Evolutionary conservation of Dazl genomic organization and its continuous and dynamic distribution throughout germline development in gynogenetic gibel carp

Abstract

To investigate germline development and germ cell specification, we identified a 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, ovary-specific expression pattern of CagDazl was confirmed in adult tissues by RT-PCR and Western blot. In addition, 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 was found, confirming that CagDazl transcript and its protein are germ plasm component and move via METRO pathway during oogenesis. Furthermore, the CagDazl is abundant and continuous throughout germline development and germ cell specification including primordial germ cell (PGC) formation, oogonium differentiation, oocyte development, and embryogenesis, and the dynamic distribution occurs at different development stages. The data suggest that maternal CagDazl might play an important role in gibel carp PGC formation. Therefore, CagDazl is a useful and specific marker for tracing germ plasm and germ cell development in the gynogenetic gibel carp. In addition, in comparison with previous studies in sexual reproduction species, the continuous and dynamic distribution of CagDazl protein in the germ plasm throughout the life cycle seems to have significant implication in sex evolution of vertebrates.

Evolutionary history of two divergent Dmrt1 genes reveals two rounds of polyploidy origins in gibel carp

“…To estimate insert size, BAC DNAs were digested with I-SceI and separated on 1% agarose gel with ramp pulse time of 5-15 s at 6 V/cm at 14°C in 0.5 Â TBE for 16 h. Two levels of BAC DNA pools were constructed for BAC clones isolation based on PCR strategy as described previously (Peng et al, 2009). CagDmrt1-1-BAC clone and CagDmrt1-2-BAC clone were completely sequenced and analyzed at Beijing Genomics Institute as described previously .…”

High male incidence and evolutionary implications of triploid form in northeast Asia Carassius auratus complex

“…Similar to other polyploid salamanders, frogs and fish (Lampert and Schartl, 2010;Schlupp, 2005;Stöck et al, 2012), the triploid form can also reproduce by sperm-dependent gynogenesis, and many diverse gynogenetic clones have been discriminated by different genetic markers, such as transferrin Yang et al, 2001, RAPD and SCAR markers (Zhou et al, 2001(Zhou et al, , 2000b, microsatellite (Guo and Gui, 2008) and mtDNA sequence (Apalikova et al, 2008;Brykov et al, 2005;Li and Gui, 2008). Interestingly, a minor but significant portion (approx 1-10%) of triploid males were found in the triploid form, and normal sperm production and their sexual reproduction ability have been demonstrated by experimental propagation and genetic analysis in the triploid form (Gui and Zhou, 2010;Peng et al, 2009;Sun et al, 2010;Zhou et al, 2000a). Moreover, a novel nucleo-cytoplasmic hybrid clone was synthesized from the sexual reproduction mating between clone D female and clone A male, and it was suggested through genetic analysis to arise via androgenesis by a mechanism of ploidy doubling of clone A sperm in clone D ooplasm through inhibiting the first mitotic division (Wang et al, 2011).…”

Expression characterization of testicular DMRT1 in both Sertoli cells and spermatogenic cells of polyploid gibel carp

“…In order to detect the specificity of anti-CagDMRT1 antiserum, preimmune serum, anti-CagDMRT1 antiserum and pre-absorbed antiserum which was pre-absorbed with purified excess CagDMRT1 protein at 4°C overnight were used for both Western blot and immunofluorescence detection as described previously (Peng et al, 2009). …”

“…The samples were embedded in O.T.C. (Tissue-Tek), then the testes were sectioned at 5 μm with a cryostat microtome (Leica) and immunofluorescence detection was performed as described (Dong et al, 2004;Peng et al, 2009;Zhai et al, 2014). At last, images were viewed and acquired under confocal microscopy (NOL-LSM 710 Carl Zeiss) as described Zhong et al, 2014).…”

“…All these features made gibel carp to become a unique case to explore the evolution of sex determination and differentiation in vertebrates. Using gibel carp as a research model for screening reproduction-related genes (Xie et al, 2001), some important genes, such as germ plasm markers vasa (Xu et al, 2005) and Dazl (Peng et al, 2009), C-type lectin (Dong et al, 2004), C1q-like (Mei et al, 2008a(Mei et al, , 2008b(Mei et al, , 2014, histone H2A variant h2af1o (Wu et al, 2009;Yue et al, 2013), and maternal-effect factor spindlin (Sun et al, 2010), had been identified and functionally characterized (Gui and Zhu, 2012), but no sex-determination related genes had been analyzed in the polyploid gibel carp. Recently, through BAC identification and sequencing, two divergent Dmrt1 genes, CagDmrt1-1 and CagDmrt1-2, have been revealed from C. auratus gibelio , however, their molecular characterization and expression pattern, especially their protein distribution in testicular cells, are still unclear.…”