Characteristics of the Vasa Gene in Silurus asotus and Its Expression Response to Letrozole Treatment

Abstract

The identification and expression of germ cells are important for studying sex-related mechanisms in fish. The vasa gene, encoding an ATP-dependent RNA helicase, is recognized as a molecular marker of germ cells and plays a crucial role in germ cell development. Silurus asotus, an important freshwater economic fish species in China, shows significant sex dimorphism with the female growing faster than the male. However, the molecular mechanisms underlying these sex differences especially involving in the vasa gene in this fish remain poorly understood. In this work, the vasa gene sequence of S. asotus (named as Savasa) was obtained through RT-PCR and rapid amplification of cDNA end (RACE), and its expression in embryos and tissues was analyzed using qRT-PCR and an in situ hybridization method. Letrozole (LT) treatment on the larvae fish was also conducted to investigate its influence on the gene. The results revealed that the open reading frame (ORF) of Savasa was 1989 bp, encoding 662 amino acids. The SaVasa protein contains 10 conserved domains unique to the DEAD-box protein family, showing the highest sequence identity of 95.92% with that of Silurus meridionalis. In embryos, Savasa is highly expressed from the two-cell stage to the blastula stage in early embryos, with a gradually decreasing trend from the gastrula stage to the heart-beating stage. Furthermore, Savasa was initially detected at the end of the cleavage furrow during the two-cell stage, later condensing into four symmetrical cell clusters with embryonic development. At the gastrula stage, Savasa-positive cells increased and began to migrate towards the dorsal side of the embryo. In tissues, Savasa is predominantly expressed in the ovaries, with almost no or lower expression in other detected tissues. Moreover, Savasa was expressed in phase I-V oocytes in the ovaries, as well as in spermatogonia and spermatocytes in the testis, implying a specific expression pattern of germ cells. In addition, LT significantly upregulated the expression of Savasa in a concentration-dependent manner during the key gonadal differentiation period of the fish. Notably, at 120 dph after LT treatment, Savasa expression was the lowest in the testis and ovary of the high concentration group. Collectively, findings from gene structure, protein sequence, phylogenetic analysis, RNA expression patterns, and response to LT suggest that Savasa is maternally inherited with conserved features, serving as a potential marker gene for germ cells in S.asotus, and might participate in LT-induced early embryonic development and gonadal development processes of the fish. This would provide a basis for further research on the application of germ cell markers and the molecular mechanisms of sex differences in S. asotus.

Keywords: Silurus asotus; gene expression; gonadal development; letrozole; vasa.

Figure 1

Nucleotide and deduced amino acid sequence of Savasa. The start codon (ATG) and stop codon (TAG) are highlighted in bold. The ORF is shown in uppercase letters, whereas the 3′ untranslated regions are indicated in lower case. The amino acid sequences are displayed underneath the ORF using single capital letter codes. The putative polyadenylation signal aataaa is in a black dashed box. The primer sequences for Savasa ORF synthesis are indicated by horizontal arrows. The ten conserved motifs for the DEAD protein family are highlighted in black boxes. The RG and RGG repeats in the N-terminal region are noted in gray boxes.

Figure 2

Multiple sequence alignment of Vasa proteins. Different colored letters indicate their degree of conservatism in different sequences. Positions of identical residues are indicated in black. Ten conserved motifs of the DEAD protein family are highlighted in black frame. The species’ names, amino acid length, and the percentage of identities for full length (FL) and DEXDc domain (DD) in other species homologues to that of SaVasa are represented at the end of alignment.

Figure 3

Phylogenetic tree analysis of Vasa proteins. The branching between S. asotus and others was deduced by MEGA 6.0 software using Poisson Correction distance based on the neighbor-joining method with 1000 bootstrap replicates. Numbers next to the branches suggest bootstrap values.

Figure 4

The spatio-temporal expression levels of Savasa using qRT-PCR analysis. (A) The relative expression of Savasa at different developmental stages. All samples were normalized against the heart beating stage. (B) The relative expression of Savasa in different adult tissues. The transcripts are predominant in the ovary. All samples were normalized against the brain. The β-actin gene was used as the reference gene. Data are shown as mean ± SD (n = 3). Different lowercase letters indicate significant differences of Savasa expression levels during different development stages or tissues, and statistical difference was set at p < 0.05.

Figure 5

Distribution of Savasa mRNA in the developmental embryos by WISH analysis. The positive cells with anti-sense vasa probe hybridization were stained purple (A–G) at different stages: (A) 2-cell; (B) 4-cell; (C) blastula; (D) gastrula; (E) eye vesicle stage; (F,F′) muscle effect stage; (G) hatching stage. Scale bars = 200 μm. WISH results revealed that the Savasa mRNA-positive signals were distinct from the 2-cell stage to hatching stage, presented by asterisk arrowheads.

Figure 6

Distribution of Savasa mRNA in the gonads by FISH. Ovarian and testicular cross sections stained for Savasa mRNA (red) and nucleus with dye DAPI (blue) were analyzed by microscopy. (A) the anti-sense probe of Savasa mRNA in the ovary; (B) the sense probe of Savasa mRNA in the ovary; (C) the anti-sense probe of Savasa mRNA in the testis; (D) the sense probe of Savasa mRNA in the testis. The sense probe did not show any detectable signals. Og, oogonia; fm, follicular membrane; yg, yolk granules; I–V, stages of oocytes; sg, spermatogonia; sc, spermatocytes; st, spermatids; sp, sperm. The scale bars are 200 μm in the ovary, and 50 μm in the testis.

Figure 7

Response of Savasa expression levels to different concentrations of Letrozole. (A) Different developmental stages, (B) brain and ovary of the female at 120 dph, (C) brain of the male, (D) testis. Bars represent the mean ± S.D. (n = 3). Different lowercase letters indicate significant differences of Savasa expression levels in different LT concentrations at the same developmental stage or tissue. Different capital letters indicate significant differences in the same LT concentration at different developmental stages or tissues (p < 0.05).