RESEARCH ARTICLE

 

Identification and expression analysis of two Wnt4 genes in the spotted scat (Scatophagus argus)

 

Jianhua Chen^a,b,c,d*,Yinglei Li^a,c, Junbin Zhang^b, Huifen Liu^b, Yongqi Li^a,c

^a Jiangsu Key Laboratory of Marine Biotechnology, Huaihai Institute of Technology, Lianyungang, China
^b College of Aquaculture and Life Science, Shanghai Ocean University, Shanghai, China
^c Co-Innovation Center of Jiangsu Marine Bio-industry Technology, Huaihai Institute of Technology, Lianyungang 222005, China
^d The Jiangsu Provincial Platform for Conservation and Utilization of Agricultural Germplasm, Nanjing, China

---

ABSTRACT

Background: WNT4 is a protein that plays a crucial role in ovarian differentiation and development in mammals, with a relatively well understood function in mammalian gonadal differentiation. The role of WNT4 in teleost fish; however, remains unclear. In the present study, cDNAs of Wnt4a and Wnt4b were cloned and characterized in the spotted scat. The expression patterns of two Wnt4 genes in the gonads at different stages of development and in fish after treatment with 17a-methyltestosterone (MT) were investigated. Results: The tissue distribution showed that Wnt4a was expressed in various tissues, including the gonads, gills, spleen, brain, and fin. Interestingly, Wnt4b not only was expressed in the gills, brain, and spleen, but also was obviously expressed in the ovary. During gonad development, Wnt4a was highly expressed in the testis at stage I and Wnt4b was mainly expressed in the ovary at stages II-III. After MT treatment, the mRNA expression of Wnt4a increased significantly up to 40 d, and the transcript level of Wnt4b decreased at 20 d. Conclusions: These results suggest that Wnt4a may be involved in gonad development and plays a role in the process of spermatogonial proliferation. Our results also demonstrate that Wnt4b is not only expressed in the nervous system, but also in the ovary and it may be involved in ovary development of the spotted scat.

Keywords: Androgen, Expression patterns, Scatophagus argus, Tissue expression, Wnt4

---

 

1. Introduction

WNT (wingless-type MMTV integration site family) is a family of secreted glycoproteins [1], which is highly conserved throughout the vertebrates [2]. The WNT signal pathway was originally found in mice [3] and the fruit fly [4], as a kind of conservative signaling pathway that plays a crucial role in regulating cell growth and differentiation [5] and in the development of the reproductive system with the formation of the Müllerian duct and regulation of follicular development in mammals [6].

WNT4 is an important member of the WNT family, and a key protein regulating the WNT/β-catenin signal pathway. In mammals, Wnt4 is considered to be an important regulatory factor for ovarian differentiation, and its function is similar to the male sex-determining gene sry (sex-determining region Y) and sox9 (SRY-related HMG-box gene 9) involved in testis differentiation [6,7]. In male mammals, WNT4 can inhibit gonadal androgen secretion, thereby inhibiting male germ cell differentiation [8]. Previous studies have shown that Wnt4 functions to repress aspects of the male pathway by blocking the migration of endothelia cells into XX gonads and repressing the proliferation of steroidogenic cells [9]. In Wnt4-knockout mice, gonad development and steroid synthesis are affected, and a female-to-male development, inadequate secretion of androgens, and the feminization sex reversal was observed with a missing Müllerian duct and of male animals [11]. In addition, WNT4 and Daxl (dosage-sensitive formation of the Wolffian duct [10]. Meanwhile, overexpression of sex reversal, adrenal hypoplasia congenital critical region on the Wnt4 in mice results in disordered testis angiogenesis, inhibited testis X-chromosome, gene 1; also called nr0b1) has been found to interact

Table 1
Primers used for fragment cloning, 5' and 3' RACE and qRT-PCR of Wnt4a and Wnt4b.

Fig. 1. a: Nucleotide and amino acid sequences of Wnt4a cDNA in spotted scat (GenBank accession number: KF914415). The start codon and stop codon were indicated with underline. b: Nucleotide and amino acid sequences of Wnt4b cDNA in spotted scat (GenBank accession number: KF914416). The start codon and stop codon were indicated with underline.

Fig. 2. Phylogenetic tree based on a deduced amino acid alignment using the neighbor-joining method for Wnt4a and Wnt4b sequences in spotted scat and other vertebrates. Spotted scat Wnt4a and Wnt4b are marked with a black triangular form and hollow triangular form, respectively. Numbers on nodes indicate bootstrap vales from 10,000 replicates.

in the same molecular pathway, where WNT4 is Dax1's inducer, and where a mutation of Wnt4 causes a decreased expression of dax1 [12]. In contrast, in humans, Wnt4 overexpression, caused by diploidy of male chromosome 1p31-P35 fragments caused the up-regulation of dax1 expression, resulting in XY males with the female phenotype [13]. This finding suggests that Wnt4 may be a sex phenotype-determining gene, and with the dax1 gene, it may have a role in controlling female development and inhibiting the formation of testis. Nevertheless, previous studies in mice have also shown that Wnt4 plays a critical role in male gonad development. In Wnt4-mutant male mice, the differentiation of testicular cells was inhibited [14]. In invertebrates such as Chlamys farreri, WNT4 is expressed predominantly in the testis [15]. In some non-mammals, such as frogs [16], studies have shown the non-dimorphic expression of Wnt4 during gonadal differentiation. Therefore, the roles of Wnt4 in different species are complex.

In teleost fish, the expression patterns and possible roles of Wnt4 involved in sex differentiation and gonad development are not fully understood, and little information is available about the Wnt4 expression. To date, studies of Wnt4 in fish have been mainly confined to several species including zebrafish [17], medaka [18], Acanthopagrus schlegelii [19], and the rainbow trout [20]. In medaka, Wnt4b (which has only been identified in fish genomes, not in other vertebrates genomes) was identified with Wnt4a, which is predominantly expressed in ovarian tissue. The Wnt4b was not detected in the gonads [18]. Similarly, two types of Wnt4a (Wnt4a1 and Wnt4a2)and Wnt4b were identified in the rainbow trout, with the expression of Wnt4a1 and Wnt4a2 displaying a slight sexual dimorphism in favor of males during early gonadal differentiation [20].

The spotted scat (Scatophagus argus) is widely distributed in coastal waters of the Indo-Pacific, South and Southeast Asia, and China. The gonadal development of male and female spotted scats is not synchronized, and the gonads of males mature earlier than those of females during artificial breeding [21], which creates difficulties in the artificial breeding of the species. Furthermore, no information regarding sexual differentiation and development is available for the spotted scat. Since Wnt4 is important in the gonad differentiation of vertebrates, to elucidate its function in the sexual differentiation of the spotted scat, we isolated the cDNA sequences of Wnt4a and Wnt4b, and analyzed their mRNA expression in adult tissues and in different stages of gonad development. In addition, we examined their expression in the gonads of the spotted scat after treatment with 17a-methyltestosterone (MT).

2. Materials and methods

2.1. Experimental fish

Spotted scat (14.3 ± 1.1 cm in length; 135 ± 8.7 g in mass) were obtained from a fish farm in Yangjiang, Guangdong Province, China. The experimental fish were held in an aquarium facility with seawater (salinity of 30 ppt, water temperature ranging from 20 to 25°C). Fish were fed commercial food three times a d.

2.2. Total RNA extraction and cDNA synthesis

Fish were euthanized with 100 mg/L MS222 (Sigma) and dissected. Total RNA was isolated from 11 tissues (liver, spleen, kidney, gills, intestine, heart, brain, fin, muscle, ovary, and testis) of adult fish (n = 8), gonads from fish of different developmental stages (n = 8) except for stage V (gonads of different developmental stages has been described by Liu et al. [22]) and gonads of MT-treated fish (n = 15). TRIZOL reagent (Invitrogen) was used for the isolation following the manufacturer's protocol. The quality of the total RNA was checked using optical density at 260 and 280 nm and verified by electrophoresis on 1% agarose gel. The samples were quantified by a GeneQuant pro UV/Vis Spectrophotometer (GE Healthcare). For each sample, 1 μg DNase I-treated total RNA was reverse transcribed using Reverse Transcriptase M-MLV (TaKaRa) with the oligo (dT)18 and random primers in a 20 μL final volume according to the manufacturer's protocol. The cDNAs were used to clone the genes and carry out the analyses of gene expression by the quantitative real-time PCR (qRT-PCR) method.

Fig. 3. Tissue-specific expression of a: Wnt4a and b: Wnt4b, in several tissues of spotted scat measured using qRT-PCR and calculated by the comparative CT method (2-ΔΔCt) with 18S as an internal standard. Data are presented as mean ± SEM (n = 8). The different lowercase letters indicate statistically significant differences (P < 0.05).

2.3. Molecular cloning ofWnt4a and Wnt4b cDNAs

To clone the Wnt4a and Wnt4b genes of the spotted scat, degenerate primers (Table 1) were designed from the conserved regions of the known fish Wnt4a and Wnt4b sequences, respectively. The core fragment cDNAs of Wnt4a and Wnt4b were amplified in a 25 μL volume containing 1 μL of cDNA template, 0.5 μLof eachspecific primer, and 1 unit of Taq DNA polymerase (TaKaRa). The PCR products were electrophoresed on 1% agarose gel, the target DNA fragments were purified using a Gel Extraction kit (OMEGA), and they were then cloned into pMD18-T vectors (TaKaRa). The clones with confirmed recombinant plasmids were sequenced by Shanghai Majorbio Bio-Pharm Technology Co., Ltd. (Shanghai, China) using an ABI 3730 Genetic Analyzer.

According to the sequence information for the core fragments of Wnt4a and Wnt4b, gene-specific primers (Table 1 ) were designed for 5'-RACE and 3'-RACE. The rapid amplification of cDNA ends (RACE) was performed with specific primers and adaptor primers. 5'-RACE and 3'-RACE were amplified with the SMARTerTM RACE cDNA Amplification Kit (Clontech) following the manufacturer's instructions. Nested 5'- and 3'-RACE PCR products of the expected size were handled and sequenced as described above.

2.4. Sequence analysis

The cDNAs and amino acid sequences were analyzed using the BLAST program at the National Center for Biotechnology Information (NCBI: www.ncbi.nim.nih.gov) and the Expert Protein Analysis (ExPASy: www.expassy.org/tools). The protein structures were predicted using online analysis tools, such as ScanProsite (http://prosite.expasy.org/ scanprosite/) and SignaIP (www.cbs.dtu.dk/services/SignaIP-4.0). The phylogenetic tree was generated using MEGA 4.0 [23]. The data was re-sampled with 1000 bootstrap replications to determine the confidence indices within the phylogenetic tree.

2.5. MT treatment and sample collection

In the pilot study, four groups that had been fed diets containing MT (Sigma-Aldrich, USA) at concentrations of 25, 50, 100, and 150 mg/kg, were established. Sex reversal was observed in the 50, 100, and 150 mg/kg group but not in the 25 mg/kg group. Anorexia was also observed in the 100 and 150 mg/kg groups, where the mortality rate increased. Generally, feeding appeared to be normal in the 25 and 50 mg/kg groups. Thus, 50 mg/kg MT was selected as the most appropriate dose [24].

Fig. 4. Expression of a: Wnt4a and b: Wnt4b during the different developmental stages of gonads in the spotted scatmeasured using qRT-PCRand calculated bythe comparative CT method (2-AACt) with 18S as an internal standard. Data are presented as mean ± S.E.M (n = 8). T indicates the testis and O indicates ovary.

MT was prepared for the spotted scat diet by dissolving it in ethanol to yield 100 mLof MT solution (50 mg/L), and then added to 100 g ofdry pellets placed on an enamel tray. Trays were left in a fume hood for 2 d to evaporate the ethanol. The control diet was prepared in the same way, using only ethanol.

To further investigate the effects of MT on Wnt4a and Wnt4b expression in gonads during the early developmental stages, the experimental design followed previously published protocols [24]. Briefly, fishes were randomly divided into two groups (total 150 fishes): the MT-treated group and the control group, in triplicate. The MT-treated group was fed a diet containing MT 50 mg/kg, and the control group was fed a diet containing 0 mg/kg. Fishes were fed experimental diets and then the gonads ( n = 15) were sampled at 20, 40, and 60 d after treatment with MT. Some of gonad tissue that was sampled from each fish was frozen in liquid nitrogen immediately, and stored at -80°C until the RNA extraction. Another part of the gonad sample from each fish was fixed in Bouin's solution for histological examination.

2.6. Expression analysis ofWnt4a and Wnt4b by real-time RT-PCR

qRT-PCR was carried out to determine the expression of Wnt4a and Wnt4b in different adult fish tissues, and at different developmental stages of the gonads (stages I to IV). Gene expression in the spotted scats that were fed experimental diets was measured by the SYBR Green I chimeric fluorescence method. The qRT-PCR primers for the target genes are listed in Table 1. The 18S rRNA was used as an internal control (Table 1). The cycling program was 95°C for 30 s, 40 cycles of 95°C for 5 s, and 58°C for 1 min. The specificity of the amplicons was verified by melting curve analysis. The efficiency of the reactions was checked by analyzing serial dilutions of cDNA. The relative expression level of the genes was calculated with the 2-AACt method [25]. All of the experimental data is shown as the mean ± SEM. Data was analyzed with a one-way ANOVA, followed by Duncan's multiple comparison test, with P < 0.05 indicating a significant difference, using SPSS 16.0 for Windows.

3. Results

3.1. Molecular characterization ofWnt4a and Wnt4b cDNA

Two types of Wnt4 genes ( Wnt4a and Wnt4b) were cloned in the spotted scat. The sequences of Wnt4a (GenBank: KF914415) (Fig. 1a) and Wnt4b (GenBank: KF914416) (Fig. 1b) contain a complete open reading frame of 1059 nucleotides encoding a putative 352 AA, and 1077 nucleotides encoding a putative 358 AA, respectively. The protein sequence analysis revealed that the amino acid sequence of the spotted scat WNT4a shares a high identity with the WNT4a of other teleost fish (over 90% identical), and the protein is highly conserved throughout the vertebrates (approximately 80%). With regard to the spotted scat WNT4b, it shares a high identity with the WNT4b of other teleost fish (>90%), but has a lower identity (57.0-61.5%) with the WNT4a or WNT4 of other vertebrates. With regard to other teleost fish, such as zebrafish and medaka, the spotted scat WNT4a has a lower identity (59.9%) compared with WNT4b. The phylogenetic analysis showed that the spotted scat WNT4b sequence forms a clade with the WNT4b sequence of other teleost fish, and formed a clade with the WNT4a sequence of teleost fish, and formed another clade with the WNT4 of all vertebrates (Fig. 2). Moreover, the WNT4a sequence of teleost fish formed a clade that branched separately from the WNT4 of other vertebrates.

3.2. Tissue expression ofWnt4a and Wnt4b

The distribution patterns of Wnt4a and Wnt4b were detected by qRT-PCR in various adult tissues, including liver, spleen, kidney, gills, intestine, heart, brain, fin, muscle, ovary, and testis. The results revealed that Wnt4a (Fig. 3a) transcripts were predominantly expressed in gills, gonads (ovary and testis), spleen, brain, and fin. Likewise, the expression pattern of Wnt4b (Fig. 3b) was abundant in gills and brain, and followed by spleen, ovary, and fin(female fish), with a very low transcript level of Wnt4b detected in other tissues.

3.3. Expression levels ofthe Wnt4a and Wnt4b at different developmental stages

The qRT-PCR expression analysis of two Wnt4 genes during different developmental stages revealed that Wnt4a (Fig. 4a) was relatively highly expressed in the testis at stage I and expressed at a lower level in the testis at stages II-IV. In the ovary, Wnt4a was mainly expressed at stages I, III, and IV. For Wnt4b (Fig. 4b), the transcript levels exhibited relatively high expression in the ovaries at stages II and III, and the expression level was very low in the testis at all stages.

 

Fig. 5. HE staining of the gonads from spotted scat treatment with MT at different time. Control group (testis: a, d and g; ovary: b, e and h) for 20,40,60 d, respectively. Treatment group (c, f and i) for 20, 40 and 60 d. OOC: Oocyte, DO: degenerating oocyte, SG: spermatogonial, OC: ovarian cavity, SP: spermatocyte.

3.4. Effects ofMT treatment on gonadal Wnt4a and Wnt4b expression

The histological sections (Fig. 5) of gonads from the spotted scat after being treated with MT showed a decreased number of oocytes and degenerated oocytes. A small number of spermatogonial were seen in the gonads (Fig. 5f) at 40 d after MT treatment. Spermatocytes were seen and a few degenerating oocytes also seen in the gonads (Fig. 5i) at 60 d after MT treatment.

The expression of Wnt4a and Wnt4b was examined by qRT-PCR in the ovaries of spotted scats treatment with MT. Fig. 6a shows that the Wnt4a mRNA expression was significantly up-regulated, compared to the control group, after 40 d of MT treatment. No significant change in the Wnt4a expression levels was seen after 20 or 60 d of MT treatment. With regard to the effects of MT on the mRNA expression of Wnt4b,the Wnt4b mRNA transcript level was lower than that of the control group after 20 d of MT treatment, and the Wnt4b mRNA expression level gradually increased following MT treatment, though no significant differences in the Wnt4b transcript levels were seen after 40 or 60 d of MT treatment (Fig. 6b).

4. Discussion

WNT, containing 350-380 amino acids, one or more N-glycosylation sites, and 23 or 24 conserved cysteine residues [26], is recognized as a key signaling molecule that can activate multiple signal pathways, playing important roles in the early development of animals and a variety of cellular activities [5,27]. According to our analysis, 24-27 conserved cysteine residues are in fish WNT4a or the WNT4 of other species, and 24 conserved cysteine residues are in fish WNT4b. The Wnt4b gene, which is only identified in fish genomes and not in other vertebrates, was also identified in the spotted scat. In the present study, we found that the Wnt4a cDNA of the spotted scat encodes a predicted protein of 352 amino acid residues, which contains 3 N-glycosylation sites and 27 conservative cysteine residues, and the Wnt4b cDNA encodes a putative 358 amino acid residues, containing 4 N-glycosylation sites and 24 conservative cysteine residues. These findings agree with the predicted proteins WNT4a or WNT4b in other fish. Protein sequence analysis revealed that the spotted scat WNT4b shares the highest homology (97.8%) with the WNT4b of Epinephelus coioides and a lower identity (approximately 60%) with the WNT4a or WNT4 of other vertebrates. These findings further demonstrate that the two Wnt4 genes (Wnt4a and Wnt4b) were successfully isolated from the spotted scat in this study.

Previous studies have shown the varied tissue distribution of Wnt4a or Wnt4 in different species [15,16,18,20,28]. In medaka, Wnt4a was detected in most tissues including the ovaries and testis, with the highest expression level detected in the ovary [18]. In addition, Wnt4 was detected in the ovary and oviduct of the garden lizard (Calotes versicolor) [28]. In the present study, the tissue-specificexpression pattern of Wnt4a in the spotted scat was similar to what was found in medaka. Nevertheless, different results were observed in Zhikong scallop (C. farreri) [15] and rainbow trout [20]. In Zhikong scallop, Wnt4 (a Wnt4a paralog) was predominantly expressed in the testis during the entire reproductive cycle. Moreover, in some non-mammalian species, such as turtles and frogs, Wnt4 was not significantly expressed in sexual dimorphism [16].These results indicate that the expression pattern of Wnt4 or Wnt4a is complex in different species. To fully understand the function of Wnt4a in gonad development of teleosts, more work must be done.

Interestingly, the Wnt4b gene was only identified in fish and the expression of Wnt4b was mostly expressed in the brain, not gonads. At present, the expression pattern of Wnt4b in fish has been detected in only a small number of species. In rainbow trout, Wnt4b was detected only in the brain and pituitary, and not in other tissues including gonads [20]. A previous report on two medaka Wnt4 genes showed that Wnt4b was not detected in gonads and expressed only in the brain, spleen, heart, kidney, gills, and intestine [18]. In the present study, Wnt4b was also expressed in the brain, gills, spleen, and fin, which is similar to the expression of Wnt4b in medaka. Curiously, Wnt4b was expressed in the ovary of the spotted scat, suggesting that WNT4b may have a role in this tissue. Further study and the functional analysis of the promoter region and gene regulatory network of Wnt4b should be conducted, especially in the ovary. In the present study, the two Wnt4 genes (Wnt4a and Wnt4b) showed high expression levels in the gills. In addition, WNT4 has been reported to play an important role in cell proliferation and cell differentiation [18, 29]. The high expression level of the two Wnt4 genes (Wnt4a and Wnt4b) in the gills may be related to the frequent cell self-renewal. Moreover, these two genes (Wnt4a and Wnt4b) were expressed in the spleen and fin, and Wnt4b was highly expressed in the brain, suggesting that WNT4b may have a vital role in nervous system development [17]. Taken together, these results suggest that WNT4, ubiquitously present in organisms and participating in various life processes, may function as a signaling molecule.

Fig. 6. Relative expression profiles of a: Wnt4a and b: Wnt4b in the spotted scat after treatment with MT using qRT-PCR and 18S as an internal standard. Data are presented as mean ± SEM (n = 15). The different both upper and lowercase letters indicate statistically significant differences (P < 0.05).

To understand the role of Wnt4a and Wnt4b in gonadal development, we analyzed their expression levels in the gonads of the spotted scat at different developmental stages. Testis at stage I showed a higher level of Wnt4a expression than that of the ovary at any stage. This is similar to the Wnt4a1 in rainbow trout, which had a high expression in the early testis [20].Jeays-Wardetal. [14] reported that Sertoli cell differentiation was compromised in Wnt4 mutant testes, and that WNT4 was involved in the mammalian testis determination pathway [14]. Consequently, the higher transcript level of Wnt4a in the testis at stage I indicates that Wnt4a may play a crucial role in testis differentiation. Nevertheless, the relatively high expression level of Wnt4b was observed in the ovariesatstages II-III, and the expression levels were very low in the testis at all stages, indicating that Wnt4b may be involved in ovarian development of the spotted scat.

Numerous experiments have demonstrated that exogenous steroids or aromatase inhibitor can affect the expression of genes related to gonad development or sex differentiation [19,30,31,32]. This may be because the exogenous sex steroid is absorbed into the fish body and it then profoundly disturbs the endocrine environment of the differentiating gonads [33]. In the present study, the Wnt4a transcript of the MT-treated spotted scat was up-regulated, and the exogenous MT may promote the expression of male differentiation genes, such as amh, which then initiates the expression of dax1 by the negative feedback regulation [33]. Previous studies in the rainbow trout have also demonstrated that androgens can up-regulate the level of dax1 transcript [34], which is consistent with the mRNA expression of dax1 in the MT-treated spotted scat [24]. These findings indicate certain correlations in the expression of Wnt4a and dax1 in the spotted scat, which are similar to previous study [35]. Furthermore, earlier studies in mammals have demonstrated that Wnt4 acts as an inducer of dax1 expression and a Wnt4 mutation results in a significantly decreased expression of dax1 [12]. Moreover, studies have shown that Wnt4 plays a role in the development of male gonads. Male mice with knockout Wnt4 were found to have defects in their Sertoli cell differentiation [14]. In an unpublished observation, Wnt4 was reported to be significantly up-regulated in the juvenile sex reversal of Paralichthys olivaceus (total length 50 mm), when the testes begin to differentiate. This suggests that Wnt4 may be involved in testis differentiation of P. olivaceus. Although Wnt4 is known to play a key role in ovarian differentiation in mammalians, it may also be necessary for the normal development of the testis. In the spotted scat, the oocytes degenerate and a small number of spermatogonial can be observed in the gonads of fish treatment with MT for 40 d. This may explain the rise of Wnt4a after 40 d of treatment, and indicate that Wnt4a plays a role in the process of spermatogonial proliferation in the spotted scat.

As far as we know, Wnt4b was mainly expressed in brain in previous studies. Until now, no studies have explored the expression pattern of Wnt4b in gonad treatment with exogenous steroids. In the present study, the expression level of Wnt4b decreased at 20 d of treatment with MT and then gradually increased for 60 d. This could be explained by a disruption of the homeostasis of hormones due to the absorption of exogenous MT, leading to an increased level of androgens in the fish body. Subsequently, the level of endogenous androgens would likely decrease, and the relative level of endogenous estrogens increase, which could inhibit the expression of Wnt4b, to maintain the appropriate endocrine homeostasis. With the clearance of MT and restoration of endogenous testosterone at 60 d of MT treatment, the expression of cyp19a1a increased (unpublished data) catalyzing the conversion of testosterone into estrogens, which may promote the expression of Wnt4b. In addition, Wnt4b was mainly expressed in the ovaries during gonad development, indicating that it might be involved in ovary development. Nevertheless, little data is available about the expression patterns of Wnt4b in the fish gonad. To better understand the molecular mechanism of gonad development in the fish, more research is needed, especially on the promoter sequences of Wnt4b and the underlying function of the gene.

5. Concluding remarks

In the present study, cDNAs of Wnt4a and Wnt4b from the spotted scat were isolated and characterized, and the tissue distributions of Wnt4a and Wnt4b identified. The expression patterns of Wnt4a and Wnt4b at different developmental stages and after MT treatment were also investigated. The expression of Wnt4a was found to be up-regulated at 40 d after MT treatment and the Wnt4b transcriptional level decreased at 20 d after MT treatment. These findings suggest that Wnt4a may be involved in the process of gonad development and play a role in spermatogonial proliferation in the spotted scat. At the same time, Wnt4b is expressed in the ovary and may be involved in its development.

Financial support

This work was supported by the Priority Academic Program Development of Jiangsu Higher Education Institutions (PAPD) (5511201401X), the Open Foundation of Jiangsu Key Laboratory of Marine Biotechnology, the Huaihai Institute of Technology (Grant No. 2013HS002) and the Science and Technology Planning Project of Lianyungang City (CN1410).

Conflict of Interest

We declare that we have no conflict of interest.

 

References

1. Dale T. Signal transduction by the Wnt family of ligands. Biochem J 1998; 329:209-23.

2. Wodarz A, Nusse R. Mechanisms of Wnt signaling in development. Annu Rev Cell Dev Biol 1998;14:59-88. http://dx.doi.org/10.1146/annurevxellbio.141.59.

3. Van Ooyen A, Nusse R. Structure and nucleotide sequence of the putative mammary oncogene int-1; proviral insertions leave the protein-encoding domain intact. Cell 1984;39:233-40. http://dx.doi.org/10.1016/0092-8674(84)90209-5.

4. Cabrera CV, Alonso MC, Johnston P, Phillips RG, Lawrence PA. Phenocopies induced with antisense RNA identify the wingless gene. Cell 1987;50:659-63. http://dx.doi.org/10.1016/0092-8674(87)90039-0.

5. Hollyday M, McMahon JA, McMahon AP. Wnt expression patterns in chick embryo nervous system. Mech Dev 1995;52:9-25. http://dx.doi.org/10.1016/0925-4773(95)00385-E.

6. Bernard P, Harley VR. Wnt4 action in gonadal development and sex determination. Int J Biochem Cell Biol 2007;39:31-43. http://dx.doi.org/10.1016/j.biocel.2006.06.007.

7. Pellegrino M, Maiorino R, Schonauer S. WNT4 signaling in female gonadal development. Endocr Metab Immune Disord Drug Targets 2010;10:168-74. http://dx.doi.org/10.2174/187153010791213074.

8. Dumic M, Lin-Su K, Leibel NI, Ciglar S, Vinci G, Lasan R, et al. Report offertility in a woman with a predominantly 46, XY karyotype in a family with multiple disorders of sexual development. J Clin Endocrinol Metab 2008;93:182-9. http://dx.doi.org/10.1210/jc.2007-2155.

9. Hsieh M, Johnson MA, Greenberg NM, Richards JS. Regulated expression of Wnts and Frizzleds at specific stages of follicular development in the rodent ovary. Endocrinology 2002;143:898-908.

10. Vainio S, Heikkilã M, Kispert A, Chin N, McMahon AP. Female development in mammals is regulated by Wnt-4 signalling. Nature 1999;397:405-9.

11. Jordan BK, Shen JHC, Olaso R, Ingraham HA, Vilain E. Wnt4 overexpression disrupts normal testicular vasculature and inhibits testosterone synthesis by repressing steroidogenic factor 1/3-catenin synergy. Proc Natl Acad Sci U S A 2003;100: 10866-71. http://dx.doi.org/10.1073/pnas.1834480100.

12. Mizusaki H, Kawabe K, Mukai T, Ariyoshi E, Kasahara M, Yoshioka H, et al. Dax-1 (dosage-sensitive sex reversal-adrenal hypoplasia congenita critical region on the X chromosome, gene 1) gene transcription is regulated by Wnt4 in the female developing gonad. Mol Endocrinol 2003;17:507-19. http://dx.doi.org/10.1210/me.2002-0362.

13. Jordan BK, Mohammed M, Ching ST, Délot E, Chen XN, Dewing P, et al. Up-regulation of Wnt-4 signaling and dosage-sensitive sex reversal in humans. Am J Hum Genet 2001;68:1102-9. http://dx.doi.org/10.1086/320125.

14. Jeays-Ward K, Dandonneau M, Swain A. Wnt4 is required for proper male as well as female sexual development. Dev Biol 2004;276:431-40. http://dx.doi.org/10.1016/j.ydbio.2004.08.049.

15. Li HL, Liu JG, Liu XL, Zhang ZF. Molecular cloning and expression analysis of wnt4 cDNA from the Zhikong scallop Chlamys farreri. J Fish Sci China 2013;20:260-8. http://dx.doi.org/10.3724/SP.J.1118.2013.00260.

16. Oshima Y, Hayashi T, Tokunaga S, Nakamura M. Wnt4 expression in the differentiating gonad of the frog Rana rugosa. Zoolog Sci 2005;22:689-93. http: //dx.doi.org/10.2108/zsj.22.689.

17. Liu A, Majumdar A, Schauerte HE, Haffter P, Drummond IA Zebrafish wnt4b expression in the floor plate is altered in sonic hedgehog and gli-2 mutants. Mech Dev 2000;91:409-13. http://dx.doi.org/10.1016/S0925-4773(99)00308-1.

18. Li JZ, Liu Q, Wang DS, Zhou LY, Sakai F, Nagahama Y. Molecular cloning and identification of two Wnt4 genes from the medaka (Oryzias latipes). Acta Hydrobiol Sin 2012;36:983-6. http://dx.doi.org/10.3724/SPJ.1035.2012.00983.

19. Wu GC, Chang CF. wnt4 Is associated with the development of ovarian tissue in the protandrous black Porgy, Acanthopagrus schlegeli. Biol Reprod 2009;81:1073-82. http://dx.doi.org/10.1095/biolreprod.109.077362.

20. Nicol B, Guerin A, Fostier A, Guiguen Y. Ovary-predominant wnt4 expression during gonadal differentiation is not conserved in the rainbow trout ( Oncorhynchus mykiss). Mol Reprod Dev 2012;79:51-63. http://dx.doi.org/10.1002/mrd.21404.

21. Lan GB, Yan B, Liao SM, Luo Y, Xie RZ. Biology of spotted scat Scatophagus argus: A review. Fish Sci 2004;24:39-11.

22. Liu H, Mu X, Gui L, Su M, Li H, Zhang G, et al. Characterization and gonadal expression of FOXL2 relative to Cyp19a genes in spotted scat Scatophagus argus. Gene 2015;561:6-14. http://dx.doi.org/10.1016/j.gene.2014.12.060.

23. Tamura K, Dudley J, Nei M, Kumar S. MEGA4: Molecular evolutionary genetics analysis (MEGA) software version 4.0. Mol Biol Evol 2007;24:1596-9. http://dx.doi.org/10.1093/molbev/msm092.

24. Chen JH, He MX, Yan BL, Zhang JB, Jin SC, Liu L. Molecular characterization of dax 1 and SF-1 and their expression analysis during sex reversal in spotted scat, Scatophagus argus. J World Aquacult Soc 2015;46:1 -19. http://dx.doi.org/10.1111/jwas.12165.

25. Livak KJ, Schmittgen TD. Analysis of relative gene expression data using real-time quantitative PCR and the 2-AACT method. Methods 2001;25:402-8. http://dx.doi. org/10.1006/meth.2001.1262.

26. Nusses R, Varmus HE. Wnt genes. Cell 1992;69:1073-87. http://dx.doi.org/10.1016/0092-8674(92)90630-U.

27. Gordon MD, Nusse R. Wnt signaling: Multiple pathways, multiple receptors, and multiple transcription factors. J Biol Chem 2006;281:22429-33. http://dx.doi.org/ 10.1074/jbc.R600015200.

28. Tripathi V, Raman R. Identification of Wnt4 as the ovary pathway gene and temporal disparity of its expression vis-a-vis testis genes in the garden lizard, Calotes versicolor. Gene 2010;449:77-84. http://dx.doi.org/10.1016/j.gene.2009.09.001.

29. Yu HS, Pask AJ, Shaw G, Renfree MB. Comparative analysis of the mammalian WNT4 promoter. BMC Genomics 2009;10:416. http://dx.doi.org/10.1186/1471-2164-10-416.

30. Jiang W, Yang Y, Zhao D, Liu X, Duan J, Xie S, et al. Effects of sexual steroids on the expression of foxl2 in Gobiocypris rarus. Comp Biochem Physiol B 2011;160: 187-93. http://dx.doi.org/10.1016/j.cbpb.2011.08.005.

31. Wang J, Liu X, Wang H, Wu T, Hu X, Qin F, et al. Expression of two cytochrome P450 aromatase genes is regulated by endocrine disrupting chemicals in rare minnow Gobiocypris rarus juveniles. Comp Biochem Physiol C 2010;152:313-20. http://dx. doi.org/10.1016/j.cbpc.2010.05.007.

32. Vizziano-Cantonnet D, Baron D, Mahe S, Cauty C, Fostier A, Guiguen Y. Estrogen treatment up-regulates female genes but does not suppress all early testicular markers during rainbow trout male-to-female gonadal transdifferentiation. J Mol Endocrinol 2008;41:277-88. http://dx.doi.org/10.1677/JME-08-0039.

33. Li M, Wang L, Wang H, Liang H, Zheng Y, Qin F, et al. Molecular cloning and characterization of amh, dax1 and cyp19a1a genes and their response to 17alpha-methyltestosterone in Pengze crucian carp. Comp Biochem Phys C 2013;157:372-81. http://dx.doi.org/10.1016/j.cbpc.2013.03.005.

34. Baron D, Houlgatte R, Fostier A, Guiguen Y. Expression profiling of candidate genes during ovary-to-testis trans-differentiation in rainbow trout masculinized by androgens. Gen Comp Endocrinol 2008;156:369-78. http://dx.doi.org/10.1016/j. ygcen.2008.01.016.

35. Zanaria E, Muscatelli F, Bardoni B, Strom TM, Guioli S, Guo W, et al. An unusual member of the nuclear hormone receptor superfamily responsible for X-linked adrenalhypoplasia congenita. Nature 1994;372:635-41. http://dx.doi.org/10.1038/372635a0.

 

---

* Corresponding author. E-mail address: chenjianhuazsu@163.com (J. Chen).

Received 15 October 2015 Accepted 22 January 2016 Available online 11 February 2016

Copyright © 2016 Pontificia Universidad Católica de Valparaíso. Production and hosting by Elsevier B.V. All rights reserved. Peer review under responsibility of Pontificia Universidad Católica de Valparaíso.