To investigate germline development and germ plasm specification, we identified a germ cell marker gene Dazl homolog (CagDazl) from gynogenetic gibel carp (Carassius auratus gibelio). Its cDNA sequence and BAC clone sequence analyses revealed the genomic organization conservation and conserved synteny of the Dazl family members and their neighborhood genes among vertebrates, especially in fish. Moreover, a polyclonal antibody specific to CagDazl was produced and used to examine its expression and distribution throughout germline development at protein level. Firstly, gonad-specific expression pattern of CagDazl was confirmed in adult tissues by RT-PCR and Western blot. And, in situ hybridization and immunofluorescence localization demonstrated its specific expression in germ cells, and both its transcript and protein were localized to germ plasm. Then, co-localization of CagDazl and mitochondrial cloud (MC) was found, confirming that CagDazl transcript and its protein are germ plasm component and move via METRO pathway during oogenesis.
During embryogenesis, vegetal cortex localized CagDazl protein move toward blastomere in cytoplasm stream before 1k-cell stage and PGC specific expression was detected in 10-somite stage. Afterward, CagDazl protein was restricted to germ cells. The CagDazl is abundant and continuous throughout germline development and germ cell specification including PGC formation, oogonium differentiation, oocyte development, and embryogenesis, and the dynamic distribution occurs at different development stages.Therefore, CagDazl is a useful and specific marker for tracing germ plasm and germ cell development in the gynogenetic gibel carp.
We traced the sex differentiation process in both gynogenetic and sexual reproduction population by immunofluorescence localization using Vasa protein as germ cell marker. The results revealed that, like the situation in zebrafish, the differentiation of gibel carp male gonad should undergo an intersex phase in juvenile fish, and the ovary differentiation is earlier than testis.