Transcriptome profiling of laser-captured germ cells and functional characterization of zbtb40 during MT-induced spermatogenesis in orange-spotted grouper (Epinephelus coioides)

Background: Spermatogenesis is an intricate process regulated by a finely organized network. The orange-spotted grouper (Epinephelus coioides) is a protogynous hermaphroditic fish, but the process of its spermatogenesis is not well-understood. In the present study, transcriptome sequencing of the male germ cells from orange-spotted grouper was performed to explore the molecular mechanisms underlying spermatogenesis. Results: In this study, the orange-spotted grouper was induced to change sex from female to male by 17alpha-methyltestosterone implantation. During the artificial spermatogenesis, different cell types from cysts containing spermatogonia, spermatocytes, spermatids, and spermatozoa were isolated by laser capture microdissection. Subsequently, transcriptomic analysis for the isolated cells were performed. A series of genes was used to verify and investigate the expression patterns in spermatogenesis. Furthermore, we also analyzed the expression of the same set of genes involved with steroid metabolism and sex throughout spermatogenesis (early-mid, late, and maturing stages) in the orange-spotted grouper. Several generally female-related genes took significantly changes in sex reversal hinted that the female-related genes in previously recognized may also play vital roles in spermatogenesis and sex reversal. In the transcriptomic data, we focused on zbtb family genes, which may be related to the process of spermatogenesis. Their expression patterns and cellular localization were examined, and the location of Eczbtb40 in different gonadal stages was investigated. We found that Eczbtb40 was expressed throughout spermatogenesis. These preliminary findings suggest that Eczbtb40 is highly conserved during vertebrate evolution and plays roles in spermatogenesis. Besides, the expression of Eczbtb40 and Eccyp17a1a overlapped in male germ cells, especially spermatogonium and spermatocyte, which suggested that Eczbtb40 might interact with Eccyp17a1a participant in spermatogenesis and sex reversal.

3 Conclusions: The present study first depicted RNA sequencing of the male germ cells from orange-spotted grouper, and identified many important functional genes and pathways involved in spermatogenesis. The Eczbtb40 gene was subjected to molecular characterization and expression pattern analysis. These results will contribute to future studies of the molecular mechanism of spermatogenesis and sex reversal.

Background
Transcriptome sequencing is a technology to catalogue all species of transcript, determine the transcriptional structure of genes, and quantify the expression of every transcript under different conditions [1]. Comparison of transcriptomes can provide both quantitative and qualitative information on genetic activity [2]. When gross tissue extracts are used as an mRNA source, tissue heterogeneity confounds assigning expressed genes to different cell populations [3]. Although some in situ technologies, such as in situ hybridization or immunohistochemistry, could confirm the spatial expression of specific gene, when numerous messages need to be examined, these technologies might not always be possible and is laborious and time consuming [4]. Hence, many researchers have tried to develop microdissection protocols to yield mRNA with sufficient quality for the subsequent transcriptomic analysis. Because of the speed, precision, and versatility, laser capture microdissection (LCM) was applied in omics , including transcriptome [5, 6] and proteome [7].
LCM is a technology first developed in the late 1990s for obtaining pure populations of targeted cells from specific microscopic regions of tissue sections for subsequent analysis.
LCM is a microscope-based technology involving sampling, sample embedding, cryosection, visualization of the target cells, adjustment of the cutting parameters, selecting the target areas, and collection of dissected cells into a tube [8]. The technology 4 was first applied to study gene expression in human cancer cells and gene expression during spermatogenesis in rodents [9][10][11]. Subsequently, many researcher used LCM to acquire different male germ cells during spermatogenesis for detecting the expression of important genes in many other species, including European sea bass [12], zebrafish [13][14][15], and African catfish [16].
We first used LCM to obtain male germ cells from different developmental stages induced by 17alpha-methyltestosterone (MT) to profile the natural process of spermatogenesis in orange-spotted grouper (Epinephelus coioides). Grouper is a protogynous hermaphroditic fish underlying sex change from female to male in the life history [17]. It has been considered as a good model for the study of sex differentiation, ovarian development, and sex reversal. Because of lack of male fish, few studies of spermatogenesis has been conducted out in orange-spotted grouper. To profile the process of spermatogenesis in grouper, the LCM protocol was optimized to obtain the four germ cell types from male grouper induced by MT implantation, including spermatogonia (SG), spermatocytes (SC), spermatids (ST), and spermatozoa (SZ).
Spermatogenesis is a developmental process in which diploid male germ cells transform into haploid functional male gametes in a tight spatial and temporal organization.
Spermatogonia develop into primary spermatocytes, which becomes haploid spermatids through two meiotic divisions without DNA replication, finally transforming into mature spermatozoa. These processes are achieved by a complex network involving many genes, the germ cells themselves, and several somatic cell types (i.e. Sertoli cells and Leydig cells) [18]. The structural and functional aspects of spermatogenesis are highly conserved among the vertebrates [19]. The release of gonadotropic hormones (follicle-stimulating hormone, luteinizing hormone, growth hormone) released from pituitary stimulate the testis to produce the androgens that initiate spermatogenesis [19]. Hormonal control is a complicated network affected by many stage-specific and cell-specific factors [13,20].
Previously, quantifying specific genes is difficult because the complex testis structure contains various cell types. Some approaches can be used to quantify and localize unique genes, including in situ hybridization [21,22], serial analysis of gene expression (SAGE) [23,24], and in vitro culture of the specific cell population [25]. However, these methods exist some problems to overcome, like the time it takes, the difficulty of the approach itself, etc. Subsequently, researchers have focused on LCM and established an optimized method to obtain specific cell types for studying spermatogenesis. Nevertheless, it is still difficult to isolate the somatic cells around the germ cells to mirror the intricate relationship between somatic cells and different male germ cells during spermatogenesis.
So in our study, transcriptome sequencing was conducted combining LCM to obtain specific cell type to explore the spermatogenesis induced by 17alpha-methyltestosterone (MT) in order to reveal the natural spermatogenesis in a new aspect. Transcriptomic analysis was performed with the goal of revealing the differential gene expression and regulatory networks and zbtb genes came into our sight. Zinc finger and BTB (broad complex, Tramtrack, and Bric-à-brac) (ZBTB) proteins are an evolutionarily conserved family of transcription factors.
Approximately 60 ZBTB proteins has been identified involving in diverse functions including development, differentiation, and oncogenesis [26][27][28]. In recent years, ZBTB16 was found to play an essential role in spermatogenesis by controlling the self-renew and differentiation of spermatogonium [29][30][31][32]. In the transcriptome data, we found that 6 several zbtb family genes was differently expressed. Further, the zbtb family genes were identified and their expression were investigated during the process of sex reversal and spermatogenesis, and the expression pattern and potential function of Eczbtb40 were determined in spermatogenesis.

Developmental stages of gonads during MT-induced sex reversal
As shown in the Fig. 1, fish oocytes in the sham group remained in the primary-growth stage throughout the experimental period. In contrast, the fish in MT-implanted group underwent sex reversion from female to male. In the first week of MT implantation, the gonads were characterized by degeneration of oocytes and simultaneous proliferation of spermatogenic cysts (Week 1; Fig. 1C). At two weeks after MT implantation, the gonads entered into the intermediate transitional stage filled with SG and SC and numerous oocytes (Week 2; Fig. 1E). At three weeks after MT implantation, the gonads transformed into functional testis, comprised mostly of ST, a small number of SZ, and a few oocytes (Week 3; Fig. 1G).

Capture of target cells
The morphology of SG, SC, and ST were characterized by hematoxylin and eosin (H&E) ( Fig.2A) and cryosection staining (Fig.2B). SZ was obtained from the functional testis.
During spermatogonia differentiation, the density of heterochromatin reached a the maximum in type B late spermatogonia [33]. When spermatid changed into sperm, the DNA was maximally compacted [18]. Thus, the sizes of the four types of male germ cells decreased (SG > SC > ST > SZ) and staining of the cell nucleus became darker. Four different male germ cells were detected in tissue slices based on these characteristics.

Validation of sample specificity
Six genes were used to examine the purity of the LCM-derived RNA samples (Fig.3A). ef1a is a reference gene and commonly used as an internal control for gene expression analysis. Its expression was detected in all four types of male germ cells at similar levels.
Vasa is a germ cell marker [34] whose expression was also found in the four cell types.
slbp2 was specifically expressed in the oocyte of orange-spotted grouper [35], while no expression was detected in any male germ cells except for in the positive control (the gonad with mainly primary-growth stage oocytes). dmrt1 was specifically expressed in the spermatogenic cells of orange-spotted grouper [36]. Here, dmrt1 was only expressed in SG and SC with no expression in the positive or negative control. A total of 253,040, 890 raw reads were obtained, and a total of 244, 984, 338 clean reads were produced after removing low-quality reads and adapter sequences ( Table 1). The average Q20 and Q30 values were 95.74% and 90.57% respectively, and the content of GC was 44.08-47.47%, reflecting the accuracy of the transcriptomic data.

Differentially expressed genes (DEGs) among the four cell typesSG, SC, ST, and SC
Fragments per kilobase million (FPKMs) were used to quantify the gene expression levels.
The FPKM values of each gene in the four cell types were compared respectively (Additional file 1). There were 16,406 up-regulated genes and 11,054 down-regulated 8 genes in SG compared to SC. A total of 15,845 up-regulated genes and 6,895 downregulated genes were identified in SC to ST. The STs had 8,320 up-regulated genes and 15,797 down-regulated genes compared to SZ. And SG had 22,599 up-regulated genes and 7,399 down-regulated genes relative to STs. There were 14,624 up-regulated genes and 12,413 down-regulated genes in SC compared to SZ. Among the four cell types, 4,483 DEGs were detected and analyzed.

GO and KEGG enrichment of DEGs
In the biological process and molecular function categories of the 4483 DEGs, cellular process (GO: 0009987) and binding (GO: 0005488) were the most enriched GO terms (Fig.   S1). And the top 20 pathways were listed from KEGG enrichment (Fig. 4A). Among them, differentiation pathways, signal pathways, and metabolism pathways, which may play crucial roles during spermatogenesis, were observed.

Expression of functional genes putatively associated with sex differentiation and steroid metabolism
From the transcriptomic data, the expression of 20 genes putatively related to sex differentiation and steroid metabolism were analyzed ( Fig.5 and 6). Eight of the sex differentiation genes (sox11a, nr0b1, era, erb, wnt9, gdf9, and bmp15) and 13 of the short-chain dehydrogenases/reductases (SDR) genes (cyp17a1, 3hd, sdr12, sdr11, p5cdh , cyp3a40, and sdr13) which are involved in steroid metabolism were expressed in different cell types with FPKM values ranging from 2.62 to 604.7. Most of these genes showed peak expression in SG and SZ (Fig. 4 B).

9
RT-PCR was conducted to validate the expression patterns of 13 genes predicted to be involved with sex differentiation and steroid metabolism (asterisk-marked genes in Fig. 4) and deduced from the transcriptomic data. As shown in Figs. 5 and 6, all of these genes were significantly changed in the process of sex reversal, which was mostly consistent with the transcriptomic data. These results indicate that the expressional analysis based on RNA-seq data was credible in the present study.

Molecular cloning and sequence analysis of Eczbtb40
Among the 4438 DEGs in four cell types, several zbtb genes including zbtb1, zbtb22, zbtb40, and zbtb44 attracted our attention. ZBTB family proteins are transcription factors that participate in various important functions. It's reported that zbtb16 ( plzf) played an important role in the self-renewal and differentiation of the undifferentiated spermatogonia [29]. Therefore, whether the zbtb genes regulate in spermatogenesis of orange-spotted grouper was examined. We studied the expression of the zbtb genes (zbtb1, zbtb22, zbtb40, and zbtb44) to determine their functions in spermatogenesis ( The open reading frame (ORF) of zbtb40 was cloned from the testis of orange-spotted grouper, which were denominated as Eczbtb40 (GenBank accession number, MN167853).
As shown in Fig. S2, Eczbtb40 consisted of a 2400-base pair ORF encoding a peptide of 799 amid acids. Amino acid sequence alignment and comparison analysis indicated that zbtb40 contains a conserved domain (Fig. S4). A phylogenetic tree was constructed based on amino acid sequences of the known zbtb40 genes (Fig. 7A). On the tree, Epinephelus coioides zbtb40 was clustered together with Larimichys crocea zbtb40. The zbtb40s contains variously conserved sites in all selected species, among them the top ten motif sites were showed (Fig. 7B). The DNA sequence of each motif site was displayed in Tab. S1.

Tissue distribution of Eczbtb40
Expression of Eczbtb40 was examined in eight tissues by semi-quantitative PCR. The results revealed very high Eczbtb40 expression in the testis, heart, and pituitary and weak expression in whole brain, head kidney, liver, and ovary (Fig.3B).

Expression profiles of Eczbtb40 in gonads during MT-induced sex reversal
The expression pattern of the Eczbtb40 during the MT-induced sex reversal process was investigated. Eczbtb40 expression showed no significant difference from the control groups (Fig. 6H). The mRNA levels in MT-treated fish were significantly higher than those in the control group in the second weeks.

In situ localization of Eczbtb40 in gonads during sex reversal
At the early stage of sex reversal, there were still a large number of primary-growth stage oocytes which wasn't detected the expression of Eczbtb40 mRNA ( Fig. 8B). At the middle stage of sex reversion, many spermatogonia and spermatocytes emerged, showing Eczbtb40 mRNA expression (Fig. 8C). At the late stage of sex reversal, there were few oocytes in the gonad and Eczbtb40 was abundantly expressed in SG, SC, ST, and SC ( Fig.   8D). In comparison, no signal was detected in the ovary from the control group and sham group (Fig. 8A).

Co-localization of Eczbtb40 and Eccyp17a1 in gonads during MT-induced sex reversal
Using the JASPAR CORE 2018 database of transcription factor DNA-binding sites, a match was found between an Eczbtb40 MEME-generated consensus sequence and the Eccyp17a1 consensus DNA binding site (Tab. S1). The subcellular localization and co-localization of Eczbtb40 and Eccyp17a in gonads cells were examined during MT-induced sex reversal by fluorescence in situ hybridization under a confocal microscope (Fig. 9). In ovary, Eczbtb40 showed little signal, while Eccyp17a1 signals were clearly present in the cytoplasm of primary growth stage oocyte ( Fig. 9A-D). In testis, the signals of Eczbtb40 in the cytoplasm of SG and SC were overlapped with the Eccyp17a1 signals ( Fig. 9E-H). Both Eczbtb40 and Eccyp17a1 showed weak signals in ST and SZ. The signals were observed nearly exclusively in the cytoplasm but were barely detectable in the nucleus.

Discussion
In present study, transcriptome analysis was used to profile the process of spermatogenesis based on the LCM technology. A large number of differently expressed genes and signal pathways related to spermatogenesis were identified in this study.

Quality and purity of the LCM-derived RNA
The whole process of LCM must produce a sufficient amount of RNA with high quality to ensure the reliability of the transcriptome results. Single-cell LCM requires a long microdissection period and the yield of RNA is limited. In this study, we modified a previous protocol [37] to handle slides and tissues, samples staining and capture the cells for preserving the RNA integrity throughout microdissection.
To verify the purity of the four target cells, several cell markers were detected. The expression of ef1a indicated the homogeneity of concentration in four cell types. Vasa, a germ cell marker [38], is required for proper germ cells development. Its expression levels were decreased gradually with the process spermatogenesis which is consistent with the expression pattern of vasa in the male germ cells of the gibel carp [38] and brownmarbled grouper [34]. As an oocyte-specific marker [35,39], slbp2 was used to distinguish the female germ cell and male germ cell. The non-expression of slbp2 in four cell types indicated that target cells didn't contaminate by female germ cells. dmrt1 was used as a spermatogenic cell marker [40] and is only expressed in SG and SC but not in ST and SZ in orange-spotted grouper. Based on the expression of these cell markers, the four target cells were considered to be pure. The quality and purity of the LCM-derived RNA met the criterion for constructing the single cell transcriptome library after a series of detection.

Transcriptome overview of spermatogenesis in orange-spotted grouper
In the present study, male germ cells during MT-induced spermatogenesis were used to explore the natural spermatogenesis and profile the expression patterns of genes related  [52]. ERα and ERβ were significantly increased after MTimplantation, likely because nr0b1 was decreased in orange-spotted grouper (Fig. 5B, C).
This suggested that estrogen and ERs are important in both male and female.
Wnt genes encode a large family of secreted factors with diverse roles in governing cell fate, proliferation, migration, polarity, and death [53]. Although few studies have evaluated the role of wnt9 in testis, the significant increase in wnt9 during sex reversal revealed its potential role in spermatogenesis (Fig. 5D). A previous study showed that the oocyte-secreted growth differentiation factor (GDF) 9 and bone morphogenetic protein 15 (BMP15) regulate the growth, differentiation, and function of granulosa and thecal cells during follicular development in oocyte development, ovulation, fertilization, and embryonic competence [54]. An increasing number of studies has focused on the function of GDF9 in the testis. GDF9 mRNA in the testis of mouse, rat, and human is specifically detected in two of the germ cell types: large spermatocytes, and round spermatids [55].
Recombinant GDF9 was reported to disrupt the inter-Sertoli tight junction permeability 14 barrier in vitro [56], suggesting that gdf9 regulates spermatogenesis in vivo. Similarly, there are several reports of the transcript or protein of BMP15 in testis and its role in testicular functions, indicating that bmp15 functions in the testis as in the ovary [57,58].
In our study, the significant changes in gdf9 and bmp15 adequately indicate their potential roles in the sex reversal or spermatogenesis of orange-spotted grouper. Overall, the significantly changes in several generally female-related genes indicate that femalerelated genes may also play vital roles in spermatogenesis and sex reversal. These genes should be further explored in the context of spermatogenesis and sex reversal.

MT-induced sex reversal
In this study, the sex reversal was artificially inducted by MT (Sigma, USA) treatment. The fabrication of the slow-release strips and steps of MT implantation were referred to our previous paper with minor modification [60]. Fish (body weight, 1.90 ± 0.65 kg; body length, 43.75 ± 9.25 cm) were divided into two groups, sham group (n = 15) and MT implantation group (n = 15). The dosage of MT was 10 mg/kg body weight. Before implantation (Week 0), gonadal tissues of five fish were collected randomly. After MT implantation, five fish were sampled randomly every week respectively from two groups until 3 weeks. For each fish, one piece of gonadal tissue was fixed in Bouin's solution for histological examination of the development stage of the gonad, another piece of the gonad was immobilized by 4% paraformaldehyde for florescence in situ hybridization (FISH), the other piece of gonadal tissue was embedded with OCT (Sakura, USA) then frozen immediately in liquid nitrogen for LCM. All the other tissues were frozen immediately in liquid nitrogen, and then stored at -80 °C until further use.

Histology analysis
Gonadal tissues were embedded in paraffin after being fixed 24 h in Bouin's solution. The embedded blocks were sectioned at 5~6 μm and stained with hematoxylin and eosin (H&E). The gonadal sections were classified by light microscopy.

Visualization of the target cells
There are several factors influencing the RNA quality in the process of visualization, including the sample cryostat sections (the quality of fresh and quick-frozen samples usually is more optimal than fixed tissues), the complex histopathology, and issues arising from the staining, and so on.

Cryostat section s of gonad for LCM
The RNase-free Membrane Slides (MMI, Switzerland) as a slide to mount the cryosections. Before sectioning, the microtome (Leica, Germany) was wiped down with RNase inhibitor (Ambion ® , USA) to avoid cross-contamination, and a new blade (Lecia, Germany) treated with RNase inhibitor was used to cut each sample. The gonad blocks were put into Leica Microtomes 30 minutes to adjust the sectioning temperature (-20 °C~ -25 °C). The ovary was cryosectioned at 7 μm, and the testis was cryosectioned at 6 μm.

Cryosections Staining
After being desiccated for one minute, the sections stained by H&E Staining Kit Plus (MMI, Switzerland). The procedures were referred to instructions of the Kit and taken some adjustments to make sure the optimization of RNA quality. However, the sections of the two groups were performed different staining protocols. The whole process was controlled in 30 minutes [61].

Laser Capture Microdissection
First of all, all facilities and tools were wiped by RNase inhibitor. General sterile glass slides were put under the Membrane Slides as supported slides. Once the stained slides exposed in air, the surface of the slide emerged massive water droplets which accelerate degradation of RNA. Thus the whole process of microdissection must control in one hour, and dry the sections quickly. Then load the slides and LCM caps (diffuser caps, MMI, Switzerland) to laser micro-cutting instrument (MMI, CellCut Plus, Switzerland). Find the cells of interest through adjusting microscope, at the same time optimize three important parameters (cell velocity, laser focus, and laser power). After circling the interesting area, the laser starts to capture the cells as many as possible. At last, unload of caps containing the captured tissue, and add 50 μl TPK Lysis Buffer (Micro Elute ® RNA Kit, Omega, USA) immediately. Extract RNA instantly or store diffuser caps in -80 °C (<2 days).

RNA extraction
The procedures of RNA extraction were referred to as instructions of Micro Elute ® RNA Kit (Omega, USA) with some adjustment.

Library preparation for transcriptome sequencing
A total amount of 1.5 µg RNA per sample was used as input material for the RNA sample preparation. NEBNext ® Ultra™ RNA Library Prep Kit were used to generate sequencing libraries for Illumina ® (NEB, USA) following the manufacturer's recommendations. And index codes were added to attribute sequences to each sample. Briefly, mRNA was purified from total RNA using poly-T oligo-attached magnetic beads.

Processing of raw reads and quantification of differential gene expression levels
Raw data (raw reads) of the fastq format were first processed through in-house Perl scripts. In this step, clean data (clean reads) were obtained by removing reads containing adapter, reads containing ploy-N and low quality reads from raw data. At the same time, Q20, Q30 and GC content the clean data were calculated. Q20 indicates that every 100 bp of sequencing reads will have an error, and Q30 indicates that every 1000 bp of sequencing reads will have an error. All the downstream analyses were based on clean data with high quality. length for the reads count at the same time, and is currently the most commonly used method for estimating gene expression levels [63].

Differential expression genes analysis
Prior to differential gene expression analysis, for each sequenced library, the read counts 20 were adjusted by edgeR program package through one scaling normalized factor. Differential expression analysis of two conditions was performed using the DEGSeq R package (1.20.0). The P values were adjusted using the Benjamini & Hochberg method.
Corrected P-value of 0.005 and log 2 (Fold change) of 1 were set as the threshold for significant differential expression.

GO and KEGG enrichment analysis of differentially expressed genes
Gene Ontology (GO) enrichment analysis of differentially expressed genes was implemented by the GOseq R package, in which gene length bias was corrected. GO terms with corrected P value less than 0.05 were considered significantly enriched by differential expressed genes. The identified DEGs were conducted for enrichment analysis subsequently by GO: Termfinder software using the hypergeometric test [64,65], and Pvalues were corrected using the Bonferroni method [57]. Being selected significantly enriched GO terms with Q-value < 0.05.
KEGG is a database resource for understanding high-level functions and utilities of the biological system, such as the cell, the organism and the ecosystem, from molecular-level information, especially large-scale molecular datasets generated by genome sequencing and other high-through put experimental technologies (http://www.genome.jp/kegg/). We used KOBAS software to test the statistical enrichment of differential expression genes in KEGG pathways.

Different expression genes analyzed by real-time PCR
To validate the accordance with those genes inferred from RNA-seq data, the relative mRNA levels of 13 differentially expressed genes (nr0b1, era, erb, wnt9, gdf9, bmp15,   cyp17a1, 3hd, sdr12, sdr11, p5cdh , cyp3a40, and sdr13) were examined by quantitative real-time PCR (RT-PCR) in sex reversal of orange-spotted grouper. Total RNA of gonad was extracted by TRIzol (Invitrogen, USA) and then 1 µg RNA from each sample was reverse transcribed with random primers by using the First Strand cDNA Synthesis Kit (Roche, USA) according to the manufacturer's instruction. The RT-PCR reaction was performed in a 10 μl reaction volume using the SYBR Green PCR master mix (Roche, USA). The amplification regime was 95 °C for 5 min, followed by 40 cycles of amplification at 95 °C for 10 s, 58 °C for 15 s and 72 °C for 20 s. The specificity of RT-PCR amplification was confirmed by melt-curve analysis, agarose gel electrophoresis, and sequencing of PCR products. All mRNA quantification data were normalized to ef1a and presented as a relative control group. The specific primers used in this study were listed in Table 3.

Open Reading Frame (ORF) cloning and sequence analysis of Eczbtb40 cDNAs
Total RNA of the gonad was extracted by TRIzol (Invitrogen, USA). RNA was reversed to cDNAs with First Strand cDNA Synthesis Kit (Roche, USA). The reverse transcription process was as follow 37 °C for 15 min, 98 °C for 5 min, 4 °C for 5 min. The amplification regime was 35 cycles of 94 °C for 20 s, 55 °C for 10 s, and 72 °C for 20 s, followed by further amplification at 72 °C for 5 min. Based on the cDNA fragments in RNA-seq data, specific upstream and downstream primers ( Table 2)  The purified product was then subcloned into the pGEM-Easy vector (Fermentas, USA).

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According to the sequencing result, the ORFs of Eczbtb40 were obtained.
The putative amino acid sequences were predicted by DNAMAN software and multiple sequence alignments of amino acids were performed in the ClustalX (1.81) software.
Meanwhile, protein phylogenetic analysis was conducted with MEGAX using the method of neighbor-joining method and the top ten motif sites were predicted by motif-based sequence analysis tools (MEME).

Tissue distribution of Eczbtb40
To detect the tissue distribution of Eczbtb40, eight tissues were dissected, including whole brain, heart, head kidney, liver, kidney, pituitary, ovary, and testis. Total RNA from eight tissues was extracted. RNA was reversed to cDNA with First Strand cDNA Synthesis Kit (Roche, USA). The reverse transcription process was as follow 37 °C for 15 min, 98 °C for 5 min, 4 °C for 5 min. The amplification regime was 35 cycles of 94 °C for 20 s, 55 °C for 10 s, and 72 °C for 20 s, followed by further amplification at 72 °C for 5 min. The specific primers used in this study were listed in Table 2.

Expression profile of Eczbtb40 in gonads during MT-induced sex reversal
The expression profiles of Eczbtb40 in the gonad were detected by RT-PCR during MTinduced sex reversal.

Duel-label in situ hybridization of Eczbtb40 and Eccyp17a1 in gonads
The protocol of dual-label in situ hybridization of Eczbtb40 and Eccyp17a1 was referred to previous study [67]. Expression of Eczbtb40 was performed using digoxigenin (DIG)labeled mRNA probes in combination with biotin-labeled Eccyp17a1 mRNA probes.

Statistical analysis
All data were expressed as mean values ± SEM. Significant differences were checked by one-way analysis of variance (ANOVA) and student′s t-test was used, and a probability level less than 0.05 (P < 0.05) was used to indicate significance. All data were performed

Consent for publication
Not applicable.

Competing interests
The authors declare that they have no competing interests.  25 The datasets used and analyzed during the current study available from the corresponding author on reasonable request.

Authors' contributions
XW and YY analyzed the sequencing data; XW, CYZ and YG collected and prepared the samples; XW wrote the manuscript; SSL and XCL supervised the study. All authors read and approved the final manuscript for publication.