Cloning and characterization of Vasa gene expression pattern in adults of the Lusitanian toadfish Halobatrachus didactylus

The Vasa gene is essential for germ cell development in eukaryotes. It encodes a RNA helicase, a member of the DEAD box protein family. Using the RACE method, we cloned the Vasa cDNA of the Lusitanian toadfish Halobatrachus didactylus, and analyzed quantitative and qualitative Vasa expression and its protein immunolocalization. We reported a main product of about 2.4 kb which encodes a protein of 615 amino acids, but other minority Vasa products were also identified by RACE-PCR. This gene is predominantly expressed in the ovaries and testes, although some relatively low extragonadal expression levels have also been identified. In situ hybridization and immunolocalization analysis during gametogenesis in the testes showed that toadfish Vasa mRNA was detected in spermatogonia, spermatocytes and spermatids. However, in the ovaries, Vasa mRNA was detected in early vitellogenic oocytes and in more advanced vitellogenic stages, showing a very weak signal in oogonia, whereas the Vasa protein was evidenced in the cytoplasm of oogonia and previtellogenic oocytes, becoming weaker as the vitellogenic and maturation processes progress. These results suggest that toadfish Vasa homologues can play an important role in gametogenesis and germ cell development, but it could also be functionally implicated in other processes that are not as well known.

one of the most highly evolved groups of marine teleosts (Modesto & Canário 2003).
The Vasa gene, also called Ddx4, is an ATP-dependent RNA helicase belonging to the DEAD (Asp-Glu-Ala-Asp)-box protein family, the largest family of helicases.These helicases are broadly conserved across all phyla, are involved in processes where RNA plays a central role, and are important factors in cell differentiation and development (Lüking et al. 1998).The Vasa gene was identified for the first time in Drosophila as a maternal-effect gene, being essential for germ cell lineage development and for proper abdomen specification (Hay et al. 1990).Vasa homologs reported from different species have also been revealed as essential genes for the development of the germ cell lineage, although they show some important differences in their regulation (Raz 2000).
The Vasa gene was the first molecular marker employed for the identification of primordial germ cells (PGCs) in fish (Yoon et al. 1997).It has also been used as a marker of testicular germ cell transplan tation in flatfish species (Pacchiarini et al. 2013a).Vasa regulatory regions have been used to control the expression of transgenes, and thus to monitor PGC migration in living fish species (Krøvel & Olsen 2002) as well as to isolate PGCs from fish (Fan et al. 2008).In this sense, it was thought that Vasa expression in fish was restricted to germ cell lineage, however Vasa extragonadal expression has been reported in several fish species, such as Onco rhynchus mykiss (Yoshizaki et al. 2000), Dicentrarchus labrax (Blázquez et al. 2011) and Solea senegalensis (Pacchiarini et al. 2013b).Many studies have reported levels and pattern changes of Vasa expression during embryogenesis, sex differentiation and larval development in various fish species (Yoon et al. 1997, Krøvel & Olsen 2004, Xu et al. 2005, Li et al. 2010, Raghuveer & Senthilkumaran 2010, Cao et al. 2012, Lin et al. 2012, Pacchiarini et al. 2013a,b), showing a differential expression during oogenesis and spermatogenesis (Cao et al. 2012).Different Vasa transcripts with different expression patterns have also been characterized in diverse fish species, such as zebrafish, tilapia and Senegalese sole (Yoon et al. 1997, Kobayashi et al. 2002, Pacchiarini et al. 2013b), and the Vasa gene is usually present as a single copy gene in the majority of chordates, e.g. in zebrafish (Krøvel & Olsen 2004).However, recently, Fujimura et al. (2011) re ported 3 Vasa gene loci in the genome of tilapia.
The present study aimed to identify the Vasa homologue from Lusitanian toadfish in a first molecular and cellular approach, using its mRNA and cDNA for quantitative and qualitative gene expres-sion in histological sections of testis, ovary, and several somatic organs and tissues.We also used a specific Vasa antibody to test for protein distribution in parallel histological sections of both gonads from adult toadfish specimens.

Biological samples
The toadfish (n = 10) were caught in the Bay of Cadiz (SW Spain) from natural populations in September 2011 and May 2012.The specimens ranged from 285 to 420 mm total length and from 628 to 1527 g total weight; the population sex ratio was close to 1:1 as previously described by Palazón-Fernández et al. (2001).After anesthetizing with 1500 ppm phenoxyethanol (Sigma), the fish were decapi tated according to REGA-ES110280000311 animal wel fare procedures (ICMAN−CSIC).The organs and tissues (ovary, testis, heart, brain, muscle, liver, gill, intestine, swim bladder, spleen and kidney) were extracted and frozen immediately in liquid nitrogen, and stored at −80°C until used.
Gonad samples for in situ hybridization (ISH) and immunohistochemical (IHC) techniques were fixed with 4% paraformaldehyde in diethyl pyrocarbonate-(DEPC) treated phosphate-buffered saline-Tween20 (PBST) solution overnight at 4°C and stored in methanol at −20°C after washing 3 times for 1 h with PBST, and then processed according to Úbeda-Manzanaro et al. (2014).Histomorphological and cell characterization in both male and female gonads was performed by haematoxylin-eosin and haematoxylin-VOF stainings.

Nucleic acid extraction, cloning and phylogenetic analysis of Vasa products
Total RNA was extracted from 100 mg of each tissue sample using TriReagent (Sigma) and DNA contamination was removed using DNase I (Fermentas), as previously described in Úbeda-Manzanaro et al. (2014).The concentration and quality of the RNA was determined by UV spectrophotometry (A260: A280 nm ratios >1.7), and total RNA integrity was measured by electrophoresis on 1% agarose-formaldehyde gel.
First-strand cDNA was synthesized using the SMARTer RACE cDNA Amplification Kit (Clontech), according to the manufacturer's instructions.To isolate a full-length cDNA sequence of Vasa, 5'-and 3'-rapid amplification of cDNA ends were performed with specific and nested primers (Table 1), designed from sequence alignment of teleost orthologs using ClustalW (www.genome.jp/tools/clustalw/).The amplification procedures were carried out in a Doppio thermocycler (VWR) according to the manufacturer's instructions, with modifications in the thermal cycling.The PCR products were purified and inserted into a pGEM-T Easy Vector System (Promega).The positive clones were sequenced at the Sequencing Service from Biomedal (Spain), and the different obtained sequences were assembled using BioEdit 7.0.9.0.(Hall 1999).
The putative amino acid sequence of the main Vasa protein product was deduced using a translate tool (http://web.expasy.org/translate/),and was aligned with other sequences from the DEAX-box protein family with ClustalW algorithm.The molecular phylogenetic analysis was conducted using the neighborjoining method (Saitou & Nei 1987) with MEGA 5.1 software (Tamura et al. 2011).Bootstrap resampling (Felsenstein 1985) was applied to assess support for individual nodes using 10 000 replicates, and evolutionary distances were computed using the Poisson correction method (Zuckerkandl & Pauling 1965), uni form rates among sites, and complete option treatment of gaps and missing data.Branches with very low bootstrap confidence values were collapsed.The Genbank accession numbers of these sequences are provided in Table S1 in the Supplement at www.intres.com/ articles/ suppl/ b021 p037_ supp.pdf.

Quantitative PCR
Total RNA (1 µg) was reverse-transcribed into cDNA using the iScript™ cDNA Synthesis Kit (Bio-Rad) according to the manufacturer's instructions.Vasa expression was analyzed by quantitative PCR (qPCR) in a Mastercycler ® ep realplex 2 S (Eppen-dorf), in a final volume of 10 µl containing 1 µl of a 1/10 dilution of cDNA, SsoFast™ EvaGreen ® Supermix (BioRad), and 300 nM of each specific primer, OLIGOVAF and OLIGOVAR (Table 1).qPCR was run for 35 cycles (95°C for 15 s, 64°C for 15 s and 72°C for 20 s).The products were verified by sequencing, and melting-curve analysis was performed for each sample to check single amplification.As the internal control, elongation factor 1 alpha (ef1-α) gene was amplified from the same set of cDNA samples using the primers EFF and EFR (Table 1).Relative quantification was performed using the 2(-Delta Delta C(T)) method described pre viously (Livak & Schmittgen 2001), using the gill as a calibrator sample.Five biological replicates of each sample were analyzed, and each PCR was performed in parallel with a technical duplicate.Negative qPCR controls using doubledistilled water instead of cDNA were included in the assays for each primer pair.
To identify statistically significant differences in Vasa tissue distribution by qPCR, 1-way ANOVA was employed, followed by a Student-Newman-Keuls (SNK) post hoc test, using SSPS 15.0 software (IBM).In each case, differences were accepted as statistically significant at p < 0.05.

ISH using Vasa sense and antisense riboprobes
RNA in situ hybridizations using digoxigenin (DIG)-labeled antisense and sense riboprobes were performed as recently described by Úbeda-Manzanaro et al. ( 2014) on histological sections of toadfish testes and ovaries, using 55°C as the temperature of hybridization.The Vasa sense and antisense riboprobes were generated from a 177-bp fragment (Table 1) cloned into pCRII Dual Promoter vector (Invitrogen).The RNA probes were produced from 1 µg of linearized plasmid using T7 (sense) or SP6 (antisense) polymerases.

IHC detection of Vasa protein
A specific toadfish anti-Vasa antibody was synthesized and tested by adequate controls (Wester, ELISA) by Biomedal.This specific antibody was designed to localize 16 amino acids (IHGDREQREREQALKD) in the toadfish Vasa protein.
IHC was performed according to Úbeda-Manzanaro et al. (2014).In parallel histological sections, preimmunized rabbit serum was used as a negative control.

Toadfish Vasa cDNA and phylogeny analysis
The full-length cDNA of the toadfish Vasa gene was 2347 bp, and has been deposited in the NCBI database with the accession number JX849133 (see Fig. S1 in the Supplement at www.int-res.com/ articles/ suppl/ b021 p037 _ supp.pdf).This sequence is comprised of an open reading frame of 1845 bp, a 5'-untranslated region (5'-UTR) of 131 bp, and a 3'-UTR of 371 bp with a poly(A) tail.Moreover, minority products were also isolated from testes and ovaries by 5'-and 3'-RACE (Genbank accession numbers JX849136-JX849142; see Fig. S2 in the Supplement).These mi nority products showed deletions/ insertions in the arginine/glycine (R/G)-rich N-terminal region or in the C-terminal region.
Phylogenetic analysis using a neighborjoining method (Fig. 1) grouped the proteins according to the protein sub-family.Toadfish Vasa protein was clustered within other teleost fish Vasa homologues apart from other vertebrates.Interestingly, the tree suggests that toadfish Vasa homologue is more closely re lated to Vasa homologues from fish species of the superorden Acanthopterygii than of Paracanthopterygii.

Vasa mRNA expression and Vasa protein distribution
Tissue analysis by qPCR showed that the Vasa gene was expressed in all studied organs and tissues (Fig. 2).The highest Vasa mRNA expression levels were found in the Fig. 1.Phylogenetic tree of DEAD-box proteins using the neighborjoining method with MEGA 5.1 testes, followed by the ovaries.Relatively low mRNA expression levels were reported in the heart, liver, spleen, kidney, swim bladder, intestine, muscle, gill and brain tissues.
ISH was used to determine the pattern of toadfish Vasa expression during gametogenesis in testis and ovary tissues from adult toadfish specimens (Fig. 3).In the testes, the antisense Vasa riboprobe showed signals in germ cells (specifically in spermatogonia, spermatocytes and spermatids).In the ovaries, the Vasa mRNA was localized in the cytoplasm of early vitellogenic oocytes, being cortically concentrated within the cytoplasm of advanced vitellogenic oocytes.A very weak Vasa expression was detected in oogonia at nuclear localization, and no Vasa mRNA expression was evidenced in previtellogenic oocytes.No detectable signal was observed using the sense probe.
IHC showed the presence of Vasa protein in the cytoplasm of spermatogonia, spermatocytes and spermatids in testis.The ovary showed strong Vasa protein immunosignals in the cytoplasm of oogonia and previtellogenic oocytes, whereas Vasa immunostaining became weaker as the vitellogenesis and maturation processes progressed (Fig. 4).

DISCUSSION
The Vasa gene, which encodes a DEAD-box RNA helicase, is the molecular marker of germ cells most documented in teleosts (Lin et al. 2012), because it shows high specificity, is widely conserved through-out the animal kingdom, and it is relatively easy to detect (Cao et al. 2012).The characterization of Vasa cDNA in Lusitanian toadfish showed a major product whose predicted protein is 615 aa long.This product contains the 8 consensus motifs of the DEAD-box protein family, and the absence of mutation in the ATP-A motif (AXXXXGKT), the ATP-B motif (DEAD), the RNA unwinding motif (SAT) and the RNA binding motif (HRIGRXXR) reveal its functionality as a helicase (Pause & Sonenberg 1992).
Different transcripts of Vasa homologues have been reported in various fish species, such as Oreochromis niloticus (Kobayashi et al. 2002), Danio rerio (Bártfai & Orbán 2003, Krøvel & Olsen 2004), Gobiocyprus rarus (Cao et al. 2012), Solea senegalensis (Pacchiarini et al. 2013b) and Scopththalmus maximus (Lin et al. 2012, Pacchiarini et al. 2013a).A single copy of the Vasa gene is the most frequent condition among vertebrates, although different isoforms can be expressed (Castrillon et al. 2000, Krøvel & Olsen 2004).However, Fujimura et al. (2011) reported 3 Vasa loci in Oreochromis niloticus (Nile tilapia), unlike other closely related East African cichlids, suggesting a lineage-specific duplication of the Vasa gene during the evolution of Nile tilapia.Among the minority Vasa products observed in the Lusitanian toadfish, the coding sequence showed up to 6 different variants (JX849136−JX849140) in the N-terminal region, which contains the arginine/glycine-rich repeats.RGG and GRG motifs have a role as a site of arginine methylation, which is an important posttranslational modification that regulates protein− protein interactions (Kirino et al. 2010).Wolke et al. (2002) suggested that the RGG repeats have also been implicated in subcellular localization of the Vasa protein in zebrafish.The high variability of the RGG-rich region observed in Lusitanian toadfish could be due to multiple alternative splicing, which suggests high variability in Vasa mRNA processing that may contribute to regulate the activity of the different Vasa transcripts.The variability in the Cterminal region of minority Vasa products was only identified from toadfish ovarian samples.One of these variants (JX849142) showed a 221 nucleotide deletion with respect to the standard Vasa sequence, resulting in the loss of the functional domains VII and VIII (Lüking et al. 1998).As a consequence, this product could not perform the specific activity of RNA-helicase and could have been generated by an abnormal or inefficient biogenesis of mRNA.The other Vasa variant (JX849141) contained all consensus motifs of the DEAD-box proteins, but lacked the conserved tryptophan (W), glutamic acid (E) and aspartic acid (D) residues in the C-terminal region; it also lacked the 3'-UTR, although it has a stop codon immediately upstream of the poly-A.Yao et al. (2012) suggested a general mechanism for production of C terminus-truncated regulatory proteins generated by polyadenylation-directed conversion of a tyrosine codon in the coding sequence to a stop codon, and this Vasa transcript (JX849141) could have been generated by a similar mechanism.However, further studies are required to confirm whether toadfish Vasa is a single copy gene or not, and also to investigate the function of different transcripts, if any.
The tissue expression profile of the Vasa gene in adult toadfish was performed by qPCR without differentiating between the transcripts, because most of these transcripts showed minimal molecular differences.Vasa expression was principally restricted to the gonads, in agreement with the role of Vasa as a translational regulator in germinal line development (Braat et al. 1999a), and as in other adult fish species (Xu et al. 2005, Ye et al. 2007, Nagasawa et al. 2009, Li et al. 2010, Blázquez et al. 2011, Cao et al. 2012, Lin et al. 2012, Presslauer et al. 2012, Xiao et al. 2013).However, the Vasa gene could also be in volved in the regulation of translation of certain mRNA, which is essential for the specification of somatic cells where Vasa protein is present (Ikenishi & Tanaka 2000).In particular, new functions of Vasa have been described in the regulation of the cell cycle in multipotent cells and tumoral cells (Gustafson & Wessel 2010, Yajima & Wessel 2011).As in the Lusitanian toadfish, relatively low extragonadal expression levels of the Vasa gene have been reported in different fish species (Yoshi zaki et al. 2000, Blázquez et al. 2011, Pacchiarini et al. 2013b), involving diverse unknown roles and functions so far.Sexual dimorphic expression patterns of the different Vasa transcripts have been reported in some teleost fish, and a switch between the Vasa transcripts was also observed during em bryonic and larval development of several fish species, suggesting that relative expression of different Vasa transcripts could be involved in sexual differentiation and/or dimorphism in these teleost species (Kobayashi et al. 2002, Krøvel & Olsen 2004, Pacchiarini et al. 2013b).Recently, a switch was reported in Solea senegalensis between the longest Vasa transcripts, which are maternally supplied, and the shortest Vasa transcripts, which are expressed de novo during the growing larvae before sexual differentiation (Pacchiarini et al. 2013b).
Different cellular distribution patterns of Vasa mRNA in gonads have been revealed in different fish species by ISH.In toadfish testes, the expression pattern of Vasa gene showing positive signals in spermatogonia, spermatocytes and spermatids was similar to the cellular distribution pattern reported in Senegalese sole (Pacchiarini et al. 2013b).However, in most fish species, Vasa gene expression was observed only in spermatogonia and spermatocytes (Kobayashi et al. 2000, Xu et al. 2005, Ye et al. 2007, Cao et al. 2012, Lin et al. 2012, Xiao et al. 2013, Pacchiarini et al. 2013a), whereas in bluefin tuna Vasa mRNA was restricted to spermatogonia only (Nagasawa et al. 2009).
Vasa gene expression during oogenesis is a very dynamic process with respect to expression levels and cellular distribution.In many fish species, Vasa mRNA ISH signals are most intense in oogonia and early vitellogenic oocytes, decreasing in more advanced vitellogenenic stages (Braat et al. 1999b, Xu et al. 2005, Nagasawa et al. 2009, Lin et al. 2012, Ye at al. 2007, Pacchiarini et al. 2013a,b).The low levels of Vasa mRNA in oogonia from toadfish ovaries contrast to the high levels of Vasa protein, which could indicate that the translation of Vasa mRNA is differentially regulated in selfrenewing germ stem cells and differentiating germ cells by meiosis, as suggested by Xu et al. (2005) in Carassius auratus gibelio.However, the absence of Vasa mRNA in the previtellogenic oocytes of Halobatrachus didactylus and Sparus aurata (Cardinali et al. 2004) could be due to the low levels of Vasa transcripts in this early stage of oogenesis in both fish species.The increased expression of Vasa mRNA in vitellogenic oocytes of Lusitanian toadfish suggests that the accumulation of Vasa maternal factor could happen when starting vitellogenesis, and also suggests a potential regulatory role for Vasa in oocyte maturation, as was suggested in tilapia (Kobayashi et al. 2000) and sea bream (Cardinali et al. 2004).On the other hand, the presence of Vasa protein in oogonia and previtellogenic oocytes could suggest an active role in the early stages of oogenesis, and the weak immunosignals observed in maturing oocytes of H. didactylus could be explained by Vasa-diluting within the total protein content of these advanced maturating oocytes.The Vasa protein may be considered a useful marker for germ cell lineage, which has an important role during gametogenesis (oogenesis and spermatogenesis), and for sex determination or sexual dimorphism of Lusitanian toadfish.

CONCLUSIONS
The Vasa homologue identified in Lusitanian toadfish was predominantly expressed in the gonads, resulting a good sex cell marker.However, other Vasa transcripts have also been identified, and the Vasa gene could be functionally involved in other physiological processes.The present study provides information useful for future research of developmental sex mechanisms, such as studies on the different Vasa transcripts and their expression, variations during the annual repro ductive cycle and during sexual determination, and differentiation.

Fig. 2 .
Fig. 2. Relative expression levels of Vasa mRNA in adult Lusitanian toadfish organs and tissues (mean ± SE, n = 5).Statistically significant differences (lower case letters: a, b, and c) were detected by ANOVA, SNK post hoc test, p < 0.05

Table 1 .
Primers used for the sequencing and quantification of Vasa mRNA levels in Halobatrachus didactylus.E: efficiency (E = 1 is 100% efficiency)