomeres into recipient loach or goldfish embryos. We found that the nucleocytoplasmic hybrids arrested at the dome stage of embryonic development and did not form any gastrula structures. The goosecoid (gsc) and no tail (ntl) genes were expressed normally before gastrulation in nucleocytoplasmic hybrids, similar to diploid loach. However, expression of the gsc and ntl genes was not maintained in nucleocytoplasmic hybrids. In chimeric embryos, blastomeres derived from nucleocytoplasmic hybrids were found to mix with the cells of recipient loach embryos at the gastrula stage. The transplanted blastomeres formed small clusters at the somitogenesis stage and, finally, small spots at the hatching stage. In contrast, when the blastomeres were transplanted into goldfish embryos, the transplanted blastomeres aggregated in the chimeric embryos. Thus, embryonic cells from nucleocytoplasmic hybrids that arrest before gastrulation could survive beyond the somitogenesis stage depending on the cytoplasmic environment in the recipient embryos.