Molecular characterization of vasa homologue in marbled goby, Oxyeleotris marmorata: Transcription and localization analysis during gametogenesis and embryogenesis.

Identification of germ cell markers is important for investigating reproduction biology in fish. Vasa is one of the most studied germ cell markers in mammals and lower vertebrates including fish. Here, we characterized a vasa homologue from the fish marbled goby (Oxyeleotris marmorata), termed omvasa. The full length of omvasa cDNA is 2344 bp and encodes 658 amino acids, sharing high identities with Vasa homologues of other vertebrates. OmVasa protein contains 15 RG/RGG repeats at N-terminus, 2 ATPase motifs, as well as RNA unwinding and RNA binding domains at C-terminus. Phylogenetic tree showed that omVasa had the closest relationship with the Vasa homologue from the fish Boleophthalmus pectinirostris, the great blue-spotted mudskipper. qRT-PCR analysis indicated that omvasa was specifically transcribed in gonads, and the transcription level was gradually increased during oocyte development. The germ cell-specific distribution of omvasa mRNA was revealed by fluorescent in situ hybridization. In ovary, the signal of omvasa RNA displayed strong-weak-strong dynamics from oogonia over pre-vitellogenic oocytes to vitellogenic oocytes. In testis, omvasa signal was strong in spermatogonia, modest in spermatocytes but undetectable in spermatids and somatic cells. During embryogenesis, the transcription of omvasa mRNA was high at blastula stage, gradually decreased from gastrula stage and maintained at a low level in later developmental stages. Whole mount in situ hybridization indicated that omvasa mRNA was specific to primordial germ cells (PGCs). In summary, marbled goby vasa is a germ cell-specific transcript during gametogenesis, and can be used as an ideal marker for tracing PGC formation and migration, which is pivotal to germ cell manipulation in this species.

Postembryonic epigenesis of Vasa-positive germ cells from aggregated hemoblasts in the colonial ascidian, Botryllus primigenus.

We investigated whether Vasa was a germline-specific marker in the colonial ascidian Botryllus primigenus, and whether it was inducible epigenetically in the adult life span. We cloned a Botryllus Vasa homologue (BpVas). The deduced open reading frame encoded 687 amino acid residues. It was expressed specifically by germline cells such as the loose cell mass, oogonia and juvenile oocytes in the ovary, and the primordial testis (compact cell mass), spermatogonia and juvenile spermatocytes in the testis. The loose cell mass, the most primitive germline cells, showed an ultrastructure of undifferentiated cells known as hemoblasts. The hemoblasts did not contain electron-dense materials or a mitochondrial assembly in the cytoplasm. These organelles appeared later in the oogonia and oocytes. When the loose cell mass and developing germ cells were eliminated by extirpating all zooids and buds from the colonies, BpVas transcripts disappeared completely from the vascularized colonies. After 14 days, when the colonies regenerated by vascular budding, BpVas-positive cells reappeared in some cases, and in 30 day colonies, BpVas-positive germ cells were observed in all the regenerated colonies. These results show that in B. primigenus, germ cells are inducible de novo from the Vasa-negative cells even at postembryonic stages.

ayk1, a novel mammalian gene related to Drosophila aurora centrosome separation kinase, is specifically expressed during meiosis.

A novel murine gene, designated ayk1, which encodes a putative serine/threonine kinase has been cloned and characterized. The predicted catalytic domain of the protein is highly similar to that of Drosophila aurora (62.9% identity), and to that of Saccharomyces Ipl1 (49.4% identity). All three proteins also have very basic calculated isoelectric points (higher than 10). aurora has been recently shown to be crucial for centrosome separation and chromosome segregation, while Ipl1 is essential for yeast viability and accurate chromosome segregation. The results of Northern analysis and in situ RNA localization support a similar role for ayk1. The gene is specifically expressed in meiotically active cells, and during spermatogenesis, ayk1 transcripts accumulate just before the first meiotic division. Much lower levels are found in mitotically active cells. We propose that Ayk1, aurora and Ipl1 belong to a distinct new subfamily of kinases. These results suggest that the pathways controlling chromosome segregation are evolutionary conserved, and that similar control mechanisms operate in mitosis and meiosis.

Ultrastructural localization of acid phosphatase in germ cells of chick embryo left ovary.

The ultracytochemical localization of acid phosphatase was studied in oogonia and oocytes of the chick embryo left ovary. The reaction products are evident in lysosomes of various types and, in some cells, in the GERL as well. Furthermore, from the onset of the meiotic prophase, the enzymatic reaction also appears in the rough endoplasmic reticulum. Non-incubated sections of the same stages were observed, with the aim of identifying and describing the structure of the organelles, in particular lysosomes which appeared positive in incubated sections. The significance of the presence of the enzyme is discussed.