Histological and sex-determining genes expression effects of 17α-methyltestosterone on mandarin fish Siniperca chuatsi gonad development

17α-methyltestosterone (MT) as a synthetic androgen has been widely used in sex reversal of fish research, but there was no published report on sex reversal of mandarin fish by MT. Moreover, comparative transcriptome analysis of testis and ovarian tissue is still lacking in mandarin fish. We used histological analyses together with RNA sequencing to characterize mandarin fish gonadal transcriptomes and investigate the effects of MT on the sex ratio, survival rate, growth, gonadal differentiation.


Conclusion
This study suggests that 17α-methyltestosterone successfully induced all-male stock and we select the part of the genes ( dmrt1, sox9, foxl2 and cyp19a1a ) related to sex determination and differentiation.

Background
The mandarin fish (Siniperca chuatsi) is an economically important fish in China.
The growth of mandarin fish shows gender dimorphism that the growth rate of female was over 15% higher than that of males [1]. Therefore, the production of allfemale stock of mandarin fish can provide important technical support for the cultivation of excellent growth character and economic character. The first step to produce all-female of mandarin fish is artificially induced sex reversal. The synthetic steroid 17α-methyltestosterone (MT) is a male-specific hormone commonly used to induce sex reversal in teleost fish [2]. The effect of MT is dependent on various factors such as timing, dose and duration of treatment [3]. MT was successfully used to masculinize genetic females into phenotypic males, such sex reversed females can produce X-type spermatozoa whose offspring would be 100% females, and the offspring can be used for actual production [4,5]. To our knowledge, the application of MT has been reported to be effective in some species such as zebrafish (Danio rerio) [6], Nile tilapia (Oreochromis niloticus) [7], orangespotted grouper (Epinephelus coioides) [8] and yellow catfish (Pelteobagrus fulvidraco) [9], but MT has been reported to have negative effects on survival, growth and/or gonadal development if a particular threshold dose is surpassed [10,11]. Therefore, the dose, timing and duration of MT used for induction of sex reversal is species-specific, and the effects of androgens need to be clarified in mandarin fish. In order to prove whether MT can successfully induce sex reversal mandarin fish to produce all-female populations, we investigated the effects of MT on the sex ratio, survival rate and growth performance. Moreover, the gonadal development of mandarin fish was determined by histological observation and levels of sex steroids testosterone (T) and 17β-estradiol (E 2 ) in serum.
Recent studies have shown that RNA-seq is a powerful strategy to quantify gene expression levels [12]. RNA-Seq has been proven more suitable to quantify transcripts expressed at low levels than microarrays or EST analysis [12][13][14]. This is because RNA-seq verifies direct transcript profiling without compromise or bias. The advantages of RNA-seq make it widely used in transcriptome profiling studies in relation to gonadal development of various fish species, including spotted knifejaw (Oplegnathus punctatus) [15], Nile tilapia [16,17] and fugu (Takifugu rubripes) [18]. However, comparative transcriptome analysis of testis and ovarian tissue is still lacking in mandarin fish.
Sex determination in fish encompasses both genetic and environmental sex determination, genetic sex determination refers to the gender of the offspring determined by the sex-determining genes on the chromosome and its interaction with the secondary genetic factors [19]. Nevertheless, few genes involved in gonadal differentiation have been identified in fish. Matsuda, et al. [20] reported that the gene named DMY (DM domain gene on Y-chromosome) was cloned from the recombination and deletion analysis of the sex-determining segment on the Y chromosome of medaka fish (Oryzias latipes). Functional analysis indicated that this gene plays an important role in sex determination of medaka fish. However, in other fish such as celebes medaka (Oryzias celebensis), rainbow trout (Oncorhynchus mykiss), Nile tilapia, zebrafish, etc., the homologous sequence could not be found [21,22]. Furthermore, sex-determining genes have yet to be discovered and identified in mandarin fish.
There are no studies with detail on sex reversal of mandarin fish by 17αmethyltestosterone. This article focused on the effect of MT on the sex ratio, survival rate, growth performance and gonadal development of mandarin fish.
Differential gene expression in female and male gonads were identified by using transcriptome sequencing technology. Characterization and expression profiles of sex-determining genes were examined during gonadal sex differentiation by MT induction in mandarin fish.

Growth status and sex ratios
The body weight and body length of mandarin fish during MT treatment were shown in Table 1. The results showed that the body weight and body length of the MTtreated group were significantly lower than those of control group (P < 0.05) from 20 dpf to 70 dpf. Among the MT treatments, the body weight and body length of M1 group were higher than M2 group with no significant difference (p > 0.05). The body weight and body length of post-treatment were summarized in Fig.1 and Fig.2. At 120 dpf, the body weight and body length of the MT treatments were significantly lower than those of control group (P<0.05). Nevertheless, no significant differences were found for the body weight and body length among the control group and MT treatments at 180 dpf and 240 dpf. There were also no significant differences in body weight and body length between the MT treatment groups at 120 dpf, 180 dpf and 240 dpf.
The survival rates of the MT treatments and control group were not significantly different (Fig.3). The MT-treated sex ratios were 100% males, while the control sex ratios were 51.11% males at 70dpf, and the ratio of females and males in the control group was about 1:1 (Table 2.).

Gonadal morphology and serum steroid hormone
At 70 dpf, the control males had testes which were populated with large volumes of spermatocytes and a few spermatogonia and spermatid and the MT-treated testes were consisted of large volumes of spermatid and a few spermatocytes and spermatogonia ( Fig. 4 a, b, c). At 120 dpf, a few spermatozoa were shown in the control testes ( Fig. 4 d), the SZ were strongly basophilic whose nuclei were stained dark blue by hematoxylin. In the seminiferous lobules away from the vas deferens, most of them were filled with spermatid. The maturity of MT-treated testes was significantly higher than that of the control testes, which testes were filled with spermatozoa ( Fig. 4 e, f). At 180 dpf, in the seminiferous lobules closed to the vas deferens of the control testes were populated with a few spermatozoa. Moreover, the lobular lumen was reduced and the lobular wall was thickened, and spermatozoa in the lobular lumen were significantly reduced or disappeared. The lobular lumen of MT-treated testes also showed signs of degeneration, but large volumes of spermatozoa were still found in the seminiferous lobules closed to the vas deferens ( Fig. 4 h, i). At 240 dpf, the lobular lumen of the MT-treated testes and the control testes were reduced to almost disappear, the lobular wall was thickened, and the lobular interstitial was developed. (Fig. 4 j, k, l). In addition, no ovarian tissue or interstitial gonads were found in MT-treated mandarin fish detected by all tissue sections.
The change in GSI of mandarin fish in control and MT treated groups were shown in Fig. 5. The results showed that GSI of the control and MT treated groups increased gradually from the 70 dpf to 240 dpf, and the highest value were at 240 dpf. GSI of MT treatments were significantly lower than that of the control group (P<0.05) at 70dpf (termination of treatment), however, the value of the control group was significantly lower than that of the MT treatments at 120 dpf (P<0.05). At 180 dpf and 240 dpf, there was no significant difference in GSI between the control and MT treated groups. During the experiment, the difference in GSI of mandarin fish in different concentrations of MT treatment group were not significant.
The changes of serum steroid hormone (T and E 2 ) in mandarin fish from 70 dpf to 240 dpf were shown in Fig. 6 and Fig.7. At termination of treatment (70dpf), the contents of T and E 2 of the control group were significantly higher than those in the MT treatment group (P<0.05), and the difference in T and E 2 of mandarin fish in different concentrations of MT treatment group were not significant. When the 17αmethyltestosterone treatment was completed, the levels of plasma T and E 2 of the MT-treated groups returned to normal, and there was no significant difference with the control group.

Illumina sequencing and unigenes annotation
Gonad cDNA libraries of nine testes and nine ovaries were sequenced. A total of 120,955,742 (97.72%) clean reads were obtained and used for de novo assembly. 60,971,396 and 59,984,346 clean reads were obtained from ovaries and testes, respectively, with a mean sequence length of 150bp (Table 3). After assembling and clustering, a total of 63,632 unigenes ranging from 201 to 21,004 bp with a mean sequence length of 1,182 bp were acquired (Fig. 8).
A total of 33,535 (52.7%) unigenes were annotated from the four databases, among which 33,471 unigenes were annotated from Nr database, 27,685 unigenes were annotated from Swissprot database, 22,501 unigenes were annotated from COG/KOG database and 20,323 unigenes were annotated from KEGG database (Fig. 9).

Analysis of differentially expressed genes
After RPKM standardized calculation, 46,391 DEGs were detected in the gonads of 120 dpf, among which 3,435 genes were highly expressed in ovaries and 42,956 in testes. When RPKM≥2, 811 and 1,530 genes were differentially expressed in ovaries and testes, respectively.  Table 4).
Identification of DEGs associated with sex development By analyzing the transcriptome data of ovaries and testes, the expression levels of 7 genes related to sex determination and differentiation were listed in Table 5. We noticed that dmrt1, amh, sox9, gsdf and dax1 expression in the testes were higher than ovaries (P < 0.05), while foxl2 and cyp19a1a expression in the ovaries were higher than testes (P<0.05).

Real-time PCR verifications
At termination of treatment (70dpf), the relative expression of dmrt1, sox9, foxl2 and cyp19a1a in the gonad were shown in fig.11 and fig.12. The relative expression of dmrt1, sox9 in M0 (♂), M1 and M2 were significantly higher than that in M0 (♀), but the relative expression of foxl2 and cyp19a1a were significantly lower than that in M0 (♀) (p<0.05). In different concentrations of MT treatment groups and M0 (♂), the relative expression of dmrt1, sox9, foxl2 and cyp19a1a were not significantly different (p>0.05).

Discussion
The anabolic effects of MT on growth have been reported in some teleost fish, but the effect of MT on growth was not the same. Some studies suggest that low concentration of MT can promote the growth of fish, such as Eurasian perch (Perca fluviatilis) [25], common carp (Cyprinus carpio L.) [26], brook trout (Salvelinus fontinalis) [27] and zebrafish [28]. This may be due to the role of androgens in stimulating appetite and accelerating protein synthesis in fish [29]. Moreover, some research holded that high concentration of MT inhibited fish growth, such as black molly (Poecilia sphenops) [30], sablefish (Anoplopoma fimbria) [31], brook trout [11] and Atlantic cod (Gadus morhua) [32]. The results of this study showed that different concentrations of MT induced marked inhibition on the growth of treatment groups, indicating that the MT concentration we selected may have exceeded the upper limit of promoting fish growth, and the treatment concentration was too high to promote the growth of mandarin fish. After hormonal treatment, growth effects as compensatory growth effects may occur in some fish [33,34]. The same phenomenon was found in our study which indicated that this may be a sign of compensatory growth. The treatment intensity of MT was related to the treatment time and concentration. When the treatment intensity of MT was low, it had no significant effect on the survival rate of the treatment group. Nevertheless, when the intensity of MT treatment was too high, the survival rate of the treatment group was significantly lower than that of the control group [35]. Research found that zebra cichlid (Cichlasoma nigrofasciatum) was feed with high-dose estradiol a few days, the mortality of treatment group was significantly higher than that of the control group [36]. Furthermore, yellow catfish was induced with low-dose 17βestradiol and 17α-methyltestosterone for a long time, and the results showed that the survival rate of the treatment group was significantly lower than that of the control group [30]. However, there was no significant difference in mortality between the treatment group and the control group when the European sea bass (Dicentrarchus labrax L.) [37] was fed with low-dose MT for a short time. In this study, we found that mandarin fish treated with dietary MT at 50 mg/kg and 100 mg/kg dosages for 2 months, the survival rate was slightly lower than that of the control group, which indicated that the concentration of MT can be used to induce the sex reversal mandarin fish, and the treatment intensity was more appropriate.
Appropriate concentration of MT treatment can promote the gonadal development and make the gonads present large volumes of spermatid in advance [28,35], which were consistent with the result of our research. It was found that MT treatment may lead to gonadal atrophy, hermaphrodites and female ratio increased [38][39][40]. For example, the GSI of the treatment group was lower than that of the control group when Nile tilapia was treated with MT at a concentration of 40 mg/kg [41]. Likewise, Phelps, et al. also found that GSI of the treatment group was lower than that of the control group in Nile tilapia [7]. In Atlantic cod, MT was successful in producing hermaphrodites (46.7%) in the groups that were treated for the longest time period In this study, we investigated the effects of MT treatment on mandarin fish gonadal transcriptomes during gonadal differentiation and screened out the genes related to the sex determination and differentiation. MT activated gonadal expression of promale genes dmrt1 and sox9 and repressed gonadal expression of pro-female genes cyp19a1a and foxl2. We found strong upregulation of dmrt1 expression in control group in males and MT-treated testes in this study, consistent with the research of honeycomb grouper (Epinephelus merra) [55]. In zabrafish, upregulation of dmrt1 expression is associated with androgen-induced gonadal masculinization [6].
Similarly, the expression of dmrt1 was found upregulation in sex reversal of rainbow trout [56] and medaka [57]. It is indicated that the dmrtl may be a key gene in the gonadal masculinisation in fish. MT treatment increased sox9 expression in mandarin fish gonads. Sox9 is an important gene associated with male sex differentiation and gonad development [58], and there are two sox9 ( sox9a and sox9b) genes in most teleost [59]. The expression of sox9 in the testis, ovary, brain, liver and heart of rainbow trout were detected and the results showed that the gene was only significantly expressed in the testis and brain [60]. Similar to rainbow trout, sox9b were found to be expressed at higher levels in the testes than ovaries in Nile tilapia [61,62].In orange-spotted grouper, the expression level of sox9 upregulated significantly and the results of tissue section showed that the gonad of it changed from ovary to testis during MT induction, indicating that sox9 may be an important factor in triggering and maintaining masculinity of orange-spotted grouper [63]. Our results suggest that sox9 may play an important role in the differentiation and development of testis. Foxl2 belongs to the forkhead transcription factors family and plays an important role in the development and maintenance of ovarian function in vertebrate animals [64]. In fish such as rainbow trout [65], Japanese flounder (Paralichthys olivaceus) [66] and Nile tilapia [67], the expression level of foxl2 in ovary was significantly higher than that in testis. In addition, in female Nile tilapia who knocked out foxl2, the E 2 levels and cyp19a1a expression levels in gonad decreased, resulting in their gonadal masculinization [68]. In our study, the expression level of foxl2 in the ovary of mandarin fish was significantly higher than that in the testis, indicating that foxl2 is a key factor in the development of ovary. Our study showed that the expression of cyp19a1a in the ovary of mandarin fish was significantly higher than that in the testis. Aromatase cytochrome P450 encoded by cyp19a1a is a key enzyme in estrogen synthesis [69,70]. In Nile tilapia [71], European sea bass [72] and Japanese eel (Anguilla japonica) [73], the expression of cyp19a1a was markedly higher in the ovary than that in testis. During the sexual differentiation of rainbow trout, cyp19a1a was highly expressed in ovaries [74]. We suggest that cyp19a1a may be one of the important gene in the development of the ovary. In a sense, the male-restricted expression of dmrt1, sox9 and no female-specific expression of cyp19a1a, foxl2 further confirmed the masculinization of mandarin fish by MT at the molecular level.

Evaluation of growth status and sex ratios
During hormone induction, the 10 dpf juvenile mandarin fish were taken every 10 days and 30 samples of each replicate were taken to measure the body weight (accurate to 0.001g) and body length (accurate to 0.01cm). At the termination of treatment, the survival rate was counted and each parallel including 30 individuals were dissected to obtain external morphology of the gonads which used to determine the gonadal sex of each fish and sex ratios for each exposure group.
At the termination of hormone induction, 300 individuals were randomly selected from the control group and MT treatment groups respectively, and then transferred into 20×20 m cement pool at water temperature of 25-30℃ with natural photoperiod. Each group consisted of 3 biological replicates comprising 900 juvenile fish each. At 120 dpf, 180 dpf, and 240 dpf, 30 individuals of the control group, M1 treatment group, and M2 treatment group were randomly selected from the cement pool, which were anesthetized by MS-222 to measure body weight and body length.

Determination of gonadal morphology and serum steroid hormone
At 70dpf (termination of treatment), 120 dpf, 180 dpf, and 240 dpf, 30 individuals were randomly selected from the control group (male gonads), M1 treatment group, and M2 treatment group respectively, which were anesthetized by MS-222 and dissected to obtain the individual gonads. After weighed body weight and gonad weight, gonads were transferred to Bouin's solution, and then gonadosomatic index (GSI) were calculated using the formula: GSI = (gonad weight/body weight) Í100.

Statistical analysis
The results were expressed as mean ± SD. The percentage data were transformed by arcsin square root transformation. Data were subjected to one-way ANOVA followed by Duncan's multiple range test for post hoc comparisons of means. SPSS version 21.0 was used for the analyses in the study. The level of significance for all statistical tests was set at 5% (p< 0.05).

Availability of data and materials
All data generated or analysed during this study are included in this published article.

Authors' contributions
SL, PX and GFL designed the experiments. SL and PX performed the treatments, sample collection and laboratory work with the assistance of YQS and HDY. SL and PX analysed and interpreted the RNASeq data with the assistance of XGL. XLC and DLG performed the mandarin fish histology. SL wrote the manuscript with contributions from SB and XPZ. All authors read and approved the final manuscript.

Ethics approval and consent to participate
Mandarin fish experiments were approved by the Institutional Animal Care and Use Committee of Sun Yat-sen University and performed in accordance with the guidelines for experimental animals established by this committee.

Consent for publication
Not applicable.

Competing interests
The authors declare no competing financial interests.  Note: *significant difference between control group and MT treatments (P<0.05).

Author details
Each experiment was repeated three times.  The body length of S. chautsi post-treatment. Data presented are expressed as mean±SD (n= The gonadosomatic index (GSI) of S. chautsi male in control and MT treated groups. Data pre Figure 6 Effects of 17α-methyltestosterone on serum T levels in S. chautsi male in control and MT trea Effects of 17α-methyltestosterone on serum E2 levels in S. chautsi male in control and MT tre Homologous genes in public databases of S.chuatsi-Unigene Effects of 17α-methyltestosterone on expression level of dmrt1, sox9 in the gonads of S. cha Effects of 17α-methyltestosterone on expression level of foxl2, cyp19a1a in the gonads of S.

Supplementary Files
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