2 Laboratory for Marine Fisheries Science and Food Production Processes, Qingdao National Laboratory for Marine Science and Technology, Qingdao 266373, China;
3 Guangdong Key Laboratory of Animal Conservation and Resource Utilization, Guangdong Public Laboratory of Wild Animal Conservation and Utilization, Guangdong Institute of Applied Biological Resources, Guangzhou 510260, China;
4 Guangdong Liangshi Aquatic Seed Industry Co., Ltd., Foshan 528100, China
In almost all vertebrates, sexual reproduction is an important biological process, essential to maintain long-term survival and procreation. In order to ensure the continuation of species, vertebrates have evolved complex patterns of sex determination and differentiation. Fish are the most abundant vertebrates, distributed nearly in all aquatic environments, which contain more than 30 000 species (Froese and Pauly, 2017). Fish have evolved all the strategies for sexual reproduction known in vertebrates, including parthenogenesis (Schartl et al., 1995), monoecism (Warner, 1984) and dioecism. The mechanisms of fish sex determination include two forms: genetic sex determination (GSD) and environmental sex determination (ESD) (Janzen, 1995). However, sex determination and differentiation remain poorly understood as they are extremely plastic and changeable, and even fish with GSD can be easily affected by environmental factors and exogenous hormones (Ospina-Álvarez and Piferrer, 2008). Currently, only a few master sex determination genes have been confirmed in several fish species: dmy in medaka (Oryzias latipes) (Matsuda et al., 2002; Nanda et al., 2002), gsdf in medaka (Oryzias luzonensis) (Myosho et al., 2012), irf9y in rainbow trout (Oncorhynchus mykiss) (Yano et al., 2012), amhr2 in fugu (Takifugu rubripes) (Kamiya et al., 2012), dmrt1 in half smooth tongue sole (Cynogossus semilaevis) (Chen et al., 2014) and amhy in Patagonian silverside (Odontesthes hatcheri) (Hattori et al., 2012), and Nile tilapia (Oreochromis niloticus) (Li et al., 2015).
With the fast development of next-generation sequencing technologies, transcriptome sequencing is increasingly used to acquire gene expression data and to understand the associated mechanisms of regulation. Gonad transcriptome analysis has been used to identify genes related to sex differentiation and determination in many fish such as Nile tilapia (Oreochromis niloticus) (Tao et al., 2013), Japanese flounder (Paralichthys olivaceus) (Fan et al., 2014), Russian sturgeon (Acipenser gueldenstaedtii) (Hagihara et al., 2014), spotted knifejaw (Oplegnathus punctatus) (Du et al., 2017), fugu (Takifugu tubripes) (Wang et al., 2017b) and channel catfish (Ictalurus punctatus) (Sun et al., 2013). These data provided a lot of gonadal transcriptomic information and identified numerous sex-related genes, facilitating further studies on fish sex determination and gonadal differentiation. Moreover, previous studies have also demonstrated that the brain controls reproduction through the hypothalamic-pituitary-gonadal (HPG) axis and it influences gonad development (Weltzien et al., 2004; Sreenivasan et al., 2008). Thus, the hypothalamus and the pituitary gland are considered the tissues of choice to study the regulation mechanisms of sex determination and gonadal differentiation.
Mandarin fish, also known as Chinese perch (Siniperca chuasti), is one of the most important commercial fish, and has been widely cultured in China. It is in great demand from Chinese market because of its high palatability and high content of essential amino acids and unsaturated fatty acids (Chu et al., 2010; Zhang et al., 2011). Mandarin fish is a demersal piscivore that feeds solely on live prey fish (Liang et al., 1998) and, although it grows fast, it is susceptible to diseases. For this reason, previous studies mainly focused on its growth (Wang et al., 2016; Tu et al., 2017), immunity (Wang et al., 2012, 2014, 2017a) and prey preference (He et al., 2013, 2018). In addition, female Mandarin fish grow faster than the males, therefore culturing all-female populations is a very effective approach to boost aquaculture production. However, only a few sexrelated genes have been cloned, such as the aromatase P450 gene (cyp19a) (Zou et al., 2017) and various growth hormone genes (Lu et al., 2008). In this study, the whole HPG axis transcriptome of Mandarin fish was sequenced using an Illumina HiSeq 2000 platform and numerous differentially expressed genes were identified in ovary and testis samples. This study was designed to provide a comprehensive understanding of the reproductive axis in Mandarin fish and to identify sex-related genes participating in sex determination and in gonadal differentiation. These data provide a valuable genomic resource and a large number of molecular markers to be used for further research on sex determination and gonadal differentiation in Mandarin fish.2 MATERIAL AND METHOD 2.1 Sampling
Samples of the hypothalamus, pituitary gland, testis, and ovary were collected from freshly caught Mandarin fish (weight>500 g), frozen in liquid nitrogen and stored at -80 ℃, to construct four independent libraries. The hypothalamus and pituitary gland samples of six Mandarin fish (3 males and 3 females) were mixed into a single sample. The testis and ovary samples from three different individuals were also mixed into one sample.2.2 RNA extraction and library construction
Total RNA was extracted from fresh frozen tissue using a TRIzol Reagent kit (Invitrogen, CA, USA) following the extraction protocol. Total RNA concentration, RIN value, 28S/18S and fragment size were measured using an Agilent 2100 Bioanalyzer (Agilent RNA 6000 Nano Kit), while purity was determined by ultraviolet spectrophotometer (NanoDropTM).
After total RNA was extracted, mRNA with polyA tail was enriched using magnetic beads with Oligo (dT), and an appropriate amount of interrupting reagent was added to the mRNA to fragment it at high temperature. One strand of cDNA was synthesized using the interrupted mRNA as template, and then double stranded cDNA was synthesized. After kit purification, recycling, and adhesive end repairing, base "A" was added to the 3ʹ end of cDNA and the joint was connected. Following fragment size selection, PCR amplification was performed. The constructed library was tested by an Agilent 2100 Bioanalyzer and ABI Step One Plus Real-Time PCR System, and by real-time PCR. Finally, the cDNA library was sequenced using Illumina HiSeq™. All procedures were performed in Beijing Genomics Institute, Shenzhen, China.2.3 De-novo assembly and functional annotation
To ensure the reliability of the results, reads with joint contamination, unknown base N content greater than 5%, and low quality were removed before data analysis, and the filtered reads were named "clean reads" and stored in FASTQ format. Then, a de novo transcriptome assembly was carried out using Trinity software.
Functional annotation was performed through homology searches against the main public databases. All unigenes were compared with the sequences in the NCBI nucleotide (NT, ftp://ftp.ncbi.nlm.nih.gov/blast/db) database using BLASTn, the NCBI nonredundant (NR, ftp://ftp.ncbi.nlm.nih.gov/blast/db) protein database, the Clusters of eukaryotic Orthologous Groups (KOG, http://www.ncbi.nlm.nih.gov/KOG) database, the Kyoto Encyclopedia of Genes and Genomes (KEGG, http://www.genome.jp/kegg) and the SwissProt (http://ftp.ebi.ac.uk/pub/databases/swissprot) protein database using BLASTx or Diamond.
Blast2GO was used to identify the NR annotation results, and the number of unigenes related to each gene ontology (GO, http://geneontology.org) was calculated based on biological processes, cell composition and molecular function. InterProScan5 was used to annotate the InterPro (http://www.ebi.ac.uk/interpro) database.2.4 Identification of differentially expressed genes (DEGs)
Based on the assembly results, clean reads of each sample were compared with each other using Bowtie2 software, and the gene expression level of each sample was calculated using RSEM. The differentially expressed genes (DEGs) between samples were detected using the PossionDis algorithm, which was defined as false discovery rate (FDR)≤0.001, and the gene with expression difference of more than 2 times. DEGs were classified and enriched for the GO function and KEGG pathway classification.2.5 Data validation by qRT-PCR
The reliability of transcriptome data was verified by qRT-PCR using LightCycler 480 SYBR GreenⅠMaster (Roche) and Light cycler 480 RealTime PCR system (Roche). One denaturation cycle was performed at 95 ℃ for 10 min, and 40 amplification cycles were performed at 95 ℃ for 10 s, 60 ℃ for 20 s and 72 ℃ for 20 s. The relative expressions of 15 DEGs in testis and ovaries, including dmrt1, spata4, fshr, nanos2, sox9, cyp19a1a, foxl2, hsd17b1, hsd17b10, hsd17b12a, hsd17b8, spata2, spata5, star3, and star5, were conformed using beta-actin as the reference gene. All samples were triplicated and double-tailed student t-test was performed in confidence value of 95% (P≤0.05) to determine the significance of gene expression. All primers were designed using Primer Premier 6 (Table 1).2.6 Identification of SSRs and SNPs
Simple sequence repeat (SSR) sequences were identified using MISAv1.0 (http://pgrc.ipkgatersleben.de/misa) in order to search for single nucleotide to hexanucleotide repeats. Parameters were set as follows: single base repeat at least 12 times, double base repeat 6 times, triple base repeat 5 times, quadruple base repeat 4 times, penta base repeat 3 times, hexa base repeat 2 times. When the distance between two microsatellites is less than 100 bp, a composite microsatellite is formed. HISAT (http://ccb.jhu.edu/software/hisat/index.shtml) was used to compare clean reads to unigenes in order to obtain single nucleotide polymorphism (SNP) sequences, and GATK (https://www.broadinstitute.org/gatk) was used to filter out low-quality SNPs with FS filter set at >30.0 and QD filter set at < 2.0.3 RESULT 3.1 Overview of transcriptome assembly quality
A total database of 27.56 Gb was analyzed using an Illumina HiSeq platform. Following the removal of assembly and redundancy, 134 124 unigenes were obtained, with total length, average length and GC content of 182 89 703 bp, 1 361 bp and 45.75%, respectively. The values of N50, N70, and N90 were 3 312 bp, 1 888 bp, and 451 bp respectively (Table 2). All unigenes were more than 300 bp in length, and 19 453 of them (14.50%) were more than 3 000 bp in length (Fig. 1).3.2 Gene function annotation
Through sequence matching against seven public databases, an annotation analysis was conducted on the remaining 134 124 non-redundant unigenes, and 82 290 sequences were annotated. The percentage of annotated unigenes in the NT database (76 329; 56.91%) was the highest, followed by the NR database (59 688; 44.50%), while the sequences annotated in the GO database were the lowest (5 241; 3.91%) (Table 3). A total of 37 028 genes were annotated in five public databases: NR, KOG, KEGG, SwissProt, and Inter Pro (Fig. 2a). The percentage of sequences consistent with all unigene BLASTx hits against other populations in the NR database showed that Larimichthys crocea (42.93%) has the largest amount of homologous sequences to Siniperca chuatsi, followed by Stegastes partitus (18.36%), Oreochromis niloticus (4.82%), and Notothenia coriiceps (3.72%) (Fig. 2b).
To predict the functional classification of genes, possible pathways, and gene interactions, all unigenes were annotated in GO, KOG, and KEGG databases.
A total of 18 247 unigenes were assigned to three GO categories: biological processes, cellular component and molecular function. In the category of biological processes, cellular (2 764) and metabolic processes (2 234) were the most represented items. In the cellular component category, the most represented terms were cell (2 100) and cell part (2 080). In addition, in the molecular function category, the most abundant terms related to binding (2 543) and catalytic activity (1 866) (Fig. 3a).
A total of 45 741 (34.10%) KOG-annotated genes were classified into 25 molecular families, with the most abundant distribution observed in "signal transduction mechanisms" (12 936 unigenes), followed by "general function prediction only" (11 266 unigenes); the smallest distribution was "coenzyme transport and metabolism", with only 296 unigenes (Fig. 3b).
A total of 45 979 (27.92%) unigenes annotated in KEGG were divided into six different functional groups, among which the three exhibiting the largest distribution were "signal transduction" (9 131 unigenes), "global and overview maps" (4 820 unigenes) and "cancer: overview" (4 638 unigenes) (Fig. 3c).3.3 Simple sequence repeats (SSRs) and single nucleotide polymorphisms (SNPs)
A total of 49 495 SSR markers were identified from 31 707 sequences. Most were mono-nucleotide (12 482 unigenes), di-nucleotide (23 987 unigenes) and tri-nucleotide repeats (11 085 unigenes). The AC/ GT motif was most abundant in dinucleotide sequences (17 494 unigenes), followed by the AG/CT motif (4 359 unigenes). The AGG/CCT motif was most abundant in trinucleotide sequences (3 317 unigenes), followed by the AGC/CTG motif (1 893 unigenes). In addition, there were 1 292 quadnucleotide, 372 penta-nucleotide, and 277 hexanucleotide sequences (Fig. 4a).
In addition, a total of 85 899 SNPs were detected in transcriptome data. 24 426, 21 923, 14 414, and 25 136 SNP sites were detected from the four transcriptional libraries derived from hypothalamus, pituitary gland, ovary, and testis samples, respectively. The frequency of A-G transition was higher than C-T, and the frequency of A-T transversion was higher than other transversion frequencies (Fig. 4b; Table 4).3.4 Differentially expressed genes in ovary and testis samples
A total of 25 324 DEGs were identified between the ovary and testis samples. Among them, 15 289 (60.37%) were significantly higher in the ovaries (ovary-biased genes) and 10 035 (39.62%) in the testes (testis-biased genes). The remaining 47 035 genes were expressed in both testes and ovaries. Enrichment analysis of the GO signaling pathway showed that the annotation of DEGs to biological processes was predominant, followed by cellular components and molecular functions (Supplementary Fig.S1). In biological processes, 13, 13, and 3 unigenes were annotated to reproduction, reproduction processes, and hormone secretion, respectively (Supplementary Table S1). Enrichment analysis of the KEGG signaling pathway showed that DEGs were mainly distributed in metabolic pathways, endocytosis, and other processes (Supplementary Fig. S2). In addition, different numbers of DEGs were annotated to the following pathways: 150 to GnRH, 259 to Wnt, and 130 to TGF-beta. Among the pathways related to steroids, estrogen signaling, ovarian teroidogenesis, steroid hormone biosynthesis, and steroid biosynthesis were 189, 77, 50, and 27, respectively (Supplementary Table S2).
Based on the comparison of the functional annotation of Mandarin fish transcriptome sequences from various databases with the published data of other species, numerous genes related to the HPG axis and sex differentiation were identified (Supplementary Table S3), such as kisspeptins (kiss), gonadotropin-releasing hormone (gnrh), gonadotropins (gths), forkhead transcription factor2 (foxl2), cytochrome P450 (cyp19a), doublesex and mab-3 related transcription factor 1 (dmrt1), SRY-box transcription factor 9 (sox9), anti-Mullerian hormone (amh).3.5 Validation of transcriptomic data
Fifteen randomly selected DEGs between ovary and testis, including dmrt1, spata4, fshr, nanos2, sox9, cyp19a1a, foxl2, hsd17b1, hsd17b10, hsd17b12a, hsd17b8, spata2, spata5, star3, and star5, were used to verify the validation of transcriptomic data by qRT-PCR, and the results were consistent with the transcriptome data (Fig. 5).4 DISCUSSION
Mandarin fish, commonly known as Chinese perch, displays obvious sexual growth dimorphism that females grow faster than males from juvenile to commercial size. This means that all-female Mandarin fish cultures have greater economic benefits and broader prospects (Wang et al., 2006). However, due to the lack of genomic and transcriptome data and studies on sex-related genes, the molecular mechanisms of sex differentiation in this species are poorly understood. In this study, 134 124 unigenes were obtained by Illumina high-throughput transcriptome sequencing with N50, N70 and N90 values of 3 312, 1 888, and 451, respectively, indicating that the transcriptome data were of high quality and reliable. A large number of sex-related genes in the HPG axis of Mandarin fish were found, providing potential candidate genes for future research on reproduction, development and sexual differentiation.
The HPG axis is the regulatory center of reproductive processes, and its signaling pathway is initiated by gonadotropin-releasing hormone (gnrh). This hormone displays dose-dependent induction by kisspeptin (kiss) (Novaira et al., 2009; Li et al., 2019b), and once it binds to the gnrh receptor (gnrhr), it stimulates the pituitary gland to secrete gonadotropins, including the follicle-stimulating hormone (fsh) and the luteinizing hormone (lh), to promote the production of sex hormones and the development of gametes. Due to the occurrence of fish-specific genome duplication (FSGD) events (Robinson-Rechavi and Laudet, 2001; RobinsonRechavi et al., 2001), most fish species have two kinds of kisspeptin, and this is also true for Mandarin fish (Selvaraj et al., 2010). On the contrary, only kiss2 was found in Solea senegalensis (Mechaly et al., 2011), Tetraodon nigroviridis and Gasterosteus aculeatus, suggesting that kiss2 may play a more important role. Previous studies confirmed that there are at least two or more gnrhs in teleost fish and that gnrh3 is unique to fish (Hildahl et al., 2011). As for gnrh receptors, the existence of two or more gnrhrs has been confirmed in teleost fish, and up to five gnrhrs have been identified in Fugu rubripes (Moncaut et al., 2005) and in Tetraodon nigroviridis (Ikemoto and Park, 2005). In the transcriptome of Mandarin fish, three types of gnrh and three types of gnrhr were found. The selectivity of GnRH receptors to the GnRH ligand varies in different species (Lethimonier et al., 2004), and the specific binding of the GnRH ligand to the receptor in Mandarin fish remains to be explored.
Gths can increase intracellular cAMP and promote the activation of the PKA subunit that regulates the expression of steroid hormone genes (Selstam et al., 1976; Reinhart et al., 1999; Stocco et al., 2005). It was suggested that fsh may play an important role in the cyp19a1a control during ovarian differentiation through the synthesis of cAMP second messenger (Yamaguchi et al., 2007; Guiguen et al., 2010). The major sex hormones active in teleost fish are testosterone (T), 11-ketotestosterone (11-kt) and 17-estradiol (E2). Their synthesis starts with cholesterol. Cholesterol is transferred from the outer mitochondrial membrane to the inner membrane under the action of a steroidogenic acute regulatory protein (star) (Miller, 2007), and then is converted to androstenedione under the catalysis of P450scc, CYP17A1 and 3β-HSD. Subsequently, 17β-HSD3 catalyzes the conversion of androstenedione to T, which is further catalyzed to 11-Ketotestosterone by CYP11 and 11β-HSD2, and CYP19 catalyzes the formation of E2 from androstenedione T (Simpson et al., 1994). Our data showed that in the process of androgen synthesis, most of the genes coding for speed-limit enzymes were highly expressed in males, including cyp11a1, cyp17a1, cyp11b, and hsd11β2. In contrast, during estrogen synthesis, CYP19 aromatase is the key rate-limiting enzyme, encoded by cyp19a1 (including cyp19a1a and cyp19a1b). In particular, cyp19a1a encodes gonadal aromatase, which is highly expressed in females and is mainly involved in estrogen production in Mandarin fish. The function of sex hormones is accomplished by binding to receptors. Several isoforms of er and ar have been found in Mandarin fish, including era, erb1, erb2, ara, and arb, which are similar to isoforms observed in other fish (Ogino et al., 2018).
Since the activity of CYP19 aromatase directly determines the male to female hormone ratio, cyp19a1a is considered the central factor for sex differentiation in fish (Li et al., 2019a). Most of the sex-related genes found in fish are related to cyp19a1a. Foxl2 is the earliest marker of ovarian determination and differentiation in vertebrates, and is highly expressed in female Mandarin fish. The transient transfection of fish showed that foxl2 not only directly activated the transcription of cyp19a1a and mediated the synthesis of estrogen (Yamaguchi et al., 2007), but it also bound to steroidogenic factor-1 (sf1/nr5a1) to enhance the transcriptional activity of cyp19a1a (Wang et al., 2007). However, no sexual specific expression of nr5a1a or nr5a1b was observed in Mandarin fish, while nr5a2 was highly expressed in the testis. Evidence proves the antagonistic effect of nr5a1 and nr5a2 (Shi et al., 2019), but the role of nr5a2 in this process remains to be explored.
Dmrt1 is a testis-biased gene that is highly conserved across animal phyla and was also highly expressed in male Mandarin fish. Previous studies have demonstrated that dmrt1 not only indirectly down-regulates the expression of cyp19a1a by inhibiting foxl2 (Li et al., 2013; Lindeman et al., 2015), but it also directly binds to the cyp19a1a promoter to down-regulate aromatase activity (Wang et al., 2010). Dmrt1 can promote the expression activity of sox9 (Wei et al., 2019), which has also an antagonistic effect on foxl2 (Nef and Vassalli, 2009; Georges et al., 2014). In Mandarin fish transcriptome data, only one sox9 gene with high testicular expression was found, unlike most teleost fish that have both sox9a and sox9b. In addition, amh, an important downstream gene related to sox9 and dmrt1 (Rodríguez-Marí et al., 2005; Webster et al., 2017), was shown to directly inhibit the expression of cyp19a1a (Rouiller-Fabre et al., 1998), which is also highly expressed in the testes of Mandarin fish.
The combination of existing studies and transcriptome data from sex related genes with differential expression, suggests that Mandarin fish might share a similar regulation pattern for sex-related genes with other teleosts (Fig. 6).5 CONCLUSION
In this study, high-throughput RNA sequencing was used to provide transcriptome data of Siniperca chuatsi. The molecular mechanisms of gene function, reproductive regulation and gametogenesis in the HPG axis of Mandarin fish were predicted. A large number of candidate genes for gender preference have been identified, and these should be further studied to better understand their exact functions in these processes. In addition, a large number of SSR and SNP markers were found, providing basic information for the marker-assisted breeding of Siniperca chuatsi.6 DATA AVAILABILITY STATEMENT
All raw reads of transcriptome sequencing data have been deposited at the NCBI Short Read Archive (SRA) database (SRA accession Nos.: SRR11743000, SRR11743001, SRR11743002, SRR11743003).Electronic supplementary material
Supplementary material (Supplementary Tables S1–S3 & Figs.S1–S2) is available in the online version of this article at https://doi.org/10.1007/s00343-020-0251-y.
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