Next Article in Journal
Influence of Different Lactation Stages on Circadian Rhythmicity of Metabolic Biomarkers in Dairy Cows: A Pilot Study
Next Article in Special Issue
A First Insight into the Gonad Transcriptome of Hong Kong Catfish (Clarias fuscus)
Previous Article in Journal
Alternatives to Piglet Castration: From Issues to Solutions
Previous Article in Special Issue
Genotyping Strategies Using ddRAD Sequencing in Farmed Arctic Charr (Salvelinus alpinus)
 
 
Search for Articles:
Title / Keyword
Author / Affiliation / Email
Journal
Article Type
 
 
Advanced Search
 
Section
Special Issue
Volume
Issue
Number
Page
 
Journals
Animals
Volume 11
Issue 4
10.3390/ani11041042
animals-logo
Submit to this Journal Review for this Journal Propose a Special Issue
► Article Menu

Article Menu

share Share announcement Help format_quote Cite question_answer Discuss in SciProfiles thumb_up
...
Endorse textsms
...
Comment
first_page
settings
Order Article Reprints
Font Type:
Arial Georgia Verdana
Font Size:
Aa Aa Aa
Line Spacing:
Column Width:
Background:
Article

Comparison of Gonadal Transcriptomes Uncovers Reproduction-Related Genes with Sexually Dimorphic Expression Patterns in Diodon hystrix

by
Huapu Chen
1,
Zhiyuan Li
1,
Yaorong Wang
1,
Hai Huang
2,
Xuewei Yang
3,
Shuangfei Li
3,
Wei Yang
4,* and
Guangli Li
1,*
1
Guangdong Research Center on Reproductive Control and Breeding Technology of Indigenous Valuable Fish Species, Key Laboratory of Marine Ecology and Aquaculture Environment of Zhanjiang, Fisheries College, Guangdong Ocean University, Zhanjiang 524088, China
2
Key Laboratory of Utilization and Conservation for Tropical Marine Bioresources, Hainan Tropical Ocean University, Ministry of Education, Sanya 572022, China
3
Shenzhen Key Laboratory of Marine Bioresource and Eco-Environmental Science, College of Life Sciences and Oceanography, Shenzhen University, Shenzhen 518060, China
4
Food and Environmental Engineering Department, Yangjiang Polytechnic, Yangjiang 529566, China
*
Authors to whom correspondence should be addressed.
Animals 2021, 11(4), 1042; https://doi.org/10.3390/ani11041042
Submission received: 7 March 2021 / Revised: 4 April 2021 / Accepted: 4 April 2021 / Published: 7 April 2021
(This article belongs to the Special Issue Aquaculture Genetics and Genomics)
Browse Figures
Review Reports Versions Notes

Abstract

:

Simple Summary

Spot-fin porcupine fish (Diodon hystrix) has been recognized as a new and emerging aquaculture species with promising economic value in south China. However, due to the lack of understanding of reproductive regulation, the management of breeding and reproduction under captivity remains a major technological barrier for the development of large-scale aquaculture of D. hystrix. In this study, the first gonad transcriptomes of D. hystrix were analyzed using Illumina HiSeq sequencing. Comparison of ovary and testis transcriptomes identified a set of differentially expressed genes (DEGs) supposed to be associated with gonadal development and gametogenesis. The conserved expression profiles of the well-known reproduction-related genes implies their similar roles in gonad differentiation and development in D. hystrix. The detailed transcriptome data can improve our understanding of the regulatory functions of sex-related genes in D. hystrix.

Abstract

Diodon hystrix is a new and emerging aquaculture species in south China. However, due to the lack of understanding of reproductive regulation, the management of breeding and reproduction under captivity remains a barrier for the commercial aquaculture of D. hystrix. More genetic information is needed to identify genes critical for gonadal development. Here, the first gonadal transcriptomes of D. hystrix were analyzed and 151.89 million clean reads were generated. All reads were assembled into 57,077 unigenes, and 24,574 could be annotated. By comparing the gonad transcriptomes, 11,487 differentially expressed genes were obtained, of which 4599 were upregulated and 6888 were downregulated in the ovaries. Using enrichment analyses, many functional pathways were found to be associated with reproduction regulation. A set of sex-biased genes putatively involved in gonad development and gametogenesis were identified and their sexually dimorphic expression patterns were characterized. The detailed transcriptomic data provide a useful resource for further research on D. hystrix reproductive manipulation.

1. Introduction

Spot-fin porcupine fish (Diodon hystrix) belonging to the family Diodontidae (Teleostei, Tetraodontiformes) is mainly distributed in the lagoons and coral reefs of tropical seawater [1,2]. Although D. hystrix is widely considered to have tetrodotoxin [3], this fish species is noted for its delicious meat and nutritious skin that is rich in collagen, especially in the Pacific Islands region and southern Hainan Island, China [4,5]. According to the assessment of nutrient value, D. hystrix has a high content of amino acids and the rate of essential amino acids is accorded with the FAO/WHO standard. Moreover, the fat contents in skin and muscle tissues are found to be negligible, contributing to a low energy value [6]. D. hystrix with such distinguished features is increasingly becoming a highly popular food fish with considerable economic value, the commercial demand is rising rapidly in south China and the market price continues to climb [5,6].
The outstanding growth performance and ease of cultivation make D. hystrix a very promising species for commercial aquaculture [6]. As a gonochoristic fish species, however, porcupine fish gonad maturates earlier in males than females under either natural or aquaculture conditions. It is, therefore, difficult, to obtain fertilized eggs using conventional techniques of artificial propagation. With the bloom of porcupine fish industry, artificial breeding unfortunately remains a significant barrier for the successful aquaculture of D. hystrix. The commodity fish still has to be captured from the sea. Due to a weak resilience of the natural population of D. hystrix, its wild stock is now gravely threatened and the fish resource has been declining greatly in recent years as a result of critical hazards, such as a sharp increase of fish catch and continued destruction of natural habitat [7]. Thus, the large-scale aquaculture of D. hystrix is absolutely in great and urgent need of artificial breeding and reproduction management.
The reproduction process, typically including gonad differentiation, development, maturation and gametogenesis, is more complex in fish than in other vertebrate species and is under the influence of genetic and environmental factors [8,9]. Years of practical experience have shown that a thorough knowledge of the mechanisms involved in reproductive regulation could strongly promote the development of efficient management of reproduction, which is a necessary prerequisite for the breeding of aquaculture species. To date, however, the only studies in D. hystrix focus mainly on systematics and zoogeography [2,4], characterization of mitochondrial genome and phylogenetic analysis [5], molecular marker development [7], parasitic diseases [10,11], tetrodotoxic poisoning mechanism [3], as well as the analysis of nutrient compositions that can provide reference data for the formulation design of special diets [6]. The research efforts on reproductive biology are severely limited and the underlying mechanism of reproduction remains poorly understood. These research gaps seriously impede the progress of artificial breeding techniques for D. hystrix. To provide useful guidance to the future practice of reproductive manipulation, it is essential to pay serious attention to delineating the regulatory mechanisms of the reproductive process. In particular, the efforts to functionally explore the key genes associated with sex differentiation, gonad development and maturation, and gametogenesis are indispensable.
Compared with other commercial aquaculture species, the genetic information available for D. hystrix is rather limited. Thus far, reproduction-related genes have been rarely reported in D. hystrix and a fundamental understanding of their expression profiles is still lacking. Before elucidating the specific roles of these genes, there is an immediate need to enrich the genomic background knowledge. With improved efficiency, next-generation sequencing (NGS) based transcriptome sequencing is an efficient gene expression profiling technology that is superior in generating a great amount of transcript sequences and mRNA expression data rapidly and cost-effectively, especially for non-model species [12,13]. It has been frequently employed in functional gene identification and gene-expression regulation analysis in aquaculture fish species [14,15,16] to provide a general representation of the genes that are expressed in specific tissues [17]. The expression patterns of reproduction-related genes generally exhibit significant differences between the sexes both in the developing and developed gonads. By comparative analysis of gonad transcriptomes, many candidate genes and pathways involved in sexual or reproductive regulation and gonad maturation have been identified in silver sillago (Sillago sihama) [18], spotted knifejaw (Oplegnathus punctatus) [19], olive flounder (Paralichthys olivaceus) [20], yellow catfish (Pelteobagrus fulvidraco) [21], Amur catfish (Silurus asotus) [22], and Amur sturgeon (Acipenser schrenckii) [23]. These studies provide helpful insights into reproduction-related genes and enable the discovery of new gene candidates.
In this study, Illumina-based gonadal RNA sequencing, Trinity de novo assembly and annotation were firstly conducted in D. hystrix. Furthermore, comparative transcriptomics was applied to reveal the expression patterns of sex-biased genes and the differences in the expression of the genes which potentially are involved in the regulation of reproduction were analyzed and discussed. This study mainly aimed i) to further enrich the available genetic and genomic data for a deeper understanding of gene expression and functional gene mining, and ii) to identify as many genes putatively related to gonad differentiation, development, maturation, and gametogenesis as possible for future research into the molecular mechanisms of reproduction in D. hystrix.

2. Materials and Methods

2.1. Ethics Statement

Animal experiments were carried out in strict accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals. The protocol was approved by the Animal Research and Ethics Committee of Guangdong Ocean University (NIH Pub. No. 85–23, revised 1996). All efforts were made to minimize animal suffering and to reduce the number of animals used.

2.2. Sample Collection and Preparation

For transcriptome sequencing, six adult D. hystrix (three males and three females) were obtained from the South China Sea (18°11′2.73′′ N, 109°18′32.66′′ E) on 12 May 2020 (see Table S1 for the specific information of the fish samples). Live fish were sacrificed by decapitation following anesthetization with a 300 mg/L tricaine methanesulfonate (MS222, Sigma, Saint Louis, MO, USA) immersion bath. After dissection, the determination of fish gender was performed by morphological observation of gonads. The gonad tissues were excised from D. hystrix individuals within 1 min from sacrifice, immediately quick-frozen in liquid nitrogen, and then stored at −80 °C until RNA extraction.

2.3. Illumina RNA Sequencing

Six gonad samples (three replicates each sex) were used for the preparation of transcriptome sequencing libraries. The RNA-Seq process was performed as described previously [24]. In brief, total RNA was isolated from female and male D. hystrix gonad tissues using a Trizol reagent kit (Life Technologies, Carlsbad, CA, USA). The isolated RNA was quantified by a Nanodrop 2000c spectrophotometer (Thermo Scientific, Wilmington, DE, USA), and its integrity was confirmed by agarose gel electrophoresis and Agilent 2100 BioAnalyzer System (Agilent Technologies, Santa Clara, CA, USA). After purifying mRNA with an Oligo-dT Beads Kit (Qiagen, Hilden, Germany), cDNA libraries were constructed using a TruSeq® Stranded mRNA Sample Preparation kit following the manufacturer’s protocol. RNA sequencing of the libraries was performed using the Illumina HiSeq™ 2000 platform (Illumina, Inc., San Diego, CA, USA) that generates paired-end (PE) reads of 125 bp length.

2.4. De Novo Assembly

By means of SOAPnuke (version 1.5.0) [25], the raw reads were pruned using the software’s quality control with the parameters “-l 10 -q 0.5 -n 0.05 -p 1 -i”. In this step, clean data were generated by removing adapter sequences, reads containing ploy-N sequences and low-quality reads from the raw data. Then, the clean data were de novo assembled by Trinity RNA-Seq Assembler (version r20140717, http://trinityrnaseq.sourceforge.net (accessed on 15 June 2015)) with default parameters [26]. The shorter redundant final linear transcripts were eliminated using CD-HIT-EST when the sequences were totally covered by other transcripts with 100% identity, and the longest ones were defined as unigenes [24].

2.5. Annotation and Classification

Annotation was conducted by aligning sequence data against public databases using BLAST 2.2.26+ software (https://blast.ncbi.nlm.nih.gov/Blast.cgi (accessed on 20 April 2016)) with an E-value threshold of 1e-5. The unigenes were subjected to the sequence homology searches against the National Center for Biotechnology Information (NCBI) non-redundant (Nr), Protein family (Pfam), Clusters of Orthologous Groups of proteins (KOG/COG/eggNOG), Swiss-Prot, Kyoto Encyclopedia of Genes and Genomes (KEGG) databases. Further analysis was performed to obtain the Gene Ontology (GO) functions using the Blast2GO package [27]. The classification of GO terms was visualized using WEGO statistical software [28]. Additionally, KOBAS v2.0 (http://kobas.cbi.pku.edu.cn/home.do (accessed on 24 July 2015)) was employed to analyze the KEGG pathway annotation data and to obtain the pathway categories [29].

2.6. Differential Expression Analysis and Functional Enrichment

By means of the expected number of fragments per kb per million reads (FPKM) method, gene expression levels were calculated using RSEM software (version 1.2.15) [30]. The DESeq2 package was used to identify differentially expressed genes (DEGs) between ovaries and testes [31]. FDR value ≤ 0.01 and |log2 (Fold Change)| ≥ 1 were used as the threshold for significantly differential expression. Additionally, GO and KEGG functional enrichment analyses were performed to determine the DEGs that were significantly enriched in GO terms and KEGG pathways at Bonferroni-corrected p-value ≤ 0.05 compared with the whole-transcriptome background. GO enrichment analysis of DEGs was implemented by the topGO package’s (version 2.28.0) Kolmogorov–Smirnov test [32]. Finally, KOBAS v2.0 was used to test the statistical enrichment of DEGs in KEGG pathways [33].

2.7. Validation of DEGs by Real-Time Quantitative PCR (RT-qPCR)

A total of 23 DEGs putatively associated with reproduction were chosen to validate the results of RNA-seq by RT-qPCR analysis. The primer pairs are listed in Supplementary Table S2. Total RNA was isolated from gonad samples using TRIzol according to the manufacturer’s instructions (Invitrogen, Carlsbad, CA, USA). The RNA was then subjected to reverse transcription using a RevertAid first-strand cDNA synthesis kit (Fermentas, Vilnius, Lithuania). RT-qPCR was performed on an ABI 7500 qPCR system (Life Technologies Inc., Carlsbad, CA, USA) using SYBR Green Real Time PCR Master Mix (TaKaRa Biotechnology, Dalian, China). The reference gene β-actin was used as an internal control to determine the relative expression. Three independent biological replicates and two technique repeats were performed for each gene. The relative gene expression levels were calculated using 2−ΔΔCt method. Analysis of Variance (ANOVA) was performed by SPSS 17.0 (SPSS Inc., Chicago, IL, USA). Values with p < 0.05 were considered significant.

3. Results

3.1. Overview of Sequencing and Assembly Results

RNA-Seq of the six libraries produced a total of 156.58 million raw reads (46.85 Gb sequencing data) with a mean of 26.10 million, ranging from 21.49 to 40.74 million per sample (Table 1). Approximately 151.89 (97.00%) million clean reads with a mean Q30 of 94.32% were filtered from the raw data (Table 1). The total size of the clean data generated from each library reached more than 6.0 Gb. The statistics of sequencing saturation distribution and gene coverage showed that the sequencing coverage was sufficient to quantitatively analyze the gene expression profiles (Figure S1). All raw sequencing data were submitted to the Sequence Read Archive (SRA, http://www.ncbi.nlm.nih.gov/sra/ (accessed on 25 October 2020)) of the NCBI database under BioProject accession number PRJNA674446.
All clean data were then imported to the Trinity package for de novo assembly using the default parameters. The high-quality clean reads were assembled into 139,628 transcripts with a N50 length of 3118 bp (Table 2). Further redundancy elimination resulted in a total of 57,077 unigenes with an average length of 1300 bp. In terms of sequence length distribution, 33,675 (59.00%) unigenes were > 500 bp in length and 19,620 (34.37%) unigenes were >1000 bp in length (Table 2). These results demonstrated the high quality of assembly.

3.2. Unigenes Annotation

Functional annotation was carried out by aligning the 57,077 unigenes to the protein sequences of public databases. A total of 24,574 (43.05%) unigenes were successfully annotated in at least one of the queried databases (Table 3). Of which, 23,114 (40.50%) and 13,488 (23.63%) unigenes had homologous sequences in the Nr and Swiss-prot protein databases, respectively. Meanwhile, 12,434 (21.78%), 15,157 (26.56%), and 13,699 (24.00%) unigenes could be annotated and classified in the GO, KOG, and KEGG databases, respectively (Table 3). Specifically, 12,434 unigenes annotated in the GO database were divided into three main subcategories and assigned into 56 2nd GO terms (Figure S2), and 15,157 unigenes were successfully clustered into 25 KOG subcategories (Figure S3). Furthermore, 13,699 (24.28%) unigenes were grouped into six main KEGG categories that include 287 secondary pathways (Figure S4).

3.3. Differential Expression Analysis

The levels of gene expression were normalized using the FPKM values. The distribution of gene expression levels was shown as a series of box plots (Figure 1A). About half of the genes were found to be expressed at extremely low levels (0.1 ≤ FPKMs ≤ 1) or not expressed at all (0 ≤ FPKMs < 0.1) in the gonads, and only a small proportion was considered to be highly expressed (FPKMs ≥ 60). To ensure if all gonad samples were reliable, the gene expression patterns of testes and ovaries were visualized via a heatmap using the Pearson correlation coefficient as distance measure (Figure 1B). The correlation analysis clearly indicated that the samples were classified into two major groups, representing testis and ovary, respectively. In addition, the result of principal component analysis (PCA) showed that three testis samples (blue circles) formed a cluster and three ovary samples (red circles) formed another distinct cluster (Figure S5).
By comparison of the unigene expression levels in gonadal transcriptomes, a total of 11,487 unigenes were detected to be differentially expressed between the ovaries and testes (FDR ≤ 0.01, |Log2 (fold change)| ≥ 1) (Table S3). Among these DEGs, 6888 testis-biased (downregulated) and 4599 ovary-biased (upregulated) transcripts with significant differences in expression levels between the sexes were obtained (Figure 2A). The further analysis indicated that 46 and 1163 DEGs were specifically expressed (0 ≤ FPKMs ≤ 0.1) in the ovaries and testes, respectively. The remaining 10,278 DEGs were expressed in both gonads (Figure 2B).

3.4. Enrichment Analysis of DEGs

GO functional analysis was performed and the obtained DEGs were finally assigned to 52 2nd level GO terms. Of these functional terms, ‘cellular process’, ‘binding’, ‘single-organism process’, and ‘metabolic process’ annotated the most DEGs (Figure 3). More important, many GO terms associated with reproduction were identified, such as ‘reproductive process’ (GO:0022414), ‘gamete generation’ (GO:0007276), ‘steroid hormone mediated signaling pathway’ (GO:0043401), and ‘oocyte differentiation’ (GO:0009994) (Table S4). GO enrichment analysis showed that 224, 62, and 107 GO terms were significantly enriched in categories ‘biological process’, ‘cellular component’, and ‘molecular function’, respectively (p < 0.05) (Table S4). The top three most significant GO terms involved in ‘biological processes’ were ‘DNA repair’ (GO:0006281), ‘tRNA processing’ (GO:0008033), and ‘lymph vessel development’ (GO:0001945). The top three most significant terms involved in ‘molecular functions’ included ‘RNA methyltransferase activity’ (GO:0008173), ‘DNA binding’ (GO:0003677), and ‘chemorepellent activity’ (GO:0045499). The top three ‘cellular components’ GO terms were ‘catalytic complex’ (GO:1902494), ‘ribosome’ (GO:0005840), and ‘cytoplasm’ (GO:0005737).
KEGG enrichment analysis was also carried out with the DEGs to uncover their functional characteristics. In total, 5598 DEGs were mapped to 198 KEGG pathways, of which ‘endocytosis’ (ko04144), ‘MAPK signaling pathway’ (ko04010), ‘focal adhesion’ (ko04510), and ‘regulation of actin cytoskeleton’ (ko04810) annotated the most genes (Table S5). Meanwhile, 20 KEGG pathways were significantly enriched (q-value < 0.05). The DEGs upregulated in ovaries and testes were involved in 17 and 12 significant pathways, respectively (Figure 4). Moreover, the enrichment analysis showed that clearly different pathways were enriched between the ovary- and testis-biased DEGs. The ovary-biased DEGs were significantly enriched in pathways such as ‘ribosome biogenesis in eukaryotes’, ‘DNA replication’, ‘pyrimidine metabolism’, ‘cell cycle’, and ‘spliceosome’ (Figure 4A), suggesting the importance of nucleic acid synthesis, protein homeostasis, and cell proliferation and differentiation for ovarian function. Whereas the testis-biased DEGs were highly enriched in pathways such as ‘focal adhesion’, ‘cytokine-cytokine receptor interaction’, ‘cell adhesion molecules (CAMs)’, ‘ECM (extracellular matrix)-receptor interaction’, and ‘MAPK signaling pathway’ (Figure 4B). Such noticeable differences imply the variation in the gene regulatory landscape between the D. hystrix ovary and testis.

3.5. Sex-Biased Genes of Interest Related to Reproduction Regulation

A number of GO terms and KEGG pathways known to be associated with gonadal development and maintenance, gametogenesis, oocyte maturation, and sperm motility were found in the present study, such as ‘reproductive process’ (GO:0022414), ‘gonad development’ (GO:0008406), ‘sexual reproduction’ (GO:0019953), ‘germ cell development’ (GO:0007281), ‘gamete generation’ (GO:0007276), ‘spermatogenesis’ (GO:0007283), ‘ovarian steroidogenesis’ (ko04913), ‘steroid hormone biosynthesis’ (ko00140), ‘Wnt signaling pathway’ (ko04310), and ‘MAPK signaling pathway’ (ko04010) (Table S6). There were 221 and 618 genes grouped into reproduction-related GO terms and KEGG pathways, respectively (Table S6). Based on the resources of annotation and enrichment analyses, 67 DEGs were obtained and these genes were determined to be involved in gonadal differentiation and development, gamete generation and maturation in vertebrates were identified, including gonadal soma-derived factor 1 (gsdf1), SRY-box transcription factor 9 (sox9), anti-Müllerian hormone (amh), doublesex- and mab-3-related transcription factor 1 (dmrt1), Cytochrome P450 aromatase (cyp19a1a), Kelch-like protein 10 (klhl10), zona pellucida sperm-binding protein 1 (zp1), and so on (Table 4). Among these DEGs, 45 were detected to be testis-biased, and 22 were ovary-biased. For example, the expressions of amh and gsdf1 unigenes were upregulated in male gonads, whereas the expressions of zygote arrest protein 1 (zar1) and zona pellucida sperm-binding protein 3 (zp3) were found to be considerably higher in the ovaries (Table 4). In addition, we found that seven genes showed gonad-specific expression patterns. Of which, one gene (cyp19a1a) showed specific expression in ovaries; And the remaining six genes, such as dmrt1, steroid 11-beta-hydroxylase (cyp11b1), transcription factor SOX6 (sox6), and forkhead box protein J3 (foxj3) showed male-specific expression patterns.

3.6. RT-qPCR Confirmation of DEGs

A total of 13 testis-upregulated and 10 ovary-upregulated DEGs were chosen and subjected to the statistical verification of expression profiles using RT-qPCR analysis. The relative expressions of these representative genes were shown in Figure 5. In general, the RT-qPCR results were found to be consistent with those of RNA-seq analysis (Figure 5). DEGs such as amh, sox9, dmrt1, and ropporin-1-like protein (ropn1l) were testis-biased (Figure 5A), whereas unigenes such as homologs of zar1, membrane-associated progesterone receptor component 1 (pgrmc1), and vasa were ovary-biased (Figure 5B). Meanwhile, a correlation analysis was conducted and the consistent tendencies of expression levels between the RNA-Seq data and RT-qPCR results (R2 = 0.8476) confirmed the reliability and accuracy of gene expression levels quantified by transcriptomic analysis (Figure 5C).

4. Discussion

Gonadal development from undifferentiated to differentiated stages and maturation is the most important determinant for the success of reproduction in fish. This highly complex biological process involves a set of functional genes that can promote the gonadal differentiation into either an ovary or a testis, and then cause a fish individual to exhibit a male or female phenotype [34]. To date, however, the molecular mechanisms underlying gonadal development have totally been unrevealed in D. hystrix. As an effective way to uncover the gene regulatory networks of gonad development and its dimorphism, transcriptome sequencing and comparative analysis between male and female gonads were employed to identify sex-related genes and to reveal their potential roles by combining differential expression data with biological pathways.

4.1. Sex-Biased Genes Involved in Steroids Synthetic Pathway

Sex steroid hormones, primarily including androgen and estrogen, influence the phenotypic sex by acting as key regulators for gonadal differentiation, development and sex maintenance in fish species [34,35]. In teleosts, the major androgen and estrogen essential to ovarian and testicular development are 11-ketotestosterone (11-KT) and 17β-estradiol (E2), respectively [36]. The syntheses of these sex steroid hormones need a series of genes encoding steroid-metabolizing enzymes. In particular, cyp19a1a, cyp11b2 encoding steroid 11-beta-hydroxylase, hsd11b1 coding for 11 beta-hydroxysteroid dehydrogenase, cyp11a1 coding for cholesterol side-chain cleavage enzyme, and hsd17bs encoding 17β-hydroxysteroid dehydrogenases with 17-ketosteroid reducing activity are considered to be the most essential.
It has been demonstrated that hsd11b1 and cyp11b2 (encodes the key enzyme that converts testosterone to 11-KT) are involved in the pivotal steps in the synthesis of 11-KT in testis, whereas cyp19a1a gene product that catalyzes the conversion of androgens to estrogens is essential for the E2 synthesis in the ovary [37]. Previous reports have indicated that cyp11b2 expression levels were comparatively higher in the testes of some teleost fishes [38,39], implicating a regulatory role for cyp11b2 gene in testicular development. In this study, cyp11b2 gene was expressed at significantly higher levels in D. hystrix testes, while cyp19a1a was found to be upregulated in ovaries compared to testes, exhibiting a similar expression pattern to other fish species [34,37,40,41]. The findings suggested that these genes play potential roles in the development of gonads and may participate in D. hystrix reproduction. Interestingly, the sexual dimorphism of serum E2 and 11-KT levels has already been observed in fish species; the levels of E2 and 11-KT are relatively higher in females and males, respectively [34,41]. In Oreochromis niloticus, both serum E2 levels and cyp19a1a expression were comparatively higher in females than in males [41]. In Oryzias latipes, mutation of cyp19a1a gene led to a marked decrease in the gonadal E2 level in female (XX) individuals [42]. Collectively, higher serum E2 levels may be attributed to the elevated ovarian cyp19a1a expression.
Steroidogenic acute regulatory protein (StAR) participates in the rate-limiting step of steroid biosynthesis by transporting cholesterol to the inner mitochondrial membrane where the cholesterol side-chain cleavage enzyme converts this substrate to pregnenolone [34,35]. In teleosts, the star gene was found to be highly expressed during spermatogenesis, oocyte maturation and ovulation [43,44]. Our study showed that the expression levels of star and cyp11a1 in the testes of D. hystrix were significantly higher than those in the ovaries. The current results showed good agreement with the experimental results in Scatophagus argus [34] and P. olivaceus [45], suggesting that the abundances of star and cyp11a1 may partially affect the rate of steroid synthesis in teleost fish. In fish gonads, sex steroids synthesis-related genes are modulated by the hypothalamic–pituitary–gonadal (HPG) axis, one of the key steps is cAMP-mediated stimulation of star expression [34].
In O. niloticus, star mRNA levels in testes were greatly enhanced by injection of human chorionic gonadotropin (hCG) [46]. Moreover, recombinant follicle stimulating hormone (FSH)/luteinizing hormone (LH) administration increased the star and hsd11b1 expressions, E2/11-KT levels, and finally promoted the ovary and testicular development in S. argus [47]. Thus, understanding the expression and endocrine regulation of steroidogenic genes would greatly help us establish effective methods for controlling reproduction in D. hystrix aquaculture, such as multiple gonadotropin-releasing hormone agonist (GnRHa) injections or implants that are commonly utilized for the artificial induction of oocyte maturation/ovulation and spermination in fish.

4.2. Candidate Genes Related to Gonad Differentiation and Development

The molecular mechanisms involved in sex determination and gonad differentiation are variable among phyla. Although the top upstream regulators in the sex determination pathway are less conserved, the downstream genes are more conserved. With rare exceptions, almost all currently identified sex-determining genes belong to one of the three protein families (Dmrt, TGF-β and its signaling pathway, and Sox) [21].
Here, regulatory genes that appear to be involved in male gonad differentiation were identified from the D. hystrix gonad transcriptomes. In particular, detection of highly expressed dmrt1 as a male-biased gene would be of great interest. Dmrt1 belonging to the Dmrt gene family generally functions as a conserved transcription factor in the sexual regulatory cascade. Dmrt1 and its paralogs have been claimed as master sex-determininggenes in some animal species [21,34], playing essential parts in the differentiation of testis and maintenance of male-specified germ cells [48]. Also, it has been understood that dmrt1 works as an essential factor in gonadal development and gametogenesis in fishes [20,49]. Dmrt genes stimulate male-specific differentiation but repress female-specific differentiation [21]. In O. latipes, the mutation of autosomal dmrt1 was found to be responsible for a male-to-female sex reversal [50]. Comparably, dmrt1-mutated testes exhibited serious testicular development defects and gradual loss of germ cells in zebrafish [51]. In this research, dmrt1 genes were determined to be specifically expressed in the testis; ovarian dmrt1 expression could not be detected by transcriptome analysis and RT-qPCR. The trend of dmrt1 expression was quite similar to those in other fishes such as O. niloticus, and Megalobrama amblycephala [40,52], suggesting that dmrt1 gene is a key player in the testis development in D. hystrix.
A series of sex-determining genes encoding transforming growth factor-β (TGF-β) signal components (e.g., GsdfY, amhy, Amhr2, Gdf6Y) have been identified in fish, suggesting that the TGF-β pathway is involved in gonad differentiation. Gonadal soma-derived factor (Gsdf), a TGF-β superfamily member, is found to be expressed specifically in fish gonads, predominantly in the Sertoli cells and neighboring spermatogonia of testes [53]. As a direct downstream gene of dmrt1, Jiang et al. found that gsdf gene transcription was regulated by dmrt1 [53]. Recently, the authors further demonstrated that dmrt1 could induce the expression of gsdf with the participation of splicing factor 1 (SF-1, also known as Nr5a1, an important activator of steroidogenic enzymes, including aromatase) [54].
Previous studies have shown that gsdf plays a key role in testicular differentiation in fish, and it is speculated that gsdf acts by suppressing the activator of cyp19a1a and inhibiting estrogen synthesis [53]. Mutation of gsdf in medaka and O. niloticus initiated male-to-female sex reversal [53,55], while overexpression of this gene induced testis differentiation in female O. niloticus [56]. A study involving Oncorhynchus mykiss showed that gsdf may act in the regulation of spermatogenesis by stimulating the proliferation of spermatogonia [57]. In teleost, it was reported that gsdf was expressed at a higher level in the testicular somatic cells compared with ovarian tissues [58]. Sf-1 was significantly upregulated during and after testicular differentiation in black porgy [59]. Similar trends of gsdf and sf-1 expressions were also observed in this study. Therefore, we could deduce that gsdf has a conserved function in the testis differentiation of D. hystrix.
Anti-Müllerian hormone (Amh) encoded by amh has also been identified as a member of the TGF-β family in fish species [18]. Amh suppresses the development of the Müllerian ducts and functions as a key regulator for differentiation of the Sertoli and granulosa cells, germ cell proliferation and steroidogenesis in Leydig cells in gonad development [34]. Lin et al. [51] found that amh mutation resulted in a female-biased sex ratio in zebrafish; the unrestrained germ cell proliferation in male amh mutants led to hypertrophic testes. In XY medaka, Amh type II receptor (amhr2) mutation could promote the sex reversal and amhr2 mutants mostly exhibited the signs of germ cell over-proliferation [60]. Our data showed that the expressions of amh and amhr2 genes were upregulated in the testes but weakly expressed in the ovaries, implicating the significance of Amh/Amhr2 pathway in the modulation of testicular differentiation and germ cell proliferation in D. hystrix.
Several members of the Sox (SRY-related HMG box) gene family has also been found to regulate the differentiation of gonads in fish; typical examples include sox9, sox8, sox5, and sox3 [18,61]. Here, the abundances of the two transcriptional factors sox9 and sox6 were detected in our transcriptome data and they were identified as male-biased genes. Classic studies have clearly demonstrated that sox9 plays vital roles in the testicular development of male gonad as an important sex-determination gene [35]. Sox9 was found to be expressed in the testes of rainbow trout [62], and channel catfish [63]. Its crucial role in sex determination of teleost fish has also been confirmed by genetic approaches [21]. Genomic studies have revealed that the sox9 gene in teleosts has undergone duplication and there are two copies (sox9a and sox9b) [34,61]. In both male and female medaka, sox9b was shown to be pivotal for the survival of germ cells [64]. Certain regulatory genes in male fish may regulate the expression of sox9b mRNA in teleost fish. A recent study demonstrated that the Nile tilapia dmrt1 gene positively regulated the transcription of sox9b by directly binding to a specific promoter cis-regulatory element [61]. Moreover, sox6 gene was reported to be specifically expressed in the testis and involved in the later stages of spermatogenesis in teleost fishes [65]. In this study, both RNA-seq and RT-qPCR analysis demonstrated that sox9 and sox6 mRNA levels were much higher in the male gonads. Such expression patterns were similar to those observed in O. latipes [66], S. Sillago [18], and S. argus [34], suggesting a highly conserved function of sox9 gene in teleosts.
As to the identification of genes that participate in the differentiation of gonads, it is necessary to explore their roles in both undifferentiated and differentiated gonad samples. Importantly, detailed information about the gonad differentiation-related genes should be collected and explored in more developmental stages, resembling what has been performed in other fish species [34]. Hence, the expression profiles of the sex-biased genes identified in this study should be carefully analyzed in the future. In this study, the sex-specific expression of dmrt1 implicates it as the most promising candidate sex-determining gene in D. hystrix. In order to further confirm the precise role of dmrt1, molecular genetic studies are firstly required to characterize the transcriptional regulation of putative downstream genes (e.g., gsdf, and sf-1). Meanwhile, the DMRT gene cluster will be cloned, and then gene structures will be comparatively analyzed, to identify any possible sex-linked polymorphic locus. With the foundation of above basic research, specific gene knockout and rescue experiments would be required to the functional confirmation of dmrt1 gene in sex determination.

4.3. Sex-Biased Genes Involved in Gametogenesis and Gamete Maturation

In fish aquaculture, the eventual aim of artificially induced breeding is to facilitate the development of gonads and then obtain mature gametes [26]. In this study, the expression of many genes associated with oogenesis, oocytes maturation, spermatogenesis and sperm motility was presented, such as vasa, zygote arrest 1 (zar1), zona pellucida sperm-binding proteins (zps), spermatogenesis-associated proteins (spatas), and spermine oxidase (smox). Vasa, specifically localized in germline cells, has been well characterized as a crucial player in germ cell formation in larval fish [35]. A previous study has pointed out that vasa gene is expressed in primordial germ cells during the first formation of the gonadal anlagen [67]. In Pacific bluefin tuna (Thunnus orientalis), vasa exhibited a high level of expression in spermatogonia, as well as oogonia and previtellogenic oocytes [68]. The D. hystrix transcriptomic data showed that vasa mRNA was present both in the male and female gonads, with a drastically higher mRNA level in the ovaries. Such result is consistent with those reported in southern bluefin tuna (Thunnus maccoyii) [69] and yellow catfish (Pelteobagrus fulvidraco) [70], implying that vasa play a certain role in female gonad development. However, the expression pattern of vasa shows large variations among different species [35]. The functional details of vasa in gonadal differentiation in D. hystrix need to be clarified in future studies.
In vertebrates, zar1 acts as an oocyte-specific maternal effect gene with an evolutionarily conserved function in the fertilization process [34,35]. The zona pellucida (ZP) encoded by zp genes is a glycoproteinaceous matrix surrounding the oocyte and has an important part in species-specific binding of sperm [18]. In zebrafish, the zp mRNA levels were enhanced notably during oogenesis, particularly at the previtellogenic stage [71]. It has been shown that zp1 participates in the generation of oocyte envelope, and zp3 acts as a major class of female-specific factors in the reproductive process [72]. In the present study, zp1 and zp3 were more highly expressed in the ovaries than in the testes, suggesting that these oocyte-specific genes appear to be involved in ovarian folliculogenesis in D. hystrix. Regarding male reproduction, sperm acrosome membrane-associated protein 6 (spaca6) can mediate the fusion between ovum and spermatozoa as a sperm membrane component [73]. It has been demonstrated that spatas and sperm surface protein Sp17 (sp17) provide vital functions for spermatogenesis and sperm motility [74]. Here, significantly higher expressions of genes spaca6, spata5, spata6, spata7, smox and sp17 were observed in the male gonad; the expression difference may be associated with the spermatogenesis in D. hystrix. Absolutely, these identified gonocyte-specific genes would be helpful for investigating the control mechanisms during oogenesis and spermatogenesis in D. hystrix. In order to guarantee the performance of reproductive management of this fish species under aquaculture conditions, more importantly, further efforts to establish an effective technique for inducing gamete maturation are highly encouraged.

5. Conclusions

This is the first study on the gonad transcriptome of D. hystrix and a number of 57,077 unigenes were assembled. A comparison of ovarian and testicular transcriptomes detected a set of DEGs supposedly involved in gonadal development and gametogenesis. The conserved expression profiles of the well-known reproduction-related genes imply their similar roles in the gonad differentiation and development of D. hystrix. Our findings offer a precious data source for further investigation into the regulatory mechanisms and molecular characteristics of the reproductive process in D. hystrix.

Supplementary Materials

The following are available online at https://www.mdpi.com/article/10.3390/ani11041042/s1, Table S1: The specific information of the fish samples, Table S2: PCR primer sequences used for RT-qPCR, Table S3: Differentially expressed genes identified between ovaries and testes, Table S4: GO enrichment analysis of DEGs, Table S5: KEGG enrichment analysis of DEGs, Table S6: GO terms and KEGG pathways known to be associated with reproduction, Figure S1: Sequencing saturation distribution analysis of gonadal RNA-Seq data, Figure S2: GO classification of the assembled unigenes, Figure S3: KOG classification of the assembled unigenes, Figure S4: KEGG classification of the assembled unigenes, Figure S5: Clustering of testis and ovary samples based on their gonadal transcriptome profiles by principal component analysis (PCA).

Author Contributions

Data curation, W.Y., Y.W., Z.L. and G.L.; Formal analysis, W.Y., S.L., and H.H.; Funding acquisition, H.H. and H.C.; Investigation, W.Y., Y.W., Z.L. and X.Y.; Methodology, W.Y. and H.C.; Project administration, H.H.; Resources, H.H. and H.C.; Supervision, H.H. and H.C.; Writing—original draft, W.Y. and H.C.; Writing—review and editing, G.L. and W.Y. All authors have read and agreed to the published version of the manuscript.

Funding

This research was funded by grants from the Key R & D Project of Hainan Province (grant number ZDYF2018225), Major Science and Technology Plan Projects of Hainan Province (grant number ZDKJ2016009), Natural Science Foundation of Guangdong Province (grant number 2019A1515010958), College of Hainan Tropical Marine Project Leader and Doctoral Research Grant 2016 (grant number RHDXB201612), The Open Project of Key Laboratory of Utilization and Protection of Tropical Marine Biological Resources (Hainan Tropical Ocean University), Ministry of Education (grant number UCTMB20201), Young Creative Talents Project of Guangdong Province Universities and Colleges (grant number 2017GkQNCX092), Guangdong Basic and Applied Basic Research Foundation (grant number 2019A1515110290), and China Postdoctoral Science Foundation (grant number 2020M682832).

Institutional Review Board Statement

Animal experiments were carried out in strict accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals. The protocol was approved by the Animal Research and Ethics Committee of Guangdong Ocean University (NIH Pub. No. 85–23, revised 1996).

Data Availability Statement

The data presented in this study are available on request from the corresponding author. The data are not publicly available due to the agreement with funding bodies.

Conflicts of Interest

The authors declare no conflict of interest.

References

  1. Martin, S.B.; Ribu, D.; Cutmore, S.C.; Cribb, T.H. Opistholobetines (Digenea: Opecoelidae) in Australian tetraodontiform fishes. Syst. Parasitol. 2018, 95, 743–781. [Google Scholar] [CrossRef]
  2. Leis, J.M. Nomenclature and distribution of the species of the porcupinefish family Diodontidae (Pisces, Teleostei). Mem. Natl. Mus. Vic. 2006, 63, 77–90. [Google Scholar] [CrossRef] [Green Version]
  3. Trevett, A.J.; Mavo, B.; Warrell, D.A. Tetrodotoxic poisoning from ingestion of a porcupine fish (Diodon hystrix) in Papua New Guinea: Nerve conduction studies. Am. J. Trop. Med. Hyg. 1997, 56, 30–32. [Google Scholar] [CrossRef]
  4. Bandyopadhyay, S.A. On the record of a spot-fin porcupine fish, Diodon hystrix (Linnaeus, 1758) from Mandarmani, Bay of Bengal Coast of West Bengal, India. Proc. Zool. Soc. 2014, 67, 175–177. [Google Scholar] [CrossRef]
  5. Huang, H.; Hong, G.; Ma, J.; Chen, Y.; Chen, H. Characterization of the complete mitochondrial genome of Diodon hystrix with phylogenetic consideration. Mitochondrial DNA Part B 2017, 2, 321–322. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  6. Chen, Y.; Huang, H.; Ma, J.; Gao, L.; Chen, P. Analysis and evaluation of nutrient composition in different tissues of the wild spot-fin porcupine fish (Diodon hystrix). Agric. Sci. Technol. 2017, 18, 2468–2476. [Google Scholar] [CrossRef]
  7. Ma, J.; Liu, J.; Jiang, Z.; Zhou, Y.; Yu, Z.; Wang, H.; Chen, Y.; Chen, P.; Huang, H. Development and identification of SSR markers based on RNA-seq data of Diodon hystrix. S. Chi. Fish. Sci. 2020, 16, 127–136. (In Chinese) [Google Scholar]
  8. Piferrer, F. Endocrine sex control strategies for the feminization of teleost fish. Aquaculture 2001, 197, 229–281. [Google Scholar] [CrossRef]
  9. Heule, C.; Salzburger, W.; Böhne, A. Genetics of sexual development: An evolutionary playground for fish. Genetics 2014, 196, 579–591. [Google Scholar] [CrossRef] [Green Version]
  10. Radhakrishnan, S.; Nair, N.B. Histopathology of the infestation of Diodon hystrix L. by Peniculisa wilsoni Radhakrishnan (Copepoda: Lernaeoceridae). J. Fish Dis. 1981, 4, 83–87. [Google Scholar] [CrossRef]
  11. Chandran, A.; Natarajan, P. Heavy infection of Diodon hystrix by the copepod Peniculisa wilsoni (Siphonostomatoida, Pennellidae). J. Fish Biol. 1994, 45, 167–168. [Google Scholar] [CrossRef]
  12. Metzker, M.L. Sequencing technologies—the next generation. Nat. Rev. Genet. 2010, 11, 31–46. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  13. Garber, M.; Grabherr, M.G.; Guttman, M.; Trapnell, C. Computational methods for transcriptome annotation and quantification using RNA-seq. Nat. Methods 2011, 8, 469–477. [Google Scholar] [CrossRef] [Green Version]
  14. Ma, D.Y.; Ma, A.J.; Huang, Z.H.; Wang, G.N.; Wang, T.; Xia, D.D.; Ma, B.H. Transcriptome analysis for identification of genes related to gonad differentiation, growth, immune response and marker discovery in the turbot (Scophthalmus maximus). PLoS ONE 2016, 11, e0149414. [Google Scholar] [CrossRef] [Green Version]
  15. Ma, K.Y.; Qiu, G.F.; Feng, J.B.; Li, J.L. Transcriptome analysis of the oriental river prawn Macrobrachium nipponense using 454 pyrosequencing for discovery of genes and markers. PLoS ONE 2012, 7, e39727. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  16. Xie, Z.Z.; Xiao, L.; Wang, D.D.; Fang, C.; Liu, Q.Y.; Li, Z.H.; Liu, X.C.; Zhang, Y.; Li, S.S.; Lin, H.R. Transcriptome analysis of the Trachinotus ovatus: Identification of reproduction, growth and immune-related genes and microsatellite markers. PLoS ONE 2014, 9, e109419. [Google Scholar] [CrossRef] [Green Version]
  17. Zhang, Y.; Miao, G.; Fazhan, H.; Waiho, K.; Zheng, H.; Li, S.; Ikhwanuddin, M.; Ma, H. Transcriptome-seq provides insights into sex-preference pattern of gene expression between testis and ovary of the crucifix crab (Charybdis feriatus). Physiol. Genom. 2018, 50, 393–405. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  18. Tian, C.; Li, Z.; Dong, Z.; Huang, Y.; Du, T.; Chen, H.; Jiang, D.; Deng, S.; Zhang, Y.; Wanida, S.; et al. Transcriptome analysis of male and female mature gonads of silver sillago (Sillago sihama). Genes 2019, 10, 129. [Google Scholar] [CrossRef] [Green Version]
  19. Du, X.; Wang, B.; Liu, X.; Liu, X.; He, Y.; Zhang, Q.; Wang, X. Comparative transcriptome analysis of ovary and testis reveals potential sex-related genes and pathways in spotted knifejaw Oplegnathus punctatus. Gene 2017, 637, 203–210. [Google Scholar] [CrossRef] [PubMed]
  20. Zhang, W.; Liu, Y.; Yu, H.; Du, X.; Zhang, Q.; Wang, X.; He, Y. Transcriptome analysis of the gonads of olive flounder (Paralichthys olivaceus). Fish. Physiol. Biochem. 2016, 42, 1581–1594. [Google Scholar] [CrossRef]
  21. Lu, J.; Luan, P.; Zhang, X.; Xue, S.; Peng, L.; Mahbooband, S.; Sun, X. Gonadal transcriptomic analysis of yellow catfish (Pelteobagrus fulvidraco): Identification of sex-related genes and genetic markers. Physiol. Genom. 2014, 46, 798–807. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  22. Shen, F.; Long, Y.; Li, F.; Ge, G.; Song, G.; Li, Q.; Qiao, Z.; Cui, Z. De novo transcriptome assembly and sex-biased gene expression in the gonads of Amur catfish (Silurus asotus). Genomics 2020, 112, 2603–2614. [Google Scholar] [CrossRef] [PubMed]
  23. Zhang, X.; Zhou, J.; Li, L.; Huang, W.; Ahmad, H.I.; Li, H.; Jiang, H.; Chen, J. Full-length transcriptome sequencing and comparative transcriptomic analysis to uncover genes involved in early gametogenesis in the gonads of Amur sturgeon (Acipenser schrenckii). Front. Zool. 2020, 17, 11. [Google Scholar] [CrossRef] [Green Version]
  24. Yang, W.; Chen, H.; Cui, X.; Zhang, K.; Jiang, D.; Deng, S.; Zhu, C.; Li, G. Sequencing, de novo assembly and characterization of the spotted scat Scatophagus argus (Linnaeus 1766) transcriptome for discovery of reproduction related genes and SSRs. J. Oceanol. Limn. 2018, 36, 1329–1341. [Google Scholar] [CrossRef]
  25. Chen, Y.; Chen, Y.; Shi, C.; Huang, Z.; Zhang, Y.; Li, S.; Li, Y.; Ye, J.; Yu, C.; Li, Z.; et al. SOAPnuke: A MapReduce acceleration-supported software for integrated quality control and preprocessing of high-throughput sequencing data. Gigascience 2018, 7, 1–6. [Google Scholar] [CrossRef] [Green Version]
  26. Grabherr, M.G.; Haas, B.J.; Yassour, M.; Levin, J.Z.; Thompson, D.A.; Amit, I.; Adiconis, X.; Fan, L.; Raychowdhury, R.; Zeng, Q.; et al. Full-length transcriptome assembly from RNA-Seq data without a reference genome. Nat. Biotechnol. 2011, 29, 644–652. [Google Scholar] [CrossRef] [Green Version]
  27. Conesa, A.; Götz, S.; García-Gómez, J.M.; Terol, J.; Talón, M.; Robles, M. Blast2GO: A universal tool for annotation, visualization and analysis in functional genomics research. Bioinformatics 2005, 21, 3674–3676. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  28. Ye, J.; Fang, L.; Zheng, H.; Zhang, Y.; Chen, J.; Zhang, Z.; Wang, J.; Li, S.; Li, R.; Bolund, L.; et al. WEGO: A web tool for plotting GO annotations. Nucleic Acids Res. 2006, 34 (Suppl. 2), 293–297. [Google Scholar] [CrossRef]
  29. Xie, C.; Mao, X.; Huang, J.; Ding, Y.; Wu, J.; Dong, S.; Kong, L.; Gao, G.; Li, C.Y.; Wei, L. KOBAS 2.0: A web server for annotation and identification of enriched pathways and diseases. Nucleic Acids Res. 2011, 39 (Suppl. 2), 316–322. [Google Scholar] [CrossRef] [Green Version]
  30. Li, B.; Dewey, C.N. RSEM: Accurate transcript quantification from RNA-Seq data with or without a reference genome. BMC Bioinform. 2011, 12, 323. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  31. Love, M.I.; Huber, W.; Anders, S. Moderated estimation of fold change and dispersion for RNA-seq data with DESeq2. Genome Biol. 2014, 15, 550. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  32. Ai, C.; Kong, L. CGPS: A machine learning-based approach integrating multiple gene set analysis tools for better prioritization of biologically relevant pathways. J. Genet. Genom. 2018, 45, 489–504. [Google Scholar] [CrossRef] [PubMed]
  33. Yu, F.; Duan, C.; Zhang, X.; Yao, D.; Si, G.; Gao, Y.; Gao, Z.; Umer, F.; Guo, X. RNA-seq analysis reveals different gene ontologies and pathways in rheumatoid arthritis and Kashin-Beck disease. Int J. Rheum Dis. 2018, 21, 1686–1694. [Google Scholar] [CrossRef] [PubMed]
  34. He, F.; Jiang, D.; Huang, Y.; Mustapha, F.U.; Yang, W.; Cui, X.; Tian, C.; Chen, H.; Shi, H.; Deng, S.; et al. Comparative transcriptome analysis of male and female gonads reveals sex-biased genes in spotted scat (Scatophagus argus). Fish. Physiol. Biochem. 2019, 45, 1963–1980. [Google Scholar] [CrossRef] [PubMed]
  35. Qin, G.; Luo, W.; Tan, S.; Zhang, B.; Ma, S.; Lin, Q. Dimorphism of sex and gonad-development-related genes in male and female lined seahorse, Hippocampus erectus, based on transcriptome analyses. Genomics 2019, 111, 260–266. [Google Scholar] [CrossRef]
  36. Ijiri, S.; Kaneko, H.; Kobayashi, T.; Wang, D.S.; Sakai, F.; Paul-Prasanth, B.; Nagahama, Y. Sexual dimorphic expression of genes in gonads during early differentiation of a teleost fish, the Nile tilapia Oreochromis niloticus. Biol. Reprod. 2008, 78, 333–341. [Google Scholar] [CrossRef] [Green Version]
  37. Yan, H.; Shen, X.; Cui, X.; Wu, Y.; Wang, L.; Zhang, L.; Liu, Q.; Jiang, Y. Identification of genes involved in gonadal sex differentiation and the dimorphic expression pattern in Takifugu rubripes gonad at the early stage of sex differentiation. Fish. Physiol. Biochem. 2018, 44, 1275–1290. [Google Scholar] [CrossRef] [PubMed]
  38. Socorro, S.; Martins, R.L.; Mylonas, C.; Canario, A.V. A cDNA for European sea bass (Dicentrachus labrax) 11β-hydroxylase: Gene expression during the thermosensitive period and gonadogenesis. Gen. Comp. Endocrinol. 2007, 150, 164–173. [Google Scholar] [CrossRef]
  39. Liu, S.; Govoroun, M.; D’Cotta, H.; Ricordel, M.J.; Lareyre, J.J.; McMeel, O.M.; Smith, T.; Nagahama, Y.; Guiguen, Y. Expression of cytochrome P45011β (11β-hydroxylase) gene during gonadal sex differentiation and spermatogenesis in rainbow trout, Oncorhynchus mykiss. J. Steroid. Biochem Mol. Biol. 2000, 75, 291–298. [Google Scholar] [CrossRef]
  40. Tao, W.; Yuan, J.; Zhou, L.Y.; Sun, L.N.; Sun, Y.L.; Yang, S.J.; Li, M.H.; Zeng, S.; Huang, B.F.; Wang, D.S. Characterization of gonadal transcriptomes from Nile tilapia (Oreochromis niloticus) reveals differentially expressed genes. PLoS ONE 2013, 8, e63604. [Google Scholar] [CrossRef] [Green Version]
  41. Jiang, D.N.; Chen, J.L.; Fan, Z.; Tan, D.J.; Zhao, J.E.; Shi, H.J.; Liu, Z.L.; Tao, W.J.; Li, M.H.; Wang, D.S. CRISPR/Cas9-induced disruption of wt1a and wt1b reveals their different roles in kidney and gonad development in Nile tilapia. Dev. Biol. 2017, 428, 63–73. [Google Scholar] [CrossRef]
  42. Nakamoto, M.; Shibata, Y.; Ohno, K.; Usami, T.; Kamei, Y.; Taniguchi, Y.; Todo, T.; Sakamoto, T.; Young, G.; Swanson, P.; et al. Ovarian aromatase loss-of-function mutant medaka undergo ovary degeneration and partial female-to-male sex reversal after puberty. Mol. Cell Endocrinol. 2018, 460, 104–122. [Google Scholar] [CrossRef]
  43. Rocha, A.; Zanuy, S.; Carrillo, M.; Gomez, A. Seasonal changes in gonadal expression of gonadotropin receptors, steroidogenic acute regulatory protein and steroidogenic enzymes in the European sea bass. Gen. Comp. Endocrinol. 2009, 162, 265–275. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  44. Sreenivasulu, G.; Sridevi, P.; Sahoo, P.K.; Swapna, I.; Ge, W.; Kirubagaran, R.; Dutta-Gupta, A.; Senthilkumaran, B. Cloning and expression of StAR during gonadal cycle and HCG-induced oocyte maturation of air-breathing catfish, Clarias gariepinus. Comp. Biochem. Physiol. B Biochem. Mol. Biol. 2009, 154, 6–11. [Google Scholar] [CrossRef] [PubMed]
  45. Fan, Z.; You, F.; Wang, L.; Weng, S.; Wu, Z.; Hu, J.; Zou, Y.; Tan, X.; Zhang, P. Gonadal transcriptome analysis of male and female olive flounder (Paralichthys olivaceus). Biomed. Res. Int. 2014, 2014, 291067. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  46. Yu, X.; Wu, L.; Xie, L.; Yang, S.; Charkraborty, T.; Shi, H.J.; Wang, D.S.; Zhou, L.Y. Characterization of two paralogous StAR genes in a teleost, Nile tilapia (Oreochromis niloticus). Mol. Cell Endocrinol. 2014, 392, 152–162. [Google Scholar] [CrossRef] [PubMed]
  47. Zhang, G.; Wang, W.; Su, M.; Zhang, J. Effects of recombinant gonadotropin hormones on the gonadal maturation in the spotted scat, Scatophagus argus. Aquaculture 2018, 483, 263–272. [Google Scholar] [CrossRef]
  48. Webster, K.A.; Schach, U.; Ordaz, A.; Steinfeld, J.S.; Draper, B.W.; Siegfried, K.R. Dmrt1 is necessary for male sexual development in zebrafish. Dev. Biol. 2017, 422, 33. [Google Scholar] [CrossRef] [PubMed]
  49. Herpin, A.; Schartl, M. Dmrt1 genes at the crossroads: A widespread and central class of sexual development factors in fish. FEBS J. 2011, 278, 1010–1019. [Google Scholar] [CrossRef]
  50. Masuyama, H.; Yamada, M.; Kamei, Y.; Fujiwara-Ishikawa, T.; Todo, T.; Nagahama, Y.; Matsuda, M. Dmrt1 mutation causes a male-to-female sex reversal after the sex determination by Dmy in the medaka. Chromosom Res. 2012, 20, 163–176. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  51. Lin, Q.; Mei, J.; Li, Z.H.; Zhang, X.; Zhou, L.; Gui, J.F. Distinct and cooperative roles of amh and dmrt1 in self-renewal and differentiation of male germ cells in zebrafish. Genetics 2017, 207, 1007–1022. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  52. Su, L.; Zhou, F.; Ding, Z.; Gao, Z.; Wen, J.; Wei, W.; Wang, Q.; Wang, W.; Liu, H. Transcriptional variants of Dmrt1 and expression of four Dmrt genes in the blunt snout bream, Megalobrama amblycephala. Gene 2015, 573, 205–215. [Google Scholar] [CrossRef]
  53. Jiang, D.N.; Yang, H.H.; Li, M.H.; Shi, H.J.; Zhang, X.B.; Wang, D.S. gsdf is a downstream gene of dmrt1 that functions in the male sex determination pathway of the Nile tilapia. Mol. Reprod. Dev. 2016, 83, 497–508. [Google Scholar] [CrossRef] [Green Version]
  54. Jiang, D.N.; Mustapha, U.F.; Shi, H.J.; Huang, Y.Q.; Si-Tu, J.X.; Wang, M.; Deng, S.P.; Chen, H.P.; Tian, C.X.; Zhun, C.H.; et al. Expression and transcriptional regulation of gsdf in spotted scat (Scatophagus argus). Comp. Biochem. Physiol. B Biochem. Mol. Biol. 2019, 233, 35–45. [Google Scholar] [CrossRef]
  55. Imai, T.; Saino, K.; Matsuda, M. Mutation of gonadal soma-derived factor induces medaka XY gonads to undergo ovarian development. Biochem. Biophys. Res. Commun. 2015, 467, 109–114. [Google Scholar] [CrossRef] [PubMed]
  56. Kaneko, H.; Ijiri, S.; Kobayashi, T.; Izumi, H.; Kuramochi, Y.; Wang, D.S.; Mizuno, S.; Nagahama, Y. Gonadal soma-derived factor (gsdf), a TGF-β superfamily gene, induces testis differentiation in the teleost fish Oreochromis niloticus. Mol. Cell Endocrinol. 2015, 415, 87–99. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  57. Sawatari, E.; Shikina, S.; Takeuchi, T.; Yoshizaki, G. A novel transforming growth factor-β superfamily member expressed in gonadal somatic cells enhances primordial germ cell and spermatogonial proliferation in rainbow trout (Oncorhynchus mykiss). Dev. Biol. 2007, 301, 266–275. [Google Scholar] [CrossRef] [Green Version]
  58. Shibata, Y.; Paul-Prasanth, B.; Suzuki, A.; Usami, T.; Nakamoto, M.; Matsuda, M.; Nagahama, Y. Expression of gonadal soma derived factor (GSDF) is spatially and temporally correlated with early testicular differentiation in medaka. Gene Expr. Patterns 2010, 10, 283–289. [Google Scholar] [CrossRef]
  59. Wu, G.C.; Tomy, S.; Chang, C.F. The expression of nr0b1 and nr5a4 during gonad development and sex change in protandrous black porgy fish, Acanthopagrus schlegeli. Biol. Reprod. 2008, 78, 200–210. [Google Scholar] [CrossRef]
  60. Nakamura, S.; Watakabe, I.; Nishimura, T.; Picard, J.Y.; Toyoda, A.; Taniguchi, Y.; di Clemente, N.; Tanaka, M. Hyperproliferation of mitotically active germ cells due to defective anti-Müllerian hormone signaling mediates sex reversal in medaka. Development 2012, 139, 2283–2287. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  61. Wei, L.; Li, X.; Li, M.; Tang, Y.; Wei, J.; Wang, D. Dmrt1 directly regulates the transcription of the testis-biased Sox9b gene in Nile tilapia (Oreochromis niloticus). Gene 2018, 687, 109–115. [Google Scholar] [CrossRef]
  62. Takamatsu, N.; Kanda, H.; Ito, M.; Yamashita, A.; Yamashita, S.; Shiba, T. Rainbow trout SOX9: cDNA cloning, gene structure and expression. Gene 1997, 202, 167–170. [Google Scholar] [CrossRef]
  63. Sun, F.Y.; Liu, S.K.; Gao, X.Y.; Jiang, Y.L.; Perera, D.; Wang, X.L.; Li, C.; Sun, L.Y.; Zhang, J.R.; Kaltenboeck, L.; et al. Male-biased genes in catfish as revealed by RNA-Seq analysis of the testis transcriptome. PLoS ONE 2013, 8, e68452. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  64. Nakamura, S.; Watakabe, I.; Nishimura, T.; Toyoda, A.; Taniguchi, Y.; Tanaka, M. Analysis of medaka sox9 orthologue reveals a conserved role in germ cell maintenance. PLoS ONE 2012, 7, e29982. [Google Scholar] [CrossRef] [PubMed]
  65. Yamashita, A.; Ito, M.; Takamatsu, N.; Shiba, T. Characterization of solt, a novel SoxLZ/Sox6 binding protein expressed in adult mouse testis. FEBS Lett. 2000, 481, 147–151. [Google Scholar] [CrossRef] [Green Version]
  66. Wei, L.; Yang, C.; Tao, W.J.; Wang, D.S. Genome-wide identification and transcriptome-based expression profiling of the Sox gene family in the Nile tilapia (Oreochromis niloticus). Int. J. Mol. Sci. 2016, 17, 270. [Google Scholar] [CrossRef] [Green Version]
  67. Kowalewska-Luczak, I.; Kmiec, M.; Terman, A. Aromatase cytochrome P450—The key enzyme of estrogen synthesis. Med. Weter. 2006, 62, 870–872. [Google Scholar] [CrossRef]
  68. Nagasawa, K.; Takeuchi, Y.; Miwa, M.; Higuchi, K.; Morita, T.; Mitsuboshi, T.; Miyaki, K.; Kadomura, K.; Yoshizaki, G. cDNA cloning and expression analysis of a vasa-like gene in Pacific bluefin tuna Thunnus orientalis. Fish. Sci. 2009, 75, 71–79. [Google Scholar] [CrossRef]
  69. Bar, I.; Cummins, S.; Elizur, A. Transcriptome analysis reveals differentially expressed genes associated with germ cell and gonad development in the Southern bluefin tuna (Thunnus maccoyii). BMC Genom. 2016, 17, 217. [Google Scholar] [CrossRef] [Green Version]
  70. Chen, S.L.; Zhang, G.J.; Shao, C.W.; Huang, Q.F.; Liu, G.; Zhang, P.; Song, W.T.; An, N.; Chalopin, D.; Volff, J.N.; et al. Whole-genome sequence of a flatfish provides insights into ZW sex chromosome evolution and adaptation to a benthic lifestyle. Nat. Genet. 2014, 46, 253. [Google Scholar] [CrossRef]
  71. Zeng, S.; Gong, Z. Expressed sequence tag analysis of expression profiles of zebrafish testis and ovary. Gene 2002, 294, 45–53. [Google Scholar] [CrossRef]
  72. Liu, X.; Wang, H.; Gong, Z. Tandem-repeated zebrafish zp3 genes possess oocyte-specific promoters and are insensitive to estrogen induction1. Biol. Reprod. 2006, 74, 1016–1025. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  73. Lorenzetti, D.; Poirier, C.; Zhao, M.; Overbeek, P.A.; Harrison, W.; Bishop, C.E. A transgenic insertion on mouse chromosome 17 inactivates a novel immunoglobulin superfamily gene potentially involved in sperm-egg fusion. Mamm. Genome 2014, 25, 141. [Google Scholar] [CrossRef] [PubMed]
  74. Yang, D.; Yin, C.; Chang, Y.; Dou, Y.; Hao, Z.; Ding, J. Transcriptome analysis of male and female mature gonads of Japanese scallop Patinopecten yessonsis. Genes Genom. 2016, 38, 1–12. [Google Scholar] [CrossRef]
Animals 11 01042 g001 550
Figure 1. Gene expression pattern analysis in gonads of D. hystrix. (A) The distribution of gene expression levels. The horizontal axis indicates sample name, whereas the vertical axis indicates log10 (FPKM) value. Box plots indicate the maximum, top quartile, median, bottom quartile, and minimum values from top to bottom. (B) Heatmap representation of the correlations between each two samples. The color represents the correlation coefficient. O, ovary; T, testis.
Figure 1. Gene expression pattern analysis in gonads of D. hystrix. (A) The distribution of gene expression levels. The horizontal axis indicates sample name, whereas the vertical axis indicates log10 (FPKM) value. Box plots indicate the maximum, top quartile, median, bottom quartile, and minimum values from top to bottom. (B) Heatmap representation of the correlations between each two samples. The color represents the correlation coefficient. O, ovary; T, testis.
Animals 11 01042 g001
Animals 11 01042 g002 550
Figure 2. Differential gene expression between the ovaries and testes of D. hystrix. (A) Volcano plot of the differences in gene expression. Red dots: upregulated, represent ovary-biased genes; Green dots: downregulated, represent testis-biased genes. (B) Venn diagram showing the distribution of testis-specific and ovary-specific DEGs.
Figure 2. Differential gene expression between the ovaries and testes of D. hystrix. (A) Volcano plot of the differences in gene expression. Red dots: upregulated, represent ovary-biased genes; Green dots: downregulated, represent testis-biased genes. (B) Venn diagram showing the distribution of testis-specific and ovary-specific DEGs.
Animals 11 01042 g002
Animals 11 01042 g003 550
Figure 3. Functional annotation of DEGs based on GO categorization. The horizontal axis indicates the GO functions, and the vertical axis represents the number of genes with GO function.
Figure 3. Functional annotation of DEGs based on GO categorization. The horizontal axis indicates the GO functions, and the vertical axis represents the number of genes with GO function.
Animals 11 01042 g003
Animals 11 01042 g004 550
Figure 4. Significantly enriched KEGG pathways of DEGs. (A) Pathway enrichment analysis of DEGs upregulated in testes. (B) Pathway enrichment analysis of DEGs upregulated in ovaries. The pathways and rich factor are shown in the vertical and the horizontal axis, respectively. The size of the point indicates the number of genes, and the color indicates the q value.
Figure 4. Significantly enriched KEGG pathways of DEGs. (A) Pathway enrichment analysis of DEGs upregulated in testes. (B) Pathway enrichment analysis of DEGs upregulated in ovaries. The pathways and rich factor are shown in the vertical and the horizontal axis, respectively. The size of the point indicates the number of genes, and the color indicates the q value.
Animals 11 01042 g004
Animals 11 01042 g005 550
Figure 5. Verification of expression profiles of 13 testis-biased (A) and 10 ovary-biased genes (B) using RT-qPCR. (C) Correlation analysis of the RNA-Seq data and RT-qPCR results.
Figure 5. Verification of expression profiles of 13 testis-biased (A) and 10 ovary-biased genes (B) using RT-qPCR. (C) Correlation analysis of the RNA-Seq data and RT-qPCR results.
Animals 11 01042 g005
Table
Table 1. Summary statistics of the gonadal RNA-Seq data for D. hystrix.
Table 1. Summary statistics of the gonadal RNA-Seq data for D. hystrix.
SampleRaw ReadsClean ReadsClean Bases (bp)GC Content (%)% ≥ Q20% ≥ Q30
Ovary 123,164,03322,312,2006,686,832,31848.6698.0694.32
Ovary 222,001,85421,207,1106,354,910,04448.9297.5393.24
Ovary 321,490,13320,679,4876,198,085,77848.8697.4793.14
Testis 123,845,59923,454,8287,010,039,93048.7498.3295.25
Testis 240,744,74439,306,61411,739,627,32848.5298.3195.16
Testis 325,338,57224,928,3067,457,433,49448.5798.1794.83
Mean26,097,48925,314,7587,574,488,14948.7197.9894.32
Total156,584,935151,888,54545,446,928,892
Table
Table 2. Summary statistics of the D. hystrix gonadal transcriptome assembly.
Table 2. Summary statistics of the D. hystrix gonadal transcriptome assembly.
Length RangeTranscriptUnigene
300–500 bp30,973 (22.18%)23,402 (41.00%)
500–1000 bp30,900 (22.13%)14,055 (24.62%)
1000–2000 bp31,636 (22.66%)8495 (14.88%)
>2000 bp46,119 (33.03%)11,125 (19.49%)
Total number139,62857,077
Total length (bp)261,271,79674,197,236
N50 length (bp)31182560
Mean length (bp)1871.21299.9
Table
Table 3. Statistics of the D. hystrix gonadal transcriptome annotation.
Table 3. Statistics of the D. hystrix gonadal transcriptome annotation.
DatabaseAnnotated NumberPercentage300 ≤ Length < 1000 bpLength ≥ 1000 bp
COG628111.00%15224759
GO12,43421.78%35708864
KEGG13,69924.00%37059994
KOG15,15726.56%404811,109
Pfam17,51930.69%420613,313
Swiss-prot13,48823.63%293310,555
eggNOG22,67139.72%743815,233
Nr23,11440.50%746915,645
All24,57443.05%882415,750
Table
Table 4. Searching for differential expression genes (DEGs) putatively involved in reproduction from the gonad transcriptome of D. hystrix.
Table 4. Searching for differential expression genes (DEGs) putatively involved in reproduction from the gonad transcriptome of D. hystrix.
Unigene IDGene AnnotationLog2 Fold Change
(Ovary/Testis)		FDR 1
Relatively higher expression in testis
c84183.graph_c0Gonadotropin-releasing hormone II receptor−3.631.23E-06
c81599.graph_c0Doublesex- and mab-3-related transcription factor 1−8.645.85E-07
c87605.graph_c0Doublesex- and mab-3-related transcription factor 3−8.344.17E-51
c69078.graph_c0Anti-Mullerian hormone−5.031.14E-11
c89331.graph_c1Anti-Muellerian hormone type-2 receptor−5.191.37E-20
c90445.graph_c0Forkhead box L3−7.137.21E-29
c83812.graph_c1Forkhead box protein J3−9.571.21E-08
c80643.graph_c0Forkhead box protein D3−8.627.63E-07
c90293.graph_c0Wilms tumor protein 1-interacting protein−2.736.19E-14
c86435.graph_c1Wilms tumor protein homolog−3.114.20E-07
c87662.graph_c6Wilms tumor protein−2.751.14E-06
c74886.graph_c0Gonadal soma derived factor 1−8.118.58E-34
c67874.graph_c1Transcription factor SOX9−4.291.13E-07
c73370.graph_c0Transcription factor SOX6−9.462.54E-08
c80342.graph_c0Steroidogenic acute regulatory protein−5.221.05E-26
c88056.graph_c017-beta-hydroxysteroid dehydrogenase 14−3.921.12E-17
c74550.graph_c03-oxo-5-beta-steroid 4-dehydrogenase−2.821.41E-03
c56757.graph_c03 beta-hydroxysteroid dehydrogenase type 1−3.759.81E-10
c79716.graph_c0Steroid 11-beta-hydroxylase−8.401.94E-11
c83337.graph_c1StAR-related lipid transfer protein 13−5.632.00E-19
c89692.graph_c0StAR-related lipid transfer protein 9−1.592.13E-05
c80342.graph_c0Steroidogenic acute regulatory protein−5.221.05E-26
c89905.graph_c0Estrogen receptor b2−2.412.43E-09
c84046.graph_c0Estrogen receptor−2.861.55E-10
c82367.graph_c0Estrogen receptor b1−3.065.18E-13
c89150.graph_c2Progesterone receptor−1.346.89E-04
c86613.graph_c1Kelch-like protein 10−9.333.60E-31
c85739.graph_c0Spermatogenesis-associated protein 5−1.951.19E-04
c87439.graph_c0Spermatogenesis-associated protein 20−2.164.59E-04
c86517.graph_c0Spermatogenesis-associated protein 7−4.854.25E-09
c83923.graph_c0Spermatogenesis-associated protein 17−6.611.43E-13
c84036.graph_c0Spermatogenesis-associated protein 6−3.165.66E-10
c81488.graph_c0Sperm surface protein Sp17−1.772.49E-03
c82755.graph_c0Sperm acrosome membrane-associated protein 6−3.092.95E-05
c42266.graph_c0Spermine oxidase−5.527.33E-09
c75610.graph_c0Ropporin-1-like protein−12.403.92E-39
c86581.graph_c5Splicing factor 1−2.218.72E-04
c87182.graph_c0Fibroblast growth factor receptor-like 1−6.424.76E-36
c86547.graph_c1Fibroblast growth factor 11−7.512.39E-10
c73147.graph_c0Fibroblast growth factor 7−3.525.76E-05
c77489.graph_c0Fibroblast growth factor-binding protein 1−3.122.17E-05
c81519.graph_c1Fibroblast growth factor 10−6.611.31E-11
c84573.graph_c2Fibroblast growth factor-binding protein 3−2.057.40E-03
c89383.graph_c0Fibroblast growth factor receptor 2−5.211.75E-13
c63200.graph_c0ATP synthase F0 subunit 6−2.866.13E-33
c78426.graph_c0Cholesterol side-chain cleavage enzyme−2.447.91E-05
Relatively higher expression in ovary
c73686.graph_c0Gonadotropin subunit beta-24.471.46E-28
c80357.graph_c1Catenin beta-11.793.63E-07
c85139.graph_c8Catenin alpha-22.647.20E-23
c80092.graph_c0Protein fem-1 homolog C2.888.07E-13
c85792.graph_c0Protein fem-1 homolog B2.267.47E-10
c83767.graph_c0Zygote arrest protein 15.762.60E-14
c82641.graph_c13-oxo-5-alpha-steroid 4-dehydrogenase 11.401.13E-03
c88743.graph_c5Steroid hormone receptor ERR22.344.22E-12
c87163.graph_c33-keto-steroid reductase1.902.24E-05
c73009.graph_c0Inactive hydroxysteroid dehydrogenase-like protein 11.113.55E-04
c85481.graph_c3Steroid hormone receptor ERR11.478.53E-04
c73497.graph_c0Hydroxysteroid dehydrogenase-like protein 21.143.41E-04
c72251.graph_c0Cytochrome P450 aromatase3.868.36E-08
c87866.graph_c0StAR-related lipid transfer protein 132.631.17E-10
c83997.graph_c0StAR-related lipid transfer protein 71.634.78E-07
c82351.graph_c1Membrane-associated progesterone receptor component 11.012.73E-03
c83997.graph_c2Membrane-associated progesterone receptor component 22.876.61E-09
c83333.graph_c2Progesterone-induced-blocking factor 11.664.04E-08
c42601.graph_c0Zona pellucida sperm-binding protein 36.292.60E-07
c85704.graph_c1Zona pellucida sperm-binding protein 13.722.40E-29
c87366.graph_c0Vasa1.331.15E-04
c78173.graph_c1Forkhead box protein H14.806.46E-41
1 False discovery rate.
Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Share and Cite

MDPI and ACS Style

Chen, H.; Li, Z.; Wang, Y.; Huang, H.; Yang, X.; Li, S.; Yang, W.; Li, G. Comparison of Gonadal Transcriptomes Uncovers Reproduction-Related Genes with Sexually Dimorphic Expression Patterns in Diodon hystrix. Animals 2021, 11, 1042. https://doi.org/10.3390/ani11041042

AMA Style

Chen H, Li Z, Wang Y, Huang H, Yang X, Li S, Yang W, Li G. Comparison of Gonadal Transcriptomes Uncovers Reproduction-Related Genes with Sexually Dimorphic Expression Patterns in Diodon hystrix. Animals. 2021; 11(4):1042. https://doi.org/10.3390/ani11041042

Chicago/Turabian Style

Chen, Huapu, Zhiyuan Li, Yaorong Wang, Hai Huang, Xuewei Yang, Shuangfei Li, Wei Yang, and Guangli Li. 2021. "Comparison of Gonadal Transcriptomes Uncovers Reproduction-Related Genes with Sexually Dimorphic Expression Patterns in Diodon hystrix" Animals 11, no. 4: 1042. https://doi.org/10.3390/ani11041042

Note that from the first issue of 2016, this journal uses article numbers instead of page numbers. See further details here.

Article Metrics

Back to TopTop