Vasa Identifies Germ Cells and Critical Stages of Oogenesis in the Asian Seabass

Abstract

Germ cells produce sperm and eggs for reproduction and fertility. The Asian seabass (Lates calcarifer), a protandrous marine fish, undergoes male-female sex reversal and thus offers an excellent model to study the role of germ cells in sex differentiation and sex reversal. Here we report the cloning and expression of vasa as a first germ cell marker in this organism. A 2241-bp cDNA was cloned by PCR using degenerate primers of conserved sequences and gene-specific primers. This cDNA contains a polyadenylation signal and a full open reading frame for 645 amino acid residues, which was designated as Lcvasa for the seabass vasa, as its predicted protein is homologous to Vasa proteins. The Lcvasa RNA is maternally supplied and specific to gonads in adulthood. By chromogenic and fluorescent in situ hybridization we revealed germ cell-specific Lcvasa expression in both the testis and ovary. Importantly, Lcvasa shows dynamic patterns of temporospatial expression and subcellular distribution during gametogenesis. At different stages of oogenesis, for example, Lcvasa undergoes nuclear-cytoplasmic redistribution and becomes concentrated preferentially in the Balbiani body of stage-II~III oocytes. Thus, the vasa RNA identifies both female and male germ cells in the Asian seabass, and its expression and distribution delineate critical stages of gametogenesis.

Identification and sexually dimorphic expression of vasa isoforms in Dabry′s sturgeon (Acipenser dabryanus), and functional analysis of vasa 3′-untranslated region

“…Three types of oogonia, og1, og2 and og3, were isolated in Asian seabass, showing cytoplasmic and nuclear expression in og1 and chiefly cytoplasmic expression in og2 and og3. The nuclear expression of vasa was possibly associated with the mechanism of intron retention (Xu et al 2014) or the function of DEAD-box proteins in pre-mRNA splicing and nuclear export (Linder 2006). Moreover, there was also another difference of vasa expression in ovary between Dabry's sturgeon and many other fish.…”

“…In the present study, the expression of Dabry's sturgeon vasa was investigated in the early developing oocytes. The signal of Advasa mRNA and protein was faint in undifferentiated oogonia (og1), drastically increased in differentiating oogonia (og2), and intense in primary oocytes, similar to some extent to that of gibel carp (Xu et al 2005) and Asian seabass (Xu et al 2014). In gibel carp, oogonia exhibited a low level of vasa mRNA but a high level of its protein, suggesting the differentially translational regulation of vasa in self-renewing germ stem cells, where a similar case was happening in primary spermatocytes of Dabry's sturgeon.…”

“…10 The visualization of medaka PGCs by co-injection GFP-Olnos3 3'UTR and RFPAdvasa 3'UTR mRNA at 32 somite stage. Bars 100 μm was strong in oogonia and early vitellogenic oocytes, and decreased with oocyte development (Braat et al 1999;Kobayashi et al 2000;Lin et al 2012b;Shinomiya et al 2000;Xu et al 2014). In the present study, the expression of Dabry's sturgeon vasa was investigated in the early developing oocytes.…”

“…A significant observation described in this study was that there was both cytoplasmic and nuclear expression of Advasa mRNA and protein in differentiating oogonia and primary oocytes. Presently, the nuclear expression of vasa in teleosts was only identified in the oogonia of Asian seabass (Xu et al 2014). Three types of oogonia, og1, og2 and og3, were isolated in Asian seabass, showing cytoplasmic and nuclear expression in og1 and chiefly cytoplasmic expression in og2 and og3.…”

BMP and RA signaling cooperate to regulate Apolipoprotein C1 expression during embryonic development

“…In situ hybridization and immunofluorescence staining were carried out as previously described Xu et al, 2014). Riboprobes for bmp2b (GenBank: BC114256) (Nikaido et al, 1997), bmp4 (GenBank: BC078423) (Chin et al, 1997) and apoc1 (GenBank: CN326047) were made by using DIG RNA labeling kit (Roche).…”