Dose-dependent role of AMH and AMHR2 signaling in male differentiation and regulation of sex determination in Spotted knifejaw (Oplegnathus punctatus) with X1X1X2X2/X1X2Y chromosome system

Zhao, Haixia; Li, Jun; Xiao, Zhizhong; Xiao, Yongshuang

doi:10.1186/s12964-025-02038-w

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Open access
Published: 01 February 2025

Dose-dependent role of AMH and AMHR2 signaling in male differentiation and regulation of sex determination in Spotted knifejaw (Oplegnathus punctatus) with X₁X₁X₂X₂/X₁X₂Y chromosome system

Haixia Zhao^1,2,3,4^na1,
Jun Li^1,2,3^na1,
Zhizhong Xiao^1,2,3 &
…
Yongshuang Xiao^1,2,3

Cell Communication and Signaling volume 23, Article number: 59 (2025) Cite this article

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Abstract

Sex determination mechanisms vary significantly across different chromosomal systems and evolutionary contexts. Nonetheless, the regulatory framework governing the multi-sex chromosome system (X₁X₁X₂X₂/X₁X₂Y) remains enigmatic. Through an examination of sex-related genes (dmrt1, hsd11b2, amh, sox9a, sox9b, foxl2, cyp19a), hormonal influences (E2, 11-KT), and histological analyses of gonadal development, we demonstrate that the critical period for sexual differentiation occurs between 35 to 60 days post-hatching (dph). Our multi-omics analysis identified amhr2 as a candidate sex-determining gene, revealing that the males possess three distinct amhr2 transcripts (amhr2ay, amhr2by, amhr2cy), whereas females express only one (amhr2a). In situ hybridization assays demonstrated that amhr2 is predominantly localized to primary spermatocyte and Sertoli cells of male testes. Notably, the specific mRNA expression of amhr2 is significantly enriched in amhr2cy, whose extracellular domain exhibits the highest binding affinity for Amh protein, with sexual expression differences manifesting as early as 5 dph. The outcomes of amhr2 interference (RNAi) experiments indicate that amhr2 knockdown leads to a reduction in the expression of male-related gene (dmrt1, amh, sox9a, sox9b), androgen synthesis genes (hsd11b2, cyp11a), and female-related genes (wnt4, foxl2, cyp19a, cyp19b). Conversely, overexpression of amhr2 yielded contrasting results. Our research supports the role of amhr2 as a pivotal candidate sex-determining gene. Furthermore, the dosage effect of amhr2, reflected in transcript abundance, mRNA expression levels, and binding efficacy, serves as a fundamental mechanism driving male differentiation and regulatory processes in Spotted knifejaw.

Introduction

Sex determination involves a signaling cascade initiated by sex-determining genes on the sex chromosomes, which directs the development of the initial gonads towards either ovaries or testes [1,2,3]. During the process of gonadal sex differentiation in vertebrates, a convergence is observed in both morphological characteristics and gene functionality [4, 5]. However, teleost fish display significant diversity in the differentiation of sex-determining genes [3]. The Y-linked gene amh has been confirmed as the sex determinant in species such as Patagonian pejerrey (Odontesthes hatcheri) [6], northern pike (Esox Lucius) [7], and threespine stickleback [8], while amhr2 plays a crucial role in tiger pufferfish (Takifugu rubripes) [9]. In yellow perch (Perca flavescens), the Y-chromosome-linked amhr2 is a strong candidate for the sex-determining gene [10]. Genes like dmrt1bY/dmy, gsdfY, and sox3 have been identified as sex determining genes in Oryzias species [11,12,13,14], while sdY (sexually dimorphic on the Y-chromosome) serves as the primary sex-determining factor in rainbow trout (Oncorhynchus mykiss) [15]. In Seriola species, sex determination is mediated by estrogen synthesized by the hsd17b1 gene located on the W chromosome [16]. Functional verification of sex-determining genes often involves gene knockout techniques. Overexpression studies can elucidate the impact of sex-determining genes on sexual development. For example, in the ayu, male-specific regions contain duplicates of the amhr2bY gene, and mutations in amhr2bY can lead to male-to-female sex reversal [17]. Similarly, in the Chinese tongue sole (Cynoglossus semilaevis), dmrt1 knockout may result in male-to-female sex reversal [18]. As systematic gene knockout is not yet feasible in most marine economic fish species, functional verification typically employs methods such as RNA interference or overexpression.

There are distinct molecular pathways governing female and male development in vertebrates [19, 20]. Female differentiation-related genes, such as foxl2, cyp19a, and cyp19b, play a critical role [21]. Foxl2 (Forkhead box L2) encodes a transcription factor essential for ovarian differentiation, contributing to the repression of testis-promoting genes in females [22, 23]. The cytochrome P450 aromatase genes, cyp19a and cyp19b, are responsible for catalyzing the conversion of androgens to estrogens [24]. Conversely, male sex differentiation is mediated by a distinct set of genes [25]. The dmrt1 (Doublesex and Mab-3 related transcription factor 1) is highly conserved across species and enhances the expression of male-specific genes while inhibiting female-specific pathways [26, 27]. The sox9 (sox9a and sox9b), encode transcription factors that support the development of Sertoli cells within the testes, a cell type essential for spermatogenesis and male reproductive function [28, 29]. Anti-Müllerian hormone (amh) and its receptor amhr2 work in concert to induce the regression of the Müllerian ducts, thereby preventing the development of female reproductive structures in males [30,31,32]. Hsd11b2 (11β-hydroxysteroid dehydrogenase type 2) plays a role in androgen biosynthesis, further contributing to masculinization and the establishment of male characteristics[33, 34].

Current methodologies for identifying sex-determining genes encompass quantitative trait locus (QTL) mapping, selective sweep analysis, genome-wide association studies (GWAS), genomic comparisons, and transcriptomics [10, 35,36,37]. The process of determining sex-determining gene regions and pinpointing specific genes often requires a multi-omics approach, which has facilitated the discovery of numerous sex-determining genes in various taxa, including mammals, birds, and fish [38]. GWAS utilizes molecular markers, such as single nucleotide polymorphisms (SNPs), across the entire genome to identify genetic variations associated with the target trait. Selective sweep analysis involves detecting regions and loci under positive selection pressure due to the hitchhiking effect, which leads to reduced polymorphism in neighboring regions. Both GWAS and selective sweep analysis are frequently used in tandem to pinpoint regions linked to sex that are also subject to positive evolutionary selection [38, 39]. Sex-determining genes typically exhibit sex-specific expression patterns early in development [40,41,42,43]. Transcriptomic analysis of gene sets identified through genomic screening serves as a crucial secondary screening tool for identifying sex-determining genes. This approach has been instrumental in studying sex determination in species such as the sea dragon [44], ayu (Plecoglossus altivelis) [17], yellow perch (Diploprion bifasciatum) [10], channel catfish (Ictalurus punctatus) [25, 45], and Atlantic salmon (Salmo salar) [16].

The Spotted knifejaw (Oplegnathus punctatus) is predominantly found in the subtropical and temperate waters of the western Pacific Ocean. In 2014, China achieved a breakthrough in the reproductive regulation and developed a comprehensive artificial breeding technology for Spotted knifejaw larvae, marking it as a significant species for marine cage and industrial aquaculture [29, 46, 47]. The Spotted knifejaw displays significant sexual dimorphism in growth, with males growing significantly larger than females and possesses multiple sex chromosome system (X₁X₁X₂X₂/X₁X₂Y) [48,49,50]. Current research on sex determination primarily focuses on the sex chromosome systems of XX/XY and ZZ/ZW [51,52,53,54,55], whereas the mechanisms governing sex determination and differentiation in the intricate multiple sex chromosome system (X₁X₁X₂X₂/X₁X₂Y) have not been systematically investigated.

This research aims to explore the sex-determining genes, and the mechanisms of sex determination within Spotted knifejaw which possesses a complex multiple sex chromosome system (X₁X₁X₂X₂/X₁X₂Y). The study employs multi-omics integrated analysis, including genomic resequencing, selective sweep analysis, GWAS, and gonadal transcriptomics, and RT-PCR to identify candidate sex-determining genes. Additionally, whole-genome scanning, RT-PCR, and molecular docking are applied to assess the binding efficiency. Further, gonadal RNA interference and juvenile Spotted knifejaw overexpression experiments are conducted to investigate the functional role in sex determination. The finding of this study could provide critical insights into the regulatory processes underlying sex determination and differentiation in fish with multiple sex chromosome system.

Materials and methods

Sample collection

To investigate the critical period of sex differentiation at the molecular, hormonal, and gonadal levels, samples of Spotted knifejaw were obtained from Yantai Laizhou Mingbo Aquatic Products Co., Ltd., with sampling conducted at intervals of every 7 days from 5 to 360 dph. The fish were anesthetized in water containing 120 mg/L MS-222. Prior to the experiments, genomic DNA was extracted from all Spotted knifejaw samples, and genetic sex was determined using sex-specific marker PCR for sex distinction. From 5 to 60 dph, the viscera of the Spotted knifejaw was directly fixed, and 12 samples were preserved in liquid nitrogen for RNA extraction to detect the sex differentiation period at the molecular level and to measure hormone levels. Twelve Spotted knifejaw were used for physiological sex identification and were fixed with bouin’s fixative solution and 4% paraformaldehyde (PFA). Serum was collected to measure hormone levels, and parts of the gonadal tissue were fixed with bouin’s fixative solution and 4% PFA, and after 24 h, the fixative was replaced with 70% ethanol. For tissue-specific expression analysis, three adult male and three adult female Spotted knifejaw (206.1g ± 6.6g) were selected as samples. Thirteen different tissues including testes, ovaries, brain, heart, eyes, spleen, stomach, intestine, kidney, gills, skin, liver, and muscle were dissected for total RNA extraction. To ensure the reliability of the results, each tissue sample was repeated biologically three times. In addition, RNA was extracted from the gonads of both female and male Spotted knifejaw individuals for transcriptomic analysis to further explore the gene expression patterns during gonadal development of the Spotted knifejaw gonads. The timeline for sex differentiation in Spotted knifejaw was plotted using Figdraw (RTOIT21552). Genome-wide association analysis and population evolutionary selective sweep analysis each utilized muscle tissue samples from 20 male and female Spotted knifejaw with both physiological and genetic sexes identified (231.41 ± 3.57g).

Hormone levels in the serum and gonads of Spotted knifejaw during early development were measured using a quantitative assay kit for 11-ketotestosterone (11-KT) and estradiol (E₂) (Sangon, Jiangsu). For tissue samples, the abdominal cavity of Spotted knifejaw individuals (5 dph to 60 dph) was dissected (removing as much muscle tissue as possible and retaining only the gonads), weighed the tissues in milligrams, and 9 times the volume of diluent was added. The samples were ground thoroughly on ice, the homogenate was ultrasonically disrupted to further lyse the tissue cells, and finally the homogenate was centrifuged at 5000 g for 5–10 min. The supernatant was collected as the sample solution to measure hormone content. A standardized protocol for blood collection and serum processing in Spotted knifejaw was followed. After anesthesia, the Spotted knifejaw was placed in a dissecting tray, and a sterile syringe was inserted 1 cm below the lateral line, at the anal fins, until the needle touched the spine. The needle was slightly withdrawn, creating negative pressure to draw blood into the syringe. The sample was stored at 4 °C for 2 h, then centrifuged at 4000 g for 10 min to obtain the serum. Serum was diluted with diluent at a ratio of 1:9. In detail, 30 μL of serum is taken per individual and diluted with 270 μL of diluent for hormone testing. Since each sample well was spiked with 50 μL of sample, and three biological replicates were performed for each individual, a total of 150μL of sample diluent was used in each hormone assay.

To investigate the function of amhr2 in sex determination in Spotted knifejaw, we employed RNA interference in gonadal tissue and overexpression techniques in the fish body to explore the function of amhr2. For the RNA interference experiment in gonadal tissue, three females (1 year, 537 ± 29 g) and three males (1 year, 573 ± 43 g) were used. The fish were anesthetized in water containing 120 mg/L MS-222, and their gonads were dissected for subsequent cell culture to perform the knockdown experiment after cell transfection. The overexpression experiment in the whole body of Spotted knifejaw was conducted at Yantai Laizhou Mingbo Aquatic Products Co., Ltd. Spotted knifejaw individuals at 55 dph were selected and divided into a control group and an experimental group, with 100 fish in each group. They were reared in a 120 L tank with flowing water. Plasmid injection was performed from 60 to 120 dph.

Immunohistochemistry of vasa

The tissue samples were prepared by initially transferring the fixed ovaries and testes from Spotted knifejaw into 70% alcohol for preservation, followed by dehydration using an ethanol gradient (70%−80%−90%−95%−100%). Subsequently, xylene was used to clear the samples and facilitate their transition to paraffin. Following embedding in paraffin, the samples were sectioned to a thickness of 5 μm. These sections underwent both Immunohistochemistry experiments and haematoxylin–eosin (H&E) staining.

Vasa is a well-established marker of primordial germ cells [56, 57]. Due to the limited number and unclear morphological structure of germ cells during Spotted knifejaw gonadal differentiation, it was essential to identify the characteristics of gonadal tissues and localize the positions of primordial germ cells. In this experiment, fluorescence immunohistochemistry was performed on gonadal tissues. The sections were blocked in normal goat serum, treated with anti-Vasa antibody (diluted 1:100) for 1 h, and then subjected to secondary antibody treatment with a 1:50 dilution of Cy3-conjugated goat anti-rabbit IgG antibody (BBI Life Science Corporation, HK). The slices were incubated with a DAPI staining solution for 5 min, washed, and observed under fluorescence microscope.

Hormone quantification in spotted knifejaw

Hormone levels in the serum and gonads of Spotted knifejaw during early development were measured using a quantitative assay kit for 11-ketotestosterone (11-KT) and estradiol (E₂) (Sangon, Jiangsu). The kits employed a competitive enzyme-linked immunosorbent assay (ELISA) with pre-coated antibodies against fish 11-ketotestosterone or estradiol (solid-phase antibodies) in microtiter plates. Calibration standards and samples were added to the wells, followed by the addition of HRP-labeled target antigens (enzyme-labeled antigens). After incubation and thorough washing to remove the unbound components, an immune complex of solid-phase antibody and enzyme-labeled antigen was formed on the surface of the microplate. Substrate A (0.01% hydrogen peroxide) and substrate B (0.1% 3,3',5,5'-tetramethylbenzidine, TMB) were added, and under the catalysis of horseradish peroxidase (HRP), a blue product was formed, which was then converted to yellow by the addition of the stopping solution. The absorbance (OD value) was measured at a wavelength of 450 nm using an enzyme immunoassay analyzer. The absorbance (OD value) was inversely proportional to the concentration of 11-ketotestosterone and estradiol in the samples. By fitting the calibration curve, the concentrations of 11-ketotestosterone and estradiol in the samples were calculated.

DNA preparation, RNA extraction, and cDNA synthesis

Muscle tissue for DNA extraction was carefully dissected from each individual. DNA extraction was performed using the TIANamp Marine Animals DNA Kit. For total RNA extraction, TRIzol Reagent (Invitrogen, USA) was utilized. The quality and concentration of the extracted RNA were assessed using an Ultramicro spectrophotometer (NanoDrop2000, USA). Complementary DNA (cDNA) synthesis was achieved using the Evo M-MLV RT Kit (Accurate Biology, Changsha, China). The resulting cDNA was stored at −20 °C and prepared for expression profiling.

Quantitative real-time PCR analysis

Quantitative real-time PCR (qRT‒PCR) was utilized to quantify the mRNA levels of sex-related genes, candidate genes for sex determination, and amhr2 transcript variants across various tissues and developmental stages of the Spotted knifejaw. The CFX96™ real-time detection system (Bio-Rad, USA) was employed for qRT‒PCR analysis. Primer sequences, detailed in Table S1, were designed using the NCBI Primer-BLAST tool (http://www.ncbi.nlm.nih.gov/tools/primer-blast). The qPCR reactions were carried out in a 50 μL PCR mixture consisting of 10 μL SYBR Green, 0.4 μL each of forward and reverse primers, 7.2 μL sterilized ddH₂O, and 2 μL cDNA. PCR conditions included an initial denaturation step at 95 °C for 30 s, followed by 39 cycles of denaturation at 95 °C for 5 s and annealing/extension at 59 °C for 30 s. Relative mRNA expression levels were determined using Pfaffl’s method [58, 59].

Transcriptome Sequencing and Analysis

Transcriptome analysis was performed on the gonads of female and male Spotted knifejaw individuals to identify genes associated with sex differentiation. The integrity of total RNA extracted from each sample was validated using a photometer (Thermo Scientific, USA). Only RNA samples meeting quality standards were prepared for library construction and subsequent RNA sequencing, conducted on Illumina HiSeq X Ten platforms. Fold change calculations based on FPKM values were utilized to discern sex-related genes with differential expression in male and female gonads. Additionally, genes known for their crucial roles in sex regulation were considered (Fig. 1G).

Whole-genome resequencing and sequence analysis

Total genomic DNA was extracted from samples and at least 3 µg of genomic DNA was used to construct paired-end libraries with an insert size of 300–400 bp using the Paired-End DNA Sample Prep kit (Illumina Inc., San Diego, CA, USA). These libraries were sequenced using NovaSeq 6000(Illumina Inc., San Diego, CA, USA) NGS platform at Genedenovo (Guangzhou, China). Quality trimming is an essential step to ensure high confidence in variant calling. Raw reads were processed to get high-quality clean reads according to four stringent filtering criteria: 1) removing reads with ≥ 10% unidentified nucleotides (N); 2) removing reads with > 50% bases having phred quality scores of ≤ 20; 3) removal of reads aligned to the barcode adapter.

To identify SNPs and InDels, the Burrows-Wheeler Aligner (BWA) was used to align the clean reads from each sample against the reference genome with the settings ‘mem 4 -k 32 -M’, − k is the minimum seed length, and − M is an option used to mark shorter split alignment hits as secondary alignments. Variant calling was performed for all samples using GATK’s Unified Genotyper. SNPs and InDels were filtered using GATK’s Variant Filtration with the following standards (-Window 4, -filter "QD < 2.0 || FS > 60.0 || MQ < 40.0 ", -G_filter "GQ < 20") and those exhibiting segregation distortion or sequencing errors were discarded. In order to determine the physical positions of each SNP, the software tool ANNOVAR, was used to align and annotate SNPs or InDels. Structural variant (SV) types, including translocations, inversions, and insertions, were identified using BreakDancer (Max1.1.2). Copy number variants (CNV) were classified by CNVnator (0.3.2). To evaluate the linkage disequilibrium (LD) pattern, we estimated the squared allele frequency correlation (r²) using Haploview 4.2 with the following settings: -maxdistance 1000 -dprime -minGeno 0.6 -minMAF 0.05 -hwcutoff 0. The LD decay graphs were plotted using R script [60].

Genome-Wide Association Mapping and Statistical Analysis Approaches

Genome-wide association mapping was performed using GCTA v1.92.2 [61], employing the following models: The simple linear model (LM or GLM). A general linear model that incorporates population structure as a fixed effect (GLM(Q)). Mixed linear model that incorporates kinship as a random effect (MLM(K)). A mixed linear model that considers both population structure and kinship as fixed and random effects, respectively (MLM(Q + K)). Population structure was adjusted using either the first five principal components or the ancestry components matrix corresponding to the best K value from ADMIXTURE results. Kinship was accounted for using the kinship matrix generated by the GCTA software.

To address the challenges posed by rare variants and highly unbalanced case–control ratios, we implemented the REGENIE and SAIGE algorithms. REGENIE used a block partitioning approach for SNPs, generating ridge regression predictions for each block. These predictions were then combined using a second ridge regression to produce a single predictor. This combined predictor was further decomposed into 23 chromosome-specific predictors for Genome-Wide Association Studies (GWAS) [62,63,64]. In contrast, SAIGE employed a saddle point approximation (SPA) [65] to the null distribution of the test statistic to derive accurate p-values [66]. In an effort to enhance the power of association tests, we selected variants based on gene regions or employed other methods for generating variant sets. We then utilized the GMMAT [67] software for gene/set-based association tests, which encompasses three tests: the burden test, the Sequence Kernel Association Test (SKAT) [68], and the Optimal Sequence Kernel Association Test (SKAT-O) [69]. The burden test collapses or summarizes variants within a set into a single value, which is subsequently tested for association with the trait of interest. SKAT applies a multiple regression model to regress the phenotype on genetic variants within a variant set and on covariates, allowing for different variants to have varying directions and magnitudes of effects. SKAT-O optimizes the weight in the burden test and SKAT statistics to maximize power, using the equation TSKAT − O = (1 − ρ)SKAT + ρTburden, where ρ is a parameter, and a grid search over ρ is conducted to determine the minimum p-value.

The significance threshold for Bonferroni correction was set at either a p-value of 0.01 divided by the number of markers or p-value = 0.05 divided by the number of markers, depending on the specific analysis. This threshold was used to identify associations considered statistically significant. Candidate genes (CAGs) located within a 50 kb region upstream or downstream of the markers showing significant associations were subsequently identified for further investigation.

Parameters calculation for Selective sweep

Regions undergoing selective sweeps were identified through the intersection of four distinct parameters: Nucleotide Diversity (π) [70], Theta Watterson (θW) [71], Tajima's D [71], π ratio (πA/πB) [72], Pairwise Fixation Index (Fst) [73], Fu and Li’s F* [74], and Fu and Li’s D* [74]. These parameters were computed using a 100kb sliding window approach with a step size of 10 kb, implemented through the PopGenome tool [75]. Graphical representations of the data were generated using R scripts. Candidate genes located within the selective sweep regions were then identified and extracted for further analysis.

Linkage disequilibrium (LD) analysis

The analysis of LD pertaining to sex-associated scaffolds involved the computation of pairwise r ² values among SNPs identified through whole-genome resequencing, specifically focusing on SNPs with minor allele frequencies exceeding 20%. These calculations were visualized using the LD heatmap R package. For evaluating associations with sex-associated scaffolds, association tests were performed using PLINK v1.9, applying Fisher’s exact test under the allelic model. To establish scaffold-wide significance, a Bonferroni correction was applied, with the significance threshold set at p < 0.05.

Integrated multi-omics for sex determining genes screening

Whole-genome association studies can identify genes with SNPs significantly associated with sexual dimorphism in the genome. Selection scans can pinpoint loci that have experienced continuous positive selection over evolutionary periods through multi-model segmented screening approaches. Additionally, gonadal transcriptomics can reveal genes with sex-specific expression patterns. By integrating gene sets derived from the resequencing of the Spotted knifejaw genome, along with those from whole-genome association analysis, selection scans, and gonadal transcriptomics, the overlap among these datasets can be visualized via a Venn diagram. This approach aids in identifying target genes that are both sexually dimorphic and subject to positive selection, providing a valuable resource for the subsequent screening and characterization of sex-determining genes.

Identification of amhr2 and its transcript variants

To examine the sex-determining gene amhr2 and the flow of genetic information from its genomic sequence to mRNA transcripts and amino acid sequences, we conducted genome resequencing to obtain the complete genomic sequence of amhr2, including both introns and exons. We employed PacBio CCS high-throughput sequencing and Hi-C chromosome assembly, leveraging full-length transcriptomes and third-generation sequencing technology, to derive the full-length mRNA sequence and alternative splicing patterns of amhr2. TBtools was used to extract genomic and exon sequences from the genomic data and SnapGene software facilitated the comparison of these sequences of various transcripts and intron–exon information. By integrating the complete genomic sequence, intron–exon data, alternative splicing patterns, and amino acid sequences of Amhr2, schematic representations were generated using the online plotting tool Figdraw (WTYIUd2d77, STUTY46884, RAWWYa2ff2).

To discern the differences in amhr2 transcript sequences between male and female Spotted knifejaw, we performed multiple sequence alignment of the Amhr2 amino acid sequences using ClustalW (https://www.genome.jp/tools-bin/clustalw). ESPript 3 (https://espript.ibcp.fr/ESPript/ESPript/) was used to visualize the alignment results, and SMART (http://smart.embl-heidelberg.de/) was employed to predict the protein domain positions. A schematic diagram was then created using the online tool Figdraw (SPRPR4bb41).

Phylogenetic and protein interaction analysis of amhr2

To explore the phylogenetic relationships of Amhr2 across various species, we analyzed 25 Amhr2 sequences, including that of the Spotted knifejaw. Phylogenetic inference was performed using the Neighbor-Joining (NJ) method. The accession numbers for the genes and proteins are provided in Supplementary Table S2. Sequence alignment was performed with MUSCLE through TBtools, and the phylogenetic tree was generated with MEGA 11 based on maximum likelihood (ML) analysis. The robustness of the tree nodes was evaluated with 1000 bootstrap replicates. The analysis of protein interactions related to Amhr2 was conducted using the online platform GeneMANIA (https://genemania.org/).

In Situ Hybridization

Synthetic RNA probes were essential for the in situ hybridization experiments. To produce these probes, specific PCR primers (Table S1) were used to amplify fragments of the target genes. The amplified fragments were cloned into the pGEM-19T Easy vector, linearized with restriction enzymes, and then used to synthesize and label sense and antisense RNA probes using the DIG RNA Labeling Kit (Roche, Germany). After purification with Sigma Spin™ columns (Sigma-Aldrich, USA), the probes were stored at −80°C. For tissue preparation, fixed ovaries and testes from Spotted knifejaw were dehydrated through an ethanol gradient, cleared with xylene, and embedded in paraffin. The tissue samples were then sectioned to 5 μm thickness and processed for in situ hybridization and hematoxylin–eosin staining.

Molecular docking of Amh/Amhr2

To analyze the structure of Amh/Amhr2 proteins and the binding efficiency between ligands and receptors, and to further elucidate the mechanism of sex determination mediated by amhr2, this study retrieved the amino acid sequences of Amh/Amhr2 using TBtools. Based on these sequences, the tertiary structures of the proteins were predicted through homology modeling using SWISS-MODEL (https://swissmodel.expasy.org/). The constructed tertiary structure models were evaluated with two parameters: GMQE (Global Model Quality Estimation) and QMEAN (Qualitative Model Energy Analysis). GMQE reflects the expected accuracy of the model aligned with the template and the coverage of the target, with higher numerical values indicating better quality. A QMEAN score close to 0 indicates good agreement between the model structure and the structures of 3D models of similar size. Considering both parameters, the model with the highest GMQE value and the closest QMEAN score to 0 was chosen as the target tertiary structure model for subsequent analysis. Protein molecular docking was performed using the online tool ClusPro 2.0 (https://cluspro.org/help.php), and the binding energy of the docking models was evaluated using a coefficient-weighted formula.

$$\text{E}=0.40\text{Erep}+-0.40\text{Eatt}+600\text{Eelec}+1.00\text{EDARS}$$

The lower the binding energy of Amh/Amhr2 proteins, the more readily the ligand-receptor complex can form, indicating higher binding efficiency. The top 10 models with the highest statistical weight coefficient scores and the lowest center binding energies were analyzed, along with information on the lowest binding energies, to evaluate the binding efficiency of Amh ligands and Amhr2 receptors translated from different transcripts.

RNAi and cell culture

The synthesis of double-stranded RNA (dsRNA) targeting amhr2 was conducted in vitro using the TranscriptAid T7 High Yield Transcription Kit (Thermo Scientific), following the manufacturer’s protocols. Gene-specific amplification primers for amhr2 were designed with the assistance of SnapDragon-dsRNA Design (http://www.flyrnai.org/cgi-bin/RNAi_find_primers.pl). The synthesis of amhr2-dsRNA involved amplifying gonad cDNA using the Op-ds.amhr2 primers (Table S1). The quality and concentration of the dsRNA were assessed using a Nanodrop 2000 spectrophotometer, while its purity and integrity were evaluated via 1% agarose gel electrophoresis. The dsRNA product was stored at −80 °C.

Three male and three female Spotted knifejaws were anesthetized in water containing 120 mg/L MS-222. Their gonads were then carefully extracted and washed with a 1 × PBS solution containing penicillin–streptomycin-amphotericin B (Solarbio). To observe the expression changes of sex-related genes following amhr2 knockdown, transfection was performed using Entranster™ R4000 in a 12-well cell culture medium plate. The concentration of dsRNA utilized for transfection was maintained at 40 μg/mL. Cell samples were collected at 12 h, 24 h, 48 h, 72 h, and 96 h intervals. qPCR analysis was employed to validate the knockdown results of amhr2 and to identify the expression of genes related to sex-regulatory pathways. A presumed sex regulatory network was constructed using Figdraw (OPUPY5c5c2).

Overexpression experiment

To investigate the function of amhr2 in Spotted knifejaw, this study achieved Overexpression of the gene through in vivo injection of a recombinant amhr2 plasmid. The coding sequence (CDS) of the amhr2 gene was retrieved using TBtools. Target gene primers for the recombinant plasmid were designed with SnapGene software. Since direct amplification of the amhr2 CDS could not completely amplify the amhr2 sequence, the gene was divided into three segments, and specific primers were designed for each segment. The recombinant plasmid was then subsequently constructed using the OK Clon DNA Ligation Kit II.

In the overexpression study of the amhr2 recombinant plasmid in Spotted knifejaw, A dosage of 5 μg/g of plasmid was administered via injection every 7 days. Sampling was conducted at 60 dph, 90 dph, 100 dph, and 120 dph. Muscle tissue was collected for genetic sex determination, while gonadal tissue was used to analyze the expression of sex-related genes after amhr2 overexpression, as well as for western blotting to validate the results. The sex determination pathway was depicted using the Figdraw (OPUPY5c5c2).

Western blot (WB) analysis

To validate the expression of Amhr2 protein in E. coli transformed with the recombinant plasmid and in the ovaries of Spotted knifejaw after in vivo injection in the amhr2 overexpression experiment, Amhr2 was detected using an anti-6 × His antibody (Sangon) due to the histidine tag present in the eukaryotic expression plasmid pcDNA 4/His A (Thermo Fisher). Total proteins were extracted from the ovaries in the amhr2 overexpression experiment and from E. coli transformed with the recombinant plasmid using animal and bacterial total protein extraction kits (Sangon), respectively. Protein sample concentrations were determined with Nanodrop2000, and samples were loaded onto a 12% SDS-PAGE gel. Proteins were then transferred to PVDF membranes using a blotting apparatus, blocked with 5% non-fat milk, and incubated overnight with the 6 × His antibody (Sangon) and β-actin antibody (Sangon). Protein-antibody complexes were visualized using West Pico chemiluminescent substrate.

Molecular sex marker based on amhr2 gene

Genome scanning of Spotted knifejaw was conducted using TBtools software to extract the amhr2 genomic sequences. The extracted amhr2 sequences were then aligned using SnapGene, and primers were designed. To validate the sex molecular markers, six male and six female Spotted knifejaw were selected for testing.

Statistical analysis

The relative expression and hormone levels (mean ± standard deviation (SD)) were analyzed utilizing one-way ANOVA and T-test in GraphPad Prism 9.0.0 software.

Results

Gonadal development and sex differentiation in the spotted knifejaw

Primordial germ cells (PGCs) migrate to the genital ridge in the Spotted knifejaw at 26 dph (Fig. 1Aa-Ac). The primordial gonads are characterized by a pear-shaped, flattened and elongated at the ends, and are suspended from the peritoneal epithelium below the bladder and above the intestines by mesentery (Fig. 1Ae). The PGCs appear rounded with light cytoplasmic staining, large nuclei, and strong basophilia. By 60 dph, increase in volume and with an increase in cell numbers of germ cells in the Spotted knifejaw (Fig. 1Ad). At 100 dph, interstitial cells are dispersed throughout the testes, with rise in cell numbers, and the primordium of the efferent duct of the spermatic duct also appears, marking the onset of anatomical differentiation in the testes (Fig. 1Ae). From 120 to 150 dph, both testicular volume and cell count continue to rise, efferent ducts are formed in the testes of the Spotted knifejaw, with a small number of secondary spermatocytes (Ssp) present by 180 dph (Fig. 1Af-Ah). In the ovaries of the Spotted knifejaw, the formation of the ovarian cavity (Oc) is observed at 90 dph, signifying the completion of ovarian differentiation (Fig. 1Ai). By 150 dph, cytological differentiation is evident, marked by the presence of primary spermatocytes (Psp) (Fig. 1Ag).

In adult Spotted knifejaw, the testes are elongated and located posterior to the kidneys, suspended from the peritoneal epithelium below the bladder and above the intestines by a mesentery. The interior of the testes exhibits a radial structure, with the outer margin of the testes composed of efferent duct (Ed) that converge towards the central efferent duct (Cd) (Fig. 1Am). The branching blind tubules are known as lobule cavities, extend to envelop the regions known as seminiferous lobules. PGCs are primarily located at the outer edge of the testes, with differentiation increasing as they approach the central efferent duct. Seminiferous lobules are composed of testicular interstitial tissue, germ cells, and capillaries. Primordial germ cells include type A spermatogonia (Aund)(ASg) and type B spermatogonia (BSg) cells (Fig. 1An-Ao). The adult gonad shown in the figure represents a stage III testis, predominantly composed of BSg cells, with primary and secondary spermatocytes being the main types of germ cells in the testes. The adult ovary is pale yellow and symmetrical on both sides, consisting of ovarian tissue enclosed by the ovarian wall (Ow) (Fig. 1Ap). A large red blood vessel is visible on the dorsal side of the ovary, a key anatomical feature indicative of ovarian differentiation. The germ cells, somatic cells of the ovary, and vascular tissue form the ovigerous lamellae (Ol) closely attached to the Ow (Fig. 1Aq). The center of the ovary forms the ovarian cavity, which serves as a significant marker of ovarian differentiation. The ovary depicted in the figure is in the late stage II or early stage III, with the germ cells consisting of oogonia and early perinucleolus stage spermatocyte.

Due to the limited number of germ cells and unclear morphological structures during the differentiation of the Spotted knifejaw, it was essential to determine the characterize the gonadal tissue and identify the location of the primordial germ cells. In this study, the zebrafish Vasa antibody was used to conduct fluorescence immunohistochemistry on the gonadal tissue (Fig. 1B). The experimental results revealed that in the testes at 120 dph, the germ cells marked by the Vasa signal were located at the outer edge of the testes, while the interior was predominantly composed of testicular somatic cells (Fig. 1Bb-d). In the ovaries at 120 dph, the germ cells indicated by the vasa signal were located on the inner side of the ovarian wall, adjacent to the ovarian cavity (Fig. 1Bf-Bh).

In this study, the enzyme-linked immunosorbent Assay (ELISA) method was used to quantify the concentrations of two key hormones involved in fish sex differentiation: the androgen 11-ketotestosterone (11-KT) and the 17β-estradiol (E₂) (Fig. 1C). The absorbance of the detection reaction was fitted to a standard curve based on the kit standards, as shown in Fig. 1C. The determination coefficients R² for E₂ and 11-KT were 0.9917 and 0.9929, respectively, both greater than 0.98, confirming the reliability of the curves for hormone quantification. In the early development of the Spotted knifejaw, the concentration of E₂ was low from 10 to 60 dph with significant differences in sex-specific concentrations emerging at 70 dph. From 70 to 120 dph, the concentration continued to rise, with females exhibiting significantly higher concentrations than males. The concentration of 11-KT in the early development of the Spotted knifejaw was low from 10 to 60 dph. The significant sex differences emerging at 60 dph. Androgen concentrations continued to rise from 60 to 120 dph, with male Spotted knifejaw having significantly higher 11-KT concentrations than females. These findings suggest that estrogen-mediated sex differentiation occurs at approximately 70 dph, while androgen-mediated differentiation initiates earlier, at around 60 dph (Fig. 1C).

To investigate the tissue specificity and early developmental expression characteristics of sex-related genes in the Spotted knifejaw, as well as the molecular stages of sex differentiation, an analysis was conducted based on the gonadal transcriptome and traditional sex differentiation pathways. Key female differentiation-related genes (foxl2, cyp19a, cyp19b) and male sex differentiation-related genes (dmrt1, sox9a, sox9b, amh, amhr2, hsd11b2) were examined for tissue specificity and early developmental expression profiles (Figure S1). The expression levels of female differentiation-related genes were significantly higher in the ovaries than in the testes. Foxl2 was expressed at lower levels in the intestine and gills, cyp19a was specifically expressed in the ovaries and at lower levels in the intestine, and cyp19b was expressed in the ovaries and brain, with significantly higher expression in the brain than in the ovaries. Male sex differentiation-related genes (dmrt1, sox9a, sox9b, amh, amhr2, hsd11b2) were significantly more expressed in the testes than in the ovaries. Dmrt1, amh, and amhr2 were primarily expressed in the gonads, with dmrt1 also highly expressed in the stomach and at lower levels in the gills and other tissues. Sox9a, sox9b, and hsd11b2 all showed a widespread expression pattern. In addition to the gonads, Sox9a was highly expressed in the intestine, gills, eyes, and liver; Sox9b was highly expressed in the brain, intestine, skin, and eyes; and hsd11b2 was highly expressed in the liver, intestine, and gills. These findings suggest that sox9a, sox9b, and hsd11b2 may have additional biological roles beyond their involvement in sex differentiation (Figure S1).

Based on the early developmental expression patterns of sex differentiation-related genes, the key period of molecular-level sex differentiation was identified (Fig. 1D). Among male sex-related genes, sox9b and sox9a exhibited the earliest sex-specific expression differences at 46 dph, while dmrt1, amh, amhr2, and hsd11b2 showed these differences at 36 dph, with significantly higher expression levels in testes than in ovaries. For female sex-related genes, foxl2 exhibited sex-specific expression differences at 46 dph, cyp19a at 36 dph, and cyp19b, which displayed lower overall expression levels, at 21 dph. The expression levels in ovaries were significantly higher than those in testes. Following the emergence of sex-specific differences, the expression levels of these genes continued to increase. These findings indicate that molecular-level sex differentiation in male Spotted knifejaw begins around 35 dph.

Genomic sex-specific clustering and variation in spotted knifejaw

Systematic phylogenetic analysis and population principal component analysis (PCA) were conducted on Spotted knifejaw samples to investigate the clustering patterns of males and females. The results demonstrated that the female Spotted knifejaw samples formed distinct branches within the phylogenetic tree. In the PCA, female samples clustered in one principal component, whereas the male samples formed a separate group. These findings suggest a clear genetic differentiation between the sexes, providing a basis for subsequent selection elimination and whole-genome association analyses.

The analysis of base quality for genome resequencing revealed a high abundance of reads, with most HQ-clean reads exhibiting an abundance greater than 6e + 07, and the reads ratio approaching 100%. The total number of variants including 5,907,637, with 5,274,809 single nucleotide polymorphism (SNP) sites and 632,828 insertion-deletion (InDel) markers. Regarding the positional distribution of SNP-InDel sites obtained from Spotted knifejaw resequencing, the number of sites in intronic regions was 3,232,356, while those located between genes included 1,928,496 sites. Additionally, there were 246,765 sites in exonic regions, 178,334 sites within 1 kb upstream of transcription start sites, and 170,131 sites within 1 kb downstream of transcription termination sites. Furthermore, there were 96,103 sites in the 3' untranslated region (UTR) and 31,101 sites in the 5' UTR, with 1,526 SNP-InDel sites located within 2 bp of variable splicing sites (Table 1).

Table 1 Information of resequencing analysis

Full size table

The Fst parameter (genetic differentiation index) of population evolutionary selective sweep analysis focuses primarily on the degree of genotype differentiation. The Fst values indicate that Chromosome 1 of the male and Chromosome 3 of the female Spotted knifejaw genomes are under significant positive selection pressure. Fst index of 5%, a total of 1,691 genes were identified (Fig. 2A).

Functional annotation of the SNP-InDel variants obtained from the resequencing of the Spotted knifejaw revealed that "revealed 129,054 synonymous mutations and 109,534 non-synonymous mutations. Of the non-synonymous mutations, 2,104 sites exhibited deletion mutations that were in multiples of three nucleotides, leading to the elimination of termination codons. Furthermore, 1,813 translation stop codons were induced by non-synonymous mutations, with 896 frameshift mutations caused by insertion events. Additionally, 292 sites where non-synonymous single nucleotide insertions, deletions, or substitutions removed termination codons, were identified, enabling translation to proceed. In terms of SNP transition and transversion types, transitions (mutations within the same nucleotide type) were the most prevalent, totaling 3,500,322, while transversions constituted 1,774,487 mutations. SNPs were mainly located on the heteromorphic chromosome (Chromosome 1), while the number of SNP sites on autosomes remained stable. The green lines represent INS (insertion-type structural variations), and the blue lines represent INV (inversion-type structural variations), both densely distributed on Chromosome 1 (Fig. 2B).

GWAS and selective sweep reveal sex-related genes in spotted knifejaw

GWAS filters out loci with rare alleles, high missing rates, and high heterozygosity rates, as these can cause anomalies in population and genome-wide association analyses, potentially leading to incorrect results. GWAS leverages the linkage disequilibrium between markers and functional genes, uncovering genes associated with trait variations. Manhattan and QQ plots for simple linear models (GLM with 'n'), generalized linear models (GLM(Q) with 'q'), mixed linear models (MLM(K) with 'k'), and mixed linear models (MLM(QK) with 'qk') are presented. The results indicate that under the q, n, k, and qk models, the QQ plot deviates upward from the reference line, suggesting a strong correlation between the genomic GWAS data and the sex of Spotted knifejaw (Figure S2). The Manhattan plot shows that SNPs associated with sex are predominantly located on Chromosome 1. Given the strong significance of SNP loci under the q model, the analysis primarily relies on linkage disequilibrium analysis of target genes under the q model. GWAS (Q model) identified 3,392 sex-related genes (Fig. 2C).

We identified four target genes on the fused chromosome (Y) of Spotted knifejaw based on selective sweep analysis, GWAS of sex-related SNP loci, differential expression of sex organs from the gonadal transcriptome, and early-expressed sex-related genes from genomic analysis. The multi-omics analysis revealed 212 sex-specific genes, 42 of which overlapped with SNP loci from GWAS, and 51 with loci from selective sweep analysis. Among loci with a significant Fst index of 5%, 1,691 genes were identified, while the GWAS (Q model) identified 3,392 sex-related genes, with 1,644 overlapping. Four sex-specific genes were located on Chromosome 1, with gdf11, aldh1l2, wnt4, and amhr2 positioned on Chromosome 1 in males and Chromosome 3 in females. gdf11, wnt4, and amhr2 were significantly associated with sex in the GWAS Q model, while all four genes were under positive selection in the selective sweep Fst model (Fig. 2A, Fig. 2C).

To explore the candidate genes for sex determination in Spotted knifejaw, we used linkage disequilibrium analysis to screen genes on scaffolds. The scaffolds containing the gdf11, wnt4, and amhr2 genes exhibited dense linkage disequilibrium blocks, indicating a strong association with sex, while the scaffold containing the aldh1l2 showed no significant loci (Fig. 2F).

Expression patterns of candidate sex-determining genes

To screen candidate sex-determining genes for functional validation, we examined the early developmental patterns and tissue specificity of genes (amhr2, aldh1l2, wnt4, gdf11) identified through multi-omics integration. Amhr2 showed specific expression in the gonads, with significantly higher expression in testes than ovaries. Aldh1l2 exhibited widespread expression, particularly in the heart, intestines, liver, kidneys, and brain. Wnt4 was predominantly expressed in the ovaries, with higher levels in the gills and eyes. Gdf11 had low gonadal expression but was more highly expressed in the eyes, brain, and intestines. During early development, amhr2, aldh1l2, and gdf11 were more highly expressed in male testes than female ovaries. Amhr2 showed the earliest sex difference in expression at 35 dph, with a significant increase in males during the critical period of sex differentiation. Aldh1l2 exhibited a significant difference at 90 dph, with continuous increases after 120 dph. Wnt4 expression was higher in females, with significant differences appearing after 120 dph. Gdf11 expression was higher in males, with significant differences observed at 45 dph. In conclusion, amhr2, with specific gonadal expression and the earliest sex difference, is a promising candidate sex-determining gene for further validation (Fig. 2D).

Transcriptome analysis reveals sex-specific gene expression in spotted knifejaw gonads

Upon transcriptome analysis of the adult Spotted knifejaw gonads, 216 genes exhibited sex-specific differential expression. Subsequent functional filtering reduced this list to 39 genes with known associations to sexual development. These genes were grouped based on their expression levels, measured by FPKM values (Fig. 2G). Notably, the expression of several genes, including cbx1, gatad2a, stard9, aldh3a2, dmrt1, cyp27a1, rsph1, sox30, cyp8b1, rsph6a, lhx5, gdf11, emx1, fgf1, aldh1a3, pou6f2, gatad2a, cyp11b, and sox9, was significantly higher in the testis than the ovary. In contrast, the expression of lhx8, hsd17b8, pou5fi, figla, gdf9, hsd17b1,bmp15, cyp26a1, wnt9b, foxl2, sox19b, hsd17b7, bmpr1b, cyp19a1, sox3, irf5 was significantly elevated in the ovary compared to the testis.

Sex-specific variants of the amhr2 Gene in Spotted knifejaw

The full-length male genomic sequence of amhr2 spans 9739 bp on heterochromosome 1 and consists of 13 exons (E1-E13), with lengths of 36, 191, 164, 101, 103, 221, 114, 157, 156, 136, 97, 82, and 10 bp. The female sequence is 9,726 bp, located on autosome 3, and also includes 13 exons. Notably, exon 11 (E11) is 67 bp in the female sequence, showing a 67.4% sequence similarity with the male. In males, E11 of amhr2 contains a 68–97 bp insertion, leading to variable sequence similarities across the exons: 94.6%, 97.9%, 97.9%, 99%, 98.1%, 96.4%, 98.3%, 96.8%, 96.8%, 92.7%, 90.36%, and 100% (Fig. 3A).

Based on the full-length genomic sequences of male and female Spotted knifejaw and the multiple sequence alignment of individual exons, we analyzed the DNA-to-mRNA transmission patterns of the amhr2 gene. In males, the amhr2 produces three transcript variants: amhr2ay, amhr2by, and amhr2cy. The amhr2ay variant, 1506 bp in length, results from splicing that removes introns and excludes exons E12 and E13, including only exons E1-E11. The amhr2by variant, 1269 bp long, is produced through splicing that eliminates introns and exons E1, E2, E12, and E13, encompassing exons E3-E11. The amhr2cy variant, 1173 bp, is generated by splicing that removes introns and exons E1, E2, E3, E12, and E13, leaving only exons E4-E11. In contrast, the amhr2 gene of the female Spotted knifejaw generates a single transcript variant, amhr2a, which shares the same length and splicing pattern as amhr2ay, excluding exons E12 and E13 and including exons E1-E11 (Fig. 3D).

Identification of sex-specific marker for amhr2 in Spotted knifejaw

Alignment analysis of the male and female Spotted knifejaw amhr2 gene sequences revealed a 127 bp deletion at the end of the male sequence. Based on the characteristic position of this deletion sequence, primers were designed to obtain sex-specific markers for male and female Spotted knifejaw (Figure S6). Gel electrophoresis validation of the sex-specific markers showed that both female and male Spotted knifejaw exhibited a longer band of 317 bp, while males displayed an additional specific band of 190 bp. The primers amplified the amhr2 gene on the X chromosome in females and on both the X and Y chromosomes in males. Female Spotted knifejaw only possesses one copy of the amhr2 gene located on the X chromosome. Therefore, the sex molecular marker validation in male Spotted knifejaw resulted in two bands, whereas female Spotted knifejaw showed only one band on electrophoresis. This gene electrophoresis result is consistent with the chromosome type X₁X₁X₂X₂/X₁X₂Y in Spotted knifejaw (Fig. 3B).

Phylogenetic evolution of amhr2 in spotted knifejaw

To investigate the phylogenetic evolution of the amhr2 gene across various species, this research selected 25 Amhr2 amino acid sequences, including those from the Spotted knifejaw, and constructed a phylogenetic tree using the neighbor-joining (NJ) method. The Amhr2 in the Spotted knifejaw demonstrated significant diversity, comprising four distinct transcript variant sequences. Species in which amhr2 functions as a sex-determining factor exhibited closer evolutionary relationships. For example, species including Plecoglossus altivelis (ayu), Hippocampus comes (sea dragon), Takifugu rubripes, Takifugu obscurus and Perca flavescens (yellow perch), which use amhr2 for sex determination, were grouped in a major evolutionary branch. In contrast, species like Homo sapiens (humans), Chelonia mydas, and various representatives of primates, reptiles, and amphibians, whose Amhr2 sequences are more evolutionarily distant from those of fish, formed a separate branch (Fig. 3C).

Transcript-specific expression of amhr2 variants in spotted knifejaw

Given the substantial homology between the mRNA transcript variants of amhr2 in the two sexes, primers were designed based on the multiple sequence alignments (Figure S8). These primers facilitated the analysis of the functionality of each transcript variant. Primer P1 targeted the amplification of amhr2a variants in females and amhr2ay in male Spotted knifejaw. Primer P2 was designed for the amplification of amhr2a in females and amhr2ay, amhr2by, and amhr2cy in males. Primer P3 specifically amplified the amhr2cy variant in male individuals, while Primer P4 targeted the amhr2a variant in females and amhr2ay and amhr2by in males. The tissue-specific expression patterns and early developmental stages are depicted in Fig. 4A.

Amhr2a shows specific expression in the testes of male Spotted knifejaw, with very low expression levels in other tissues. Sexual dimorphism in amhr2a expression is observed at 35 dph, increase after 90 dph, peaking at 360 dph. Amhr2ay/amhr2by/amhr2cy collectively show specific expression in the testes.. The amhr2ay/amhr2by/amhr2cy variants show specific expression in the testes, with sexual dimorphism appearing earlier, as early as 5 dph. Expression levels increase rapidly after 90 dph, peaking at 360 dph. Amhr2cy shows higher expression levels in the testes of males, and exhibits relatively high expression levels in the kidney, intestine, and heart. Significant sexual dimorphism in amhr2cy expression is observed at 40 dph, with expression levels increasing rapidly after 150 dph, peaking at 360 dph. The overall expression of amhr2ay/amhr2by, showing specific expression in the testes of male Spotted knifejaw. It also exhibits relatively high expression levels in the kidney, intestine, and heart. Sexual dimorphism in the expression of amhr2ay/amhr2by is observed at 40 dph, with expression levels increasing rapidly after 120 dph, peaking at 360 dph.

In situ hybridization of amhr2 in spotted knifejaw gonads

In situ hybridization was performed to examine the distribution of amhr2 genes in the Spotted knifejaw (Fig. 4B). The testes, consisting of seminiferous tubules lined with Sertoli and Leydig cells essential for spermatogenesis, showed positive amhr2 signals mainly in primary (Psp) and secondary (Ssp) spermatocytes, with stronger signals in Psp than Ssp. Weaker signals appeared in the Sertoli cell. No signals were detected in the testes labeled with the sense probe, serving as a control. The ovaries were mostly composed of ovarian follicles with early perinucleolus stage oocytes (stage II) surrounded by follicular cells. No signals were observed for amhr2, or the sense probe controls in the ovaries (Fig. 4B).

Binding efficiency and structural analysis of amhr2 variants

To investigate the binding efficiency of the receptor complexes formed by the male and female Spotted knifejaw Amhr2 transcript variants with Amh, protein models for Amhr2ay, Amhr2by, and Amhr2cy were constructed (Fig. 5A). Through online software analysis, the binding energies of the receptor complexes formed by these three transcript variants with the Amh molecule were assessed (considering the top 10 optimal scoring models), followed by binding efficiency (Fig. 5B). The protein model of Amhr2ay closely resembles that of Amhr2a, while the models for Amhr2by and Amhr2cy primarily differ in the extracellular domain structure. Docking energy scores revealed that Amhr2cy had the lowest binding energies and the highest binding efficiency, as it most easily bound to the Amh model's docking center. In contrast, Amhr2by and Amhr2ay showed higher docking energy scores for their extracellular domains and overall structures, indicating lower binding efficiency compared to Amhr2cy.

Based on the amino acid sequences of the male and female Spotted knifejaw amhr2 transcript variants, we conducted predictive analysis to assess the protein structures of each transcript variant (Fig. 5C). The Amhr2 transcript variants' amino acid sequences consist of a transmembrane region and a STYKc domain (serine/threonine kinase domain). The region from the N-terminus to the transmembrane region is termed the extracellular domain (ECD), while the region from the transmembrane region to the C-terminus is referred to as the intracellular domain. The main differences in amino acid sequences among male Amhr2 transcript variants are observed in the extracellular domain. The amino acid sequence of the female Amhr2a is nearly identical to the male variants, while Amhr2by has a deletion of 79 amino acids at the N-terminus compared to Amhr2ay. The Amhr2cy transcript variant has the shortest amino acid sequence among the variants and exhibits significant differences.

Protein–protein interaction analysis of amhr2

To further understand the function of Amhr2 and its interactions with related proteins, a protein–protein interaction analysis was conducted (Figure S9). The results showed that Amhr2 shares the same protein domains with Acvr1 and Tgfbr1. Amhr2 was predicted to have strong interactions with its ligand Amh, as well as with Fkbp1a and Tgfbr1. Amh and Amhr2 were found to be closely related in terms of regulatory pathways, physical interactions, co-localization, and co-expression. Other proteins such as Bmpr1b, Acvrl1, Gdf5, Ly6g6e, Bmp3, Gdf1, Gdf2, Grm2, Gdf3, Gdf9, Bmp15, Tll2, Gdf10, Gdf11, Mstn, and Tll1 were also identified in the interaction network, suggesting potential roles in sex development (Table S3). Gdf11, located on the heterochromosome, was found to be associated with sex in GWAS analysis. Selective elimination analysis indicated that Gdf11 underwent positive selection during evolution, suggesting its involvement in the regulation of Spotted knifejaw sex development.

Effects of amhr2 gene knockdown in spotted knifejaw

After knocking down the amhr2 gene in the testicular tissues of male Spotted knifejaw, the expression level of amhr2 in the dsRNA-treated group significantly decreased, confirming the effectiveness of the knockdown. The expression levels of male sex differentiation-related genes, including dmrt1, amh, sox9a, and the androgen synthesis rate-limiting gene hsd11b2, were significantly reduced in the dsRNA-treated group compared to the NC-treated group. Conversely, among the female sex differentiation-related genes, the expression levels of wnt4, foxl2, cyp19a, and cyp19b were significantly increased in the dsRNA-treated group compared to the NC-treated group. Similarly, in the ovarian tissues of female Spotted knifejaw, the expression level of amhr2 significantly decreased after amhr2 gene knockdown. Furthermore, in the dsRNA-treated group, the expression levels of male sex differentiation-related genes, including dmrt1, amh, sox9a, sox9b, and the androgen synthesis rate-limiting gene hsd11b2, were significantly reduced compared to the NC-treated group, while the expression levels of female sex differentiation-related genes, such as wnt4, foxl2, cyp19a, and cyp19b, were significantly increased. These results indicate that knocking down the amhr2 gene in male Spotted knifejaw leads to a decrease in the expression of male sex differentiation-related genes and a decrease in the expression of female sex differentiation-related genes (Fig. 6, Figure S10).

Effects of amhr2 gene overexpression in spotted knifejaw

Recombinant plasmids for eukaryotic expression were introduced into E. coli strains, and the targeted amhr2 band, measuring 1554 bp, was successfully verified. Western blot (WB) analyses of the transformed bacterial clones harboring the recombinant plasmids, as well as the overexpressed testes from the Spotted knifejaw, detected protein bands that matched the histidine tag of the recombinant plasmids, indicating efficient amplification and expression of the Amhr2 protein (Fig. 6Bb).

After overexpressing the amhr2 gene in the testicular tissues of male Spotted knifejaw, the expression level of amhr2 in the amhr2 overexpression group significantly increased, indicating the effectiveness of the amhr2 gene overexpression experiment. Among the male sex differentiation-related genes, the expression levels of dmrt1, amh, sox9a, sox9b, as well as the androgen synthesis rate-limiting genes hsd11b2, cyp11a, and cyp11a, were significantly higher in the amhr2 overexpression group compared to the control group. Similarly, among the female sex differentiation-related genes, the expression levels of wnt4, foxl2, cyp19a, and cyp19b were significantly increased in the amhr2 overexpression group compared to the control group. In the ovarian tissues of female Spotted knifejaw after knocking down the amhr2 gene, the expression level of amhr2 significantly increased in the amhr2 overexpression group. Furthermore, in the amhr2 overexpression group, the expression levels of male sex differentiation-related genes, including dmrt1, amh, sox9a, sox9b, as well as the androgen synthesis rate-limiting genes hsd11b2, cyp11a, and cyp11a, were significantly higher compared to the control group. Similarly, among the female sex differentiation-related genes, the expression levels of wnt4, foxl2, cyp19a, and cyp19b were significantly increased in the amhr2 overexpression group compared to the control group. These results suggest that overexpressing the amhr2 gene in male Spotted knifejaw not only elevated the expression of male sex differentiation-related genes but also promoted the expression of female sex differentiation-related genes (Fig. 6Bc).

The sex determination regulatory pathway

Based on the functional validation results in Spotted knifejaw, schematic diagrams were created to illustrate the dose compensation effect of amhr2 (RRTAUfbf2b) (Fig. 7A) and the sex determination regulatory pathway (PTAAP82549) (Fig. 7B). In Spotted knifejaw, Amh, secreted by germ cells, binds to the Amhr2 receptor on the supporting cell membrane of undifferentiated gonads, driving sex differentiation. In males, the amhr2y gene on the Y chromosome of supporting cells promotes the transcription of three mRNA transcripts (amhr2ay, amhr2by, amhr2cy, each encoding a corresponding receptor. Additionally, the copy of the amhr2 gene on the X chromosome promotes the transcription of amhr2a in supporting cells to synthesize the amhr2a receptor. On the other hand, female Spotted knifejaw supporting cells only have the amhr2a transcript due to the absence of the Y chromosome. Therefore, under the dose compensation effect, amhr2 promotes the process of male sex differentiation. The Amh/amhr2 system is positioned upstream in the sex control pathway. Due to the presence of three mRNA transcripts (amhr2ay, amhr2by, amhr2cy) in male Spotted knifejaw amhr2, as opposed to only one transcript (amhr2a) in female Spotted knifejaw, the dose compensation effect greatly enhances the expression of male-related genes (sox9b, dmrt1) in the testes during early Spotted knifejaw development. This subsequently increases the expression of the key gene hsd11b2 involved in androgen synthesis, promoting the conversion of testosterone to 11-KT, thus facilitating the process of male sex differentiation in Spotted knifejaw. Amhr2 also plays a regulatory role in females. Its expression in the early development of Spotted knifejaw can enhance the expression of female-related genes (wnt4, foxl2), which in turn increases the expression of key genes cyp19a/b involved in estrogen synthesis. This promotes the conversion of testosterone and facilitates the process of female sex differentiation in Spotted knifejaw.

Discussion

The critical period of sexual differentiation in spotted knifejaw

The timing of gonadal differentiation in fish species is variable and can be affected by water temperature, age, and photoperiod [77,78,79,80]. The key criteria for sex determination in early developmental and differentiation include the presence of specific gonadal structures, such as the ovarian cavity or efferent ducts, as well as differences in the number of germ cells and the occurrence of meiotic divisions in ovarian cells [81, 82]. The Spotted knifejaw demonstrates a late timing of gonadal differentiation, with the initial formation of gonads at 26 dph. In female Spotted knifejaws, differentiation of the ovarian cavity is apparent by 90 dph, whereas, in males, the efferent duct differentiation is evident at 100 dph, signifying the completion of gonadal maturation. This contrasts with certain fish species, such as smaller fish like medaka and zebrafish, in which sex differentiation occurs during the embryonic stage [83, 84]. In zebrafish, primordial germ cell migration concludes 24 h post-fertilization, and by day 5 days post-fertilization, these cells form primitive gonads. By 8 days post-fertilization, the expression of genes related to sex differentiation is detectable. Anatomical evidence of sex differentiation becomes apparent as early as 30 days post-fertilization, with ovaries exhibiting a significantly greater volume than testes [85, 86]. Many fish species undergo gonadal differentiation between 40 and 90 dph. For instance, in Japanese flounder, ovarian cavity differentiation occurs at 65 dph, while testicular differentiation begins in juveniles at 50 dph [87,88,89,90,91]. Gonadal differentiation in common carp starts at 80 dph, whereas in farmed Chinese sturgeon, gonadal differentiation is completed at 9 months post-fertilization [92, 93]. The barred knifejaw (Oplegnathus fasciatus), exhibits gonadal differentiation times similar to the Spotted knifejaw, with efferent ducts and a few germ cells appearing in male testes at 90–100 dph, indicating the start of testicular differentiation. Ovarian cavity differentiation occurs around 80 dph [94, 95].

The process of sex determination in fish involve a bidirectional potential of undifferentiated gonads to develop into either testes or ovaries. Once sex determination is initiated by sex-determining genes, a conserved genetic network for sex determination and differentiation is activated. Downstream sex-related genes in this network can regulate the expression of sex steroids, thereby controlling the final differentiation of gonads into functional gonads that correspond to the phenotypic sex [34, 96, 97]. Therefore, the process of sex differentiation exhibits temporal variation between gene expression, hormone levels, and gonadal differentiation [34, 98, 99]. This study explored the key time points of sex differentiation in the Spotted knifejaw by analyzing gene expression patterns during early development, hormone level dynamics, and gonadal differentiation characteristics (Fig. 1E). At the gene level, Differentiation occurs earliest, with stable differences in male–female expression observed at 36 dph. The timing of sex differentiation-related genes differs between females (foxl2, cyp19a, cyp19b) and males (dmrt1, sox9a, sox9b, amh, amhr2, hsd11b2). Transcription and translation of sex-related genes regulate the rise in male-effect hormone (11-KT) and female-effect hormone (E2) levels. Stable male–female differences in the hormone 11-KT in the Spotted knifejaw are observed at 60 dph, while stable male–female differences in estrogen occur at 70 dph. The changes in male–female hormone levels in the Spotted knifejaw ultimately lead to differences in gonadal differentiation. The Spotted knifejaw exhibits a relatively late timing of gonadal differentiation, with ovarian cavity differentiation observed in females at 90 dph and efferent duct differentiation observed in males at 100 dph, indicating completion of gonadal differentiation. Significant differentiation time points of sex-related genes in fish are often used as a means of selecting sex-determining genes.

Identification and validation of sex chromosome and sex-specific gene

Four candidate target genes were identified on the fusion chromosome (Y) of the Spotted knifejaw by integrating datasets from evolutionary positive selection gene sets identified through selective sweep analysis, sex-related SNP loci gene sets from genome-wide association studies, gene sets showing sex-specific expression differences in gonadal transcriptomes, and genes expressed early in relation to genomic sex. Typically, the determination of sex determination gene regions and the identification of the genes involved require a multi-omics approach. A total of 212 sex-specific genes were identified from the transcriptome, with 42 overlapping with SNP loci from GWAS and 51 with loci from selective sweep analysis. At a 5% significance level of the selective sweep Fst index, 1691 loci under positive selection were identified, and GWAS (Q model) revealed 3392 sex-related genes, with 1644 common to both. An intersection of gene sets from the three omics analyses led to the identification of four sex-specific genes on the heteromorphic chromosome 1 (amhr2, aldh1l2, wnt4, gdf11). This method has been instrumental in the discovery of sex determination genes in mammals, birds, and fish. In the case of Atlantic salmon, GWAS was employed to pinpoint the sex determination regions to three distinct genomic areas, and a unique single-copy region specific to males was identified, elucidating the sex determination mechanism in Salmonidae [84]. Similarly, in channel catfish, genetic linkage and GWAS analysis based on whole genome sequencing of YY individuals localized the sex determination region to a segment smaller than 0.5cm and 8.9 Mb in length. BCAR1 was identified as a potential sex determination gene through the transcriptome analysis of early gonadal development in both sexes [41]. In the Chinese tongue sole, high-throughput sequencing first provided whole genome sequences of ZZ males and ZW females. Comparative genomic analysis following chromosome assembly identified candidate genes associated with sex development, and subsequent expression and functional validation determined dmrt1 as the male determinant [18]. The resequencing of the Spotted knifejaw genome on chromosome 1 revealed numerous InDel loci, with genome-wide association analysis indicating that male sex-associated loci were concentrated on chromosome 1. Selective sweep analysis further confirmed that loci under positive selection were exclusively located on chromosome 1, providing compelling evidence for its role as a sex chromosome.

Male sex determination genes share common features: they are located on sex chromosomes, expressed during the early stages of sexual development, and exhibit specific expression in the gonads (testes) [36, 100]. These genes frequently display sex-specific expression patterns in the early animal development. Transcriptomic analysis or RT-PCR serves as a crucial secondary screening approach for identifying potential sex determination genes. This technique has been utilized in sex determination research across various species, including the sea dragon [101], ayu [17], yellow perch [10], and channel catfish [48]. To refine the selection of candidate genes, we investigated their expression patterns during early development and tissue-specificity. Our findings indicate that amhr2 is exclusively expressed in the gonads, with significantly higher levels in the testes compared to the ovaries. In contrast, the other three genes (aldh1l2, wnt4, and gdf11) manifested broader tissue expression. Aldh1l2 was ubiquitously expressed and particularly abundant in the heart, intestine, liver, kidney, and brain. Wnt4 was specifically expressed in the ovaries and was also highly expressed in gill and eye tissues. Gdf11 expression was minimal in the gonads but was elevated in the eye, brain, and intestinal tissues. During early development, amhr2, aldh1l2, and gdf11 showed higher expression in the male gonads than in the female ovaries. Notably, amhr2 demonstrated the earliest detectable sex-specific expression difference, which was significantly higher in males than in females at 35 dph. Based on these findings, amhr2, with its specific expression in the testes and early sex-specific expression difference, emerges as a prime candidate for a sex determination gene.

Male sex determination in spotted knifejaw: insights into amhr2 dosage compensation

Sex chromosomes harbor genes responsible for sex determination, which trigger the process of gonadal differentiation during early development [37, 101]. Bony fish exhibit diverse sex determination systems and are pivotal models for investigating sex chromosomes and the genes involved in this process. The majority of sex determination genes identified in bony fish are members of the sox or dmrt gene families, or they belong to the TGF-β signaling pathway [5]. Several genes within the TGF-β pathway, including amh, amhr2, gsdf, gdf6, and bmpr1b, have been implicated in sex determination across approximately 16 fish species [45, 101]. While most bony fish possess homomorphic sex chromosomes, only around 10% have been identified with heteromorphic sex chromosomes. Despite the ability of genetic analysis to map sex determination genes to specific chromosomal regions, their identification in the majority of bony fish remains arduous. To date, sex determination genes have been discovered in fewer than 25 species, representing a mere fraction of the 20,000 known bony fish species [37, 102]. This research is the first to delineate the male sex determination mechanism in the Spotted knifejaw, driven by amhr2 dosage compensation within the X₁X₁X₂X/X₁X₂Y system. It examines aspects such as chromosome gene cloning, mRNA transcript abundance, expression level variances among different transcripts, and receptor antibody binding efficiency. The male Spotted knifejaw's amhr2 genomic sequence (9739 bp) is situated on chromosome 1 (heteromorphic) and comprises 13 exons (E1-E13), while the female (9726 bp) is located on chromosome 3 (proto-X) and also includes 13 exons (E1-E13), with a high degree of similarity in exon 11 sequence between sexes (67.4%). The male amhr2 sequence gives rise to three distinct transcripts (amhr2ay, amhr2by, amhr2cy), with amhr2ay (1506 bp) consisting of exons E1-E11, amhr2by (1269 bp) of exons E3-E11, and amhr2cy (1173 bp) of exons E4-E11. In contrast, the amhr2 genomic DNA in females yields a single transcript, amhr2a (1506 bp), covering exons E1-E11. The amino acid sequence variations in male amhr2 transcripts predominantly reside in the extracellular domain, whereas the amhr2a transcript exhibits near identity between sexes.

The principle of structure dictating function is vividly illustrated by the variable binding efficiency observed between Amh and its receptor, Amhr2, a variability influenced by the sequence structure of Amhr2 [103, 104]. To evaluate the binding efficiency of Amhr2 proteins derived from different transcripts, molecular docking techniques were employed. Lower binding energy scores indicated a stronger propensity for receptor-antibody complex formation. While the protein models for Amhr2ay and Amhr2a were highly similar, notable differences were observed in the models for Amhr2by and Amhr2cy, particularly in the extracellular domain. Amhr2cy exhibited the lowest binding energy and the highest likelihood of interacting with the Amh model, followed by Amhr2by, whereas Amhr2ay exhibited the highest binding energy and the least efficient binding. These findings suggest that under conditions of equivalent mRNA expression and translation, Amhr2cy has the greatest potential for binding with Amh and mediating its functions, followed by Amhr2by, with Amhr2a and Amhr2ay showing the least potential. Differential expression patterns among transcripts were investigated using fluorescent quantitative PCR, revealing distinct tissue expression profiles. Despite minor sequence variations, primers P1-P4 were designed to specifically amplify the respective transcripts (P1: amhr2a, amhr2ay; P2: amhr2a, amhr2ay, amhr2by, amhr2cy; P3: amhr2cy; P4: amhr2a, amhr2ay, amhr2by). Tissue-specific expression was primarily observed for amhr2a and amhr2ay/amhr2by/amhr2cy in the testes of the Spotted knifejaw, with lower levels in other tissues. Notably, amhr2by and amhr2cy were also expressed in the kidney, intestine, and heart, suggesting potential additional functions. Analysis of early developmental patterns revealed significant sexual expression differences in amhr2a expression at 35 dph, and at 40 dph for amhr2a and amhr2ay/amhr2by. The expression of amhr2ay/amhr2by/amhr2cy differentiated between sexes as early as 5 dph, with expression levels increasing rapidly post-90 dph and peaking at 360 dph. These data implicate amhr2cy as a primary contributor to sexual expression disparities. Utilizing consistent samples as the baseline and internal control, and given the uniform primer amplification efficiency, comparative expression level analysis identified amhr2cy as the most highly expressed transcript, representing a significant factor in sexual expression differences. A comprehensive analysis encompassing chromosomal gene cloning, mRNA transcript abundance, receptor-antibody binding efficiency, and transcript expression level disparities indicates that the male-specific amhr2y gene on the Y chromosome, generating three transcripts, outperforms its X-chromosomal counterpart in terms of expression, translation, and binding efficiency, thus serving as a principal determinant of sexual dimorphism.

The Amh/Amhr2 signaling pathway is pivotal in the sex determination of various bony fish species [106]. Despite the functional distinctions between amhr2y and the autosomal amhr2 remain largely unexplored. To ascertain the role of amhr2 in sex determination, RNA interference were utilized in gonadal and overexpression techniques in fish to validate the function of amhr2 bidirectionally. The knockdown of the amhr2 gene in the testes of male Spotted knifejaw led to a marked reduction in amhr2 expression in the dsRNA treatment group, confirming the efficacy of the gene knockdown. This knockdown decreased the expression of genes associated with male sex differentiation (dmrt1, amh, sox9a, sox9b) and androgen synthesis (hsd11b2, cyp11a, cyp11a), while it increased the expression of genes related to female sex differentiation (wnt4, foxl2, cyp19a, cyp19b). Conversely, the overexpression of amhr2 in Spotted knifejaw augmented the expression of genes involved in male sex differentiation and androgen synthesis, as well as those related to female sex differentiation. These findings suggest that amhr2 is positioned upstream in the sex regulation pathway and acts as a sex determination gene, directly influencing the male sex fate and promoting the expression of female-related genes by increasing the amhr2a transcript levels in female. The proposed sex determination regulatory pathway involves the amh/amhr2 system located upstream, where the dosage compensation effect leads to enhanced male-related gene expression in the early development, thereby promoting the male sex differentiation process. Amhr2 also plays a regulatory role in females, where its early expression can enhance female-related gene expression, promoting estrogen synthesis and female sex differentiation. In bony fish, the diversification of sex determination genes is significant, with sex determining genes such as dmrt1bY/dmy, gsdfY, sdY and sox3 were identified. Gene knockout methods are commonly used for functional verification, and overexpression can promote the expression of the corresponding sex regulation pathway. In ayu, male-specific regions with duplicate amhr2bY copies lead to sex reversal [17], and in Chinese tongue sole, dmrt1 knockout can cause male sex reversal.

Dosage compensation is a common form of sex determination, as seen in the Chinese tongue sole, where dmrt1 on the Z chromosome differs in copy number between sexes. In ayu, the gene dosage of amhr2 may be crucial for sex determination [17]. In southern catfish, the sequence differences between amhr2 and amhr2y suggest potential functional differences, possibly in ligand affinity [107]. Male-specific amhr2 repeats have been identified in various bony fish, and phylogenetic analysis indicates that these repeats are independent in each lineage and cluster species with amhr2 as the sex determination gene, showing a relatively conserved evolutionary process [108].

Conclusions

In conclusion, the critical period for sexual differentiation is between 35 and 60 dph, during which exhibits stable molecular-level dimorphism in gene expression. After 60 dph, differences in hormone levels emerge. Amhr2, as one of the target genes identified through multi-omics screening, is located on a heteromorphic chromosome, specifically expressed in testes, and exhibits distinct expressions in a critical period of sex differentiation (35 dph) indicating amhr2 is a candidate gene for male sex determination. Three transcripts of the amhr2 gene (amhr2ay, amhr2by, amhr2cy) are present in male Spotted knifejaw, whereas females possess only one (amhr2a), closely resembling the male amhr2ay (91.6%). Amhr2cy's extracellular domain exhibits the highest Amh protein binding capacity. The testes exhibit specific amhr2 mRNA expression, with amhr2cy being the most abundant. Sexual expression differences start as early as 5 dph. Amhr2 gene knockdown reduced male-related gene expression (dmrt1, amh, sox9a, sox9b), androgen synthesis genes (hsd11b2, cyp11a, cyp11a), and female-related genes (wnt4, foxl2, cyp19a, cyp19b). Conversely, amhr2 overexpression increased male-related gene and androgen synthesis gene expression, along with promoting female-related genes. The sexually dimorphic amhr2 gene transcription results in significant differences in amhr2 transcript quantity, mRNA expression levels, and binding efficiency to the Amh ligand protein, and leads to cascade dosage compensation, influencing distinct fates in the sex differentiation regulatory network of fish with an X₁X₁X₂X₂/X₁X₂Y sex determination system.

Data availability

No datasets were generated or analysed during the current study.

References

Bachtrog D, Mank JE, Peichel CL, Kirkpatrick M, Otto SP, Ashman TL, et al. Sex determination: why so many ways of doing it? Plos Biol. 2014;12(7):e1001899.
Kikuchi K, Hamaguchi S. Novel sex-determining genes in fish and sex chromosome evolution. Dev Dyn. 2013;242(4):339–53.
Article CAS PubMed Google Scholar
Schwanz LE, Ezaz T, Gruber B, Georges A. Novel evolutionary pathways of sex-determining mechanisms. J Evol Biol. 2013;26(12):2544–57.
Article CAS PubMed Google Scholar
Liu H, Todd EV, Lokman PM, Lamm MS, Godwin JR, Gemmell NJ. Sexual plasticity: A fishy tale. Mol Reprod Dev. 2017;84(2):171–94.
Article CAS PubMed Google Scholar
Pan QW, Kay T, Depince A, Adolfi M, Schartl M, Guiguen Y, et al. Evolution of master sex determiners: TGF-beta signalling pathways at regulatory crossroads. Philos Trans R Soc Lond B Biol Sci. 2021;376(1832):20200091.
Hattori RS, Murai Y, Oura M, Masuda S, Majhi SK, Sakamoto T, et al. A Y-linked anti-Mullerian hormone duplication takes over a critical role in sex determination. Proc Natl Acad Sci USA. 2012;109(8):2955–9.
Article CAS PubMed PubMed Central Google Scholar
Pan Q, Feron R, Yano A, Guyomard R, Jouanno E, Vigouroux E, et al. Identification of the master sex determining gene in Northern pike (Esox lucius) reveals restricted sex chromosome differentiation. PLoS Genet. 2019;15(8):e1008013.
Kitano J, Peichel CL. Turnover of sex chromosomes and speciation in fishes. Environ Biol Fishes. 2012;94(3):549–58.
Article PubMed Google Scholar
Kamiya T, Kai W, Tasumi S, Oka A, Matsunaga T, Mizuno N, et al. A trans-species missense SNP in amhr2 is associated with sex determination in the tiger pufferfish, Takifugu rubripes (Fugu). PLoS Genet. 2012;8(7):e1002798.
Feron R, Zahm M, Cabau C, Klopp C, Roques C, Bouchez O, et al. Characterization of a Y-specific duplication/insertion of the anti-Mullerian hormone type II receptor gene based on a chromosome-scale genome assembly of yellow perch. Perca flavescens Mol Ecol Resour. 2020;20(2):531–43.
Article CAS PubMed Google Scholar
Matsuda M, Nagahama Y, Shinomiya A, Sato T, Matsuda C, Kobayashi T, et al. DMY is a Y-specific DM-domain gene required for male development in the medaka fish. Nature. 2002;417(6888):559–63.
Article CAS PubMed Google Scholar
Nanda I, Kondo M, Hornung U, Asakawa S, Winkler C, Shimizu A, et al. A duplicated copy of dmrt1 in the sex-determining region of the Y chromosome of the medaka, Oryzias latipes. Proc Natl Acad Sci USA. 2002;99(18):11778–83.
Article CAS PubMed PubMed Central Google Scholar
Myosho T, Otake H, Masuyama H, Matsuda M, Kuroki Y, Fujiyama A, et al. Tracing the emergence of a novel sex-determining gene in medaka. Oryzias luzonensis Genetics. 2012;191(1):163–70.
Article CAS PubMed Google Scholar
Takehana Y, Matsuda M, Myosho T, Suster ML, Kawakami K, Shin-I T, et al. Co-option of Sox3 as the male-determining factor on the Y chromosome in the fish Oryzias dancena. Nat Commun. 2014;5:4157.
Yano A, Guyomard R, Nicol B, Jouanno E, Quillet E, Klopp C, et al. An immune-related gene evolved into the master sex-determining gene in rainbow trout. Oncorhynchus mykiss Current Biology. 2012;22(15):1423–8.
Article CAS PubMed Google Scholar
Koyama T, Nakamoto M, Morishima K, Yamashita R, Yamashita T, Sasaki K, et al. A SNP in a steroidogenic enzyme is associated with phenotypic sex in Seriola fishes. Curr Biol. 2019;29(11):1901–909.e8.
Article CAS PubMed Google Scholar
Nakamoto M, Uchino T, Koshimizu E, Kuchiishi Y, Sekiguchi R, Wang L, et al. A Y-linked anti-Mullerian hormone type-II receptor is the sex-determining gene in ayu, Plecoglossus altivelis. PLoS Genet. 2021;17(8):e1009705.
Cui ZK, Liu Y, Wang WW, Wang Q, Zhang N, Lin F, et al. Genome editing reveals dmrt1 as an essential male sex-determining gene in Chinese tongue sole (Cynoglossus semilaevis). Sci Rep. 2017;7:42213.
Devlin RH, Nagahama Y. Sex determination and sex differentiation in fish: an overview of genetic, physiological, and environmental influences. Aquaculture. 2002;208(3–4):191–364.
Article CAS Google Scholar
Capel B. Vertebrate sex determination: evolutionary plasticity of a fundamental switch. Nat Rev Genet. 2017;18(11):675–89.
Guiguen Y, Fostier A, Piferrer F, Chang C-F. Ovarian aromatase and estrogens: A pivotal role for gonadal sex differentiation and sex change in fish. Gen Comp Endocrinol. 2010;165(3):352–66.
Article CAS PubMed Google Scholar
Baron D, Batista F, Chaffaux S, Cocquet J, Cotinot C, Cribiu E, De Baere E, Guiguen Y, Jaubert F, Pailhoux E, et al. Foxl2 gene and the development of the ovary: a story about goat, mouse, fish and woman. Reprod Nutr Dev. 2005;45:377–82.
Article CAS PubMed Google Scholar
Zhang X, Li M, Ma H, Liu X, Shi H, Li M, Wang D. Mutation of foxl2 or cyp19a1a Results in Female to Male Sex Reversal in XX Nile Tilapia. Endocrinology. 2017;158:2634–47.
CAS PubMed Google Scholar
Cheshenko K, Pakdel F, Segner H, Kah O, Eggen RIL. Interference of endocrine disrupting chemicals with aromatase CYP19 expression or activity, and consequences for reproduction of teleost fish. Gen Comp Endocrinol. 2008;155:31–62.
Article CAS PubMed Google Scholar
Li XY, Mei J, Ge CT, Liu XL, Gui JF. Sex determination mechanisms and sex control approaches in aquaculture animals. Sci China-Life Sci. 2022;65(6):1091–122.
Article PubMed Google Scholar
Webster KA, Schach U, Ordaz A, Steinfeld JS, Draper BW, Siegfried KR. Dmrt1 is necessary for male sexual development in zebrafish. Dev Biol. 2017;422:33–46.
Article CAS PubMed Google Scholar
Romano S, Kaufman OH, Marlow FL. Loss of dmrt1 restores zebrafish female fates in the absence of cyp19a1a but not rbpms2a/b. Development. 2020;147(18):dev190942.
Yokoi H, Kobayashi T, Tanaka M, Nagahama Y, Wakamatsu Y, Takeda H, Araki K, Morohashi KI, Ozato K. Sox9 in a teleost fish, medaka (Oryzias latipes): Evidence for diversified function of Sox9 in gonad differentiation. Mol Reprod Dev. 2002;63:5–16.
Article CAS PubMed Google Scholar
Hett AK, Pitra C, Jenneckens I, Ludwig A. Characterization of Sox9 in European Atlantic sturgeon (Acipenser sturio). J Hered. 2005;96:150–4.
Article CAS PubMed Google Scholar
di Clemente N, Racine C, Pierre A, Taieb J. Anti-mullerian hormone in female reproduction. Endocr Rev. 2021;42:753–82.
Article PubMed Google Scholar
Howard JA, Hart KN, Thompson TB. Molecular mechanisms of amh signaling. Front Endocrinol. 2022;13:927824.
Josso N, Picard JY. Genetics of anti-Mullerian hormone and its signaling pathway. Best Pract Res Clin Endocrinol Metab. 2022;36(1):101634.
Alderman SL, Vijayan MM. 11β-Hydroxysteroid dehydrogenase type 2 in zebrafish brain: a functional role in hypothalamus-pituitary-interrenal axis regulation. J Endocrinol. 2012;215:393–402.
Article CAS PubMed Google Scholar
Theodoridi A, Dinarello A, Badenetti L, Pavlidis M, Dalla Valle L, Tsalafouta A. Knockout of the hsd11b2 gene extends the cortisol stress response in both zebrafish larvae and adults. Int J Mol Sci. 2021;22:12525.
Duan W, Gao FX, Chen ZW, Gao Y, Gui JF, Zhao Z, et al. A sex-linked SNP mutation in amhr2 is responsible for male differentiation in obscure puffer (Takifugu obscurus). Mol Biol Rep. 2021;48(8):6035–46.
Article CAS PubMed Google Scholar
Song W, Xie Y, Sun M, Li X, Fitzpatrick CK, Vaux F, et al. A duplicated amh is the master sex-determining gene for Sebastes rockfish in the Northwest Pacific. Open Biol. 2021;11(7):210063.
Touati R, Tajouri A, Mesaoudi I, Oueslati AE, Lachiri Z, Kharrat M. New methodology for repetitive sequences identification in human X and Y chromosomes. Biomed Signal Process Control. 2021;64: 102207.
Article PubMed Google Scholar
Pan Q, Kay T, Depincé A, Adolfi M, Schartl M, Guiguen Y, et al. Evolution of master sex determiners: TGF-β signalling pathways at regulatory crossroads. Philosophical Transactions of the Royal Society B: Biological Sciences. 1832;2021(376):20200091.
Google Scholar
Li M, Zhang R, Fan G, Xu W, Zhou Q, Wang L, et al. Reconstruction of the origin of a neo-Y sex chromosome and its evolution in the Spotted knifejaw, Oplegnathus punctatus. Molecular Biology and Evolution. 2021.
Bao L, Tian C, Liu S, Zhang Y, Elaswad A, Yuan Z, et al. The Y chromosome sequence of the channel catfish suggests novel sex determination mechanisms in teleost fish. BMC Biology. 2019;17(1):6.
Odet F, Gabel SA, Williams J, London RE, Goldberg E, Eddy EM. Lactate dehydrogenase C and energy metabolism in mouse sperm. Biol Reprod. 2011;85(3):556–64 (Epub 2011/05/14).
Article CAS PubMed PubMed Central Google Scholar
Matsuda M, Nagahama Y, Kobayashi T, Matsuda C, Hamaguchi S, Sakaizumi M. The sex determining gene of medaka: a Y-specific DM domain gene (dmy) is required for male development. Fish Physiol Biochem. 2003;28(1–4):135–9.
Article CAS Google Scholar
Shao C, Chen S. Sex-determining genes and its association with mechanism of sex chromosome evolution in vertebrate. J Agr Biotech. 2012;20(12):1463–74.
Kobayashi Y, Nagahama Y, Nakamura M. Diversity and plasticity of sex determination and differentiation in fishes. Sexual Development. 2013;7(1–3):115–25.
Article CAS PubMed Google Scholar
Hattori RS, Kumazawa K, Nakamoto M, Nakano Y, Yamaguchi T, Kitano T, et al. Y-specific amh allele, I, is the master sex-determining gene in Japanese flounder Paralichthys olivaceus. Front Genet. 2022;13:1007548.
Qu M, Liu YL, Zhang YH, Wan SM, Ravi V, Qin G, et al. Seadragon genome analysis provides insights into its phenotype and sex determination locus. Sci Adv. 2021;7(34):eabg5196.
Xu Y, Zhong ZW, Zhang ZP, Feng Y, Zhao LP, Jiang YH, et al. Establishment and characterization of the gonadal cell lines derived from large yellow croaker (Larimichthys crocea) for gene expression studies. Aquaculture. 2022;546:737300.
Wang Y, Xiao Z, Liu Q, Zhai J, Pang Z, Ma W, et al. Allometric growth pattern during early ontogeny of Spotted knifejaw (Oplegnathus punctatus). Mar Sci. 2016;40(5):43–8.
CAS Google Scholar
Wang F, Gao Y, Gao Y, Li M, Zhang B, Guan C, et al. Preliminary study on land- sea alternate aquaculture of Spotted knifejaw Oplegnathus punctatus. Fish Mod. 2022;49(4):8–14.
Du XX, Liu XB, Zhang K, Liu YX, Cheng J, Zhang QQ. Discovery and functional characterization of microRNAs and their potential roles for gonadal development in Spotted knifejaw, Oplegnathus punctatus. Comp Biochem Physiol D-Genomics Proteomics. 2018;28:1–8.
Article CAS Google Scholar
de Oliveira RR, Feldberg E, dos Anjos MB, Zuanon J. Occurrence of multiple sexual chromosomes (XX/XY₁Y₂ and Z₁Z₁Z₂Z₂/Z₁Z₂W₁W₂) in catfishes of the genus Ancistrus (Siluriformes: Loricariidae) from the Amazon basin. Genetica. 2008;134(2):243–9.
Article CAS PubMed Google Scholar
Sember A, Nguyen P, Perez MF, Altmanova M, Rab P, Cioffi MdB. Multiple sex chromosomes in teleost fishes from a cytogenetic perspective: state of the art and future challenges. Philos Trans R Soc B Biol Sci. 2021;376(1833):20200098.
Vidal VPI, Chaboissier MC, de Rooij DG, Schedl A. Sox9 induces testis development in XX transgenic mice. Nat Genet. 2001;28(3):216–7.
Article CAS PubMed Google Scholar
Sutton E, Hughes J, White S, Sekido R, Tan J, Arboleda V, et al. Identification of sox3 as an XX male sex reversal gene in mice and humans. J Clin Investig. 2011;121(1):328–41.
Article CAS PubMed Google Scholar
Ou M, Chen KC, Luo Q, Liu HY, Wang YP, Chen BX, et al. Performance evaluation of XY all-male hybrids derived from XX female Channa argus and YY super-males Channa maculate. Aquacult Rep. 2021;20:100768.
Xu HY, Gui JF, Hong YH. Differential expression of vasa RNA and protein during spermatogenesis and oogenesis in the gibel carp (Carassius auratus gibelio), a bisexually and gynogenetically reproducing vertebrate. Dev Dyn. 2005;233:872–82.
Article CAS PubMed Google Scholar
Yu M, Wang FY, Li MZ, Wang Y, Gao XZ, Zhang HH, Liu ZZ, Zhou ZC, Zhao DQ, Zhang M, et al. Characteristics of the vasa gene in silurus asotus and its expression response to letrozole treatment. Genes. 2024;15(6):756.
Takehana Y, Hamaguchi S, Sakaizumi M. Different origins of ZZ/ZW sex chromosomes in closely related medaka fishes, Oryzias javanicus and O. hubbs. Chromosome Research. 2008;16(5):801–11.
Article CAS PubMed Google Scholar
Sember A, Nguyen P, Perez MF, Altmanová M, Ráb P, Cioffi MDB. Multiple sex chromosomes in teleost fishes from a cytogenetic perspective: state of the art and future challenges. Philosophical Transactions of the Royal Society B: Biological Sciences. 1833;2021(376):20200098.
Google Scholar
Li M, Xu H, Xu WT, Zhou Q, Xu XW, Zhu Y, et al. Isolation of a Male-specific molecular marker and development of a genetic sex identification technique in Spotted knifejaw (Oplegnathus punctatus). Mar Biotechnol. 2020;22(4):467–74.
Article CAS Google Scholar
Nasilowska-Adamska B, Solarska I, Paluszewska M, Malinowska I, Jedrzejczak WW, Warzocha K. FLT3-ITD and MLL-PTD influence the expression of MDR-1, MRP-1, and BCRP mRNA but not LRP mRNA assessed with RQ-PCR method in adult acute myeloid leukemia. Ann Hematol. 2014;93(4):577–93.
Article CAS PubMed Google Scholar
Yamaguchi T, Kitano T. Amh/Amhr2 signaling causes masculinization by inhibiting estrogen synthesis during gonadal sex differentiation in japanese flounder Paralichthys olivaceus). Int J Mol Sci. 2023;24(3):13.
Article Google Scholar
Zhang C, Dong S-S, Xu J-Y, He W-M, Yang T-L. PopLDdecay: a fast and effective tool for linkage disequilibrium decay analysis based on variant call format files. Bioinformatics. 2019;35(10):1786–8.
Article CAS PubMed Google Scholar
Jiang L, Zheng Z, Qi T, Kemper KE, Wray NR, Visscher PM, et al. A resource-efficient tool for mixed model association analysis of large-scale data. Nat Genet. 2019;51(12):1749–55.
Article CAS PubMed Google Scholar
Mbatchou J, Barnard L, Backman J, Marcketta A, Kosmicki JA, Ziyatdinov A, et al. Computationally efficient whole-genome regression for quantitative and binary traits. Nat Genet. 2021;53(7):1097–103.
Dey R, Schmidt EM, Abecasis GR, Lee S. A fast and accurate algorithm to test for binary phenotypes and its application to PheWAS. Am J Hum Genet. 2017;101(1):37–49.
Zhou W, Nielsen JB, Fritsche LG, Dey R, Gabrielsen ME, Wolford BN, et al. Efficiently controlling for case-control imbalance and sample relatedness in large-scale genetic association studies. Nat Genet. 2018;50(9):1335–41.
Article CAS Google Scholar
Chen H, Huffman JE, Brody JA, Wang C, Lee S, Li Z, et al. Efficient variant set mixed model association tests for continuous and binary traits in large-scale whole-genome sequencing studies. Am J Hum Genet. 2019;104(2):260–74.
Article CAS PubMed PubMed Central Google Scholar
Urrutia E, Lee S, Maity A, Zhao N, Shen J, Li Y, et al. Rare variant testing across methods and thresholds using the multi-kernel sequence kernel association test (MK-SKAT). Stat Interface. 2015;8(4):495–505.
Article PubMed PubMed Central Google Scholar
Lee S, Wu MC, Lin X. Optimal tests for rare variant effects in sequencing association studies. Biostatistics. 2012;13(4):762–75.
Article PubMed PubMed Central Google Scholar
Nei M, Li WH. Mathematical-model for studying genetic-variation in terms of restriction endonucleases. Proc Natl Acad Sci USA. 1979;76(10):5269–73.
Article CAS PubMed PubMed Central Google Scholar
Li J, Lu J, Jing R, Wang Q, Chang C, editors. Research on neutral mutation algorithm in dna polymorphism testing based on OpenMP. IEEE Advanced Information Technology, Electronic and Automation Control Conference (IAEAC); 2015 2015. Dec 19–20; Chongqing, PEOPLES R CHINA2015.
Lin T, Zhu G, Zhang J, Xu X, Yu Q, Zheng Z, et al. Genomic analyses provide insights into the history of tomato breeding. Nat Genet. 2014;46(11):1220–6.
Article CAS PubMed Google Scholar
Hudson RR, Slatkin M, Maddison WP. Estimation of levels of gene flow from DNA-sequence data. Genetics. 1992;132(2):583–9.
Article CAS PubMed PubMed Central Google Scholar
Fu YX, Li WH. Statistical tests of neutrality of mutations. Genetics. 1993;133(3):693–709.
Article CAS PubMed PubMed Central Google Scholar
Pfeifer B, Wittelsbuerger U, Ramos Onsins S, Lercher M. PopGenome: An efficient swiss army knife for population genomic analyses in R. Human Heredity. 2013;76(2):109.
Google Scholar
Nakamura M, Kobayashi T, Chang XT, Nagahama Y. Gonadal sex differentiation in teleost fish. J Exp Zool. 1998;281(5):362–72.
Article Google Scholar
Brown-Peterson NJ, Wyanski DM, Saborido-Rey F, Macewicz BJ, Lowerre-Barbieri SK. A standardized terminology for describing reproductive development in fishes. Marine and Coastal Fisheries. 2011;3(1):52–70.
Article Google Scholar
Berbejillo J, Martinez-Bengochea A, Bedo G, Vizziano-Cantonnet D. Expression of dmrt1 and sox9 during gonadal development in the Siberian sturgeon (Acipenser baerii). Fish Physiol Biochem. 2013;39(1):91–4.
Article CAS PubMed Google Scholar
Gao Z, Wang H-P, Rapp D, O’Bryant P, Wallat G, Wang W, et al. Gonadal sex differentiation in the bluegill sunfish Lepomis macrochirus and its relation to fish size and age. Aquaculture. 2009;294(1–2):138–46.
Article Google Scholar
Yang F, Ye H, Takeuchi Y, Liu F, Xu D. Characterization of the sex differentiation and gonadal development in small yellow croaker (Larimichthys polyactis) and its hybrid (L. polyactis ♀ x L. crocea ♂). Fish Physio Biochem. 2021;47(5):1467–76.
Article CAS Google Scholar
Satoh N, Egami N. Sex differentiation of germ-cells in teleost, Oryzias-latipes, during normal embryonic-development. J Embryology Exp Morph. 1972;29:385.
Google Scholar
Aoki Y, Nagao I, Saito D, Ebe Y, Kinjo M, Tanaka M. Temporal and spatial localization of three germline-specific proteins in medaka. Dev Dyn. 2008;237(3):800–7.
Article CAS PubMed Google Scholar
Nakamura S, Aoki Y, Saito D, Kuroki Y, Fujiyama A, Naruse K, et al. Sox9b/sox9a2-EGFP transgenic medaka reveals the morphological reorganization of the gonads and a common precursor of both the female and male supporting cells. Mol Reprod Dev. 2008;75(3):472–6.
Article CAS PubMed Google Scholar
Wang XG, Bartfai R, Sleptsova-Freidrich I, Sreeinivasan R, Cai M, Orban L. Analyzing gonad transformation in a vas:egfp transgenic zebrafish line. Mechanisms of Development. 2005;122:S56-S.
Google Scholar
Santos EM, Workman VL, Paull GC, Filby AL, Van Look KJW, Kille P, et al. Molecular basis of sex and reproductive status in breeding zebrafish. Physiol Genomics. 2007;30(2):111–22.
Article CAS PubMed Google Scholar
Kossack ME, Draper BW. Genetic regulation of sex determination and maintenance in zebrafish (Danio rerio). Curr Top Dev Biol. 2019;134:119–49.
Kitano T, Takamune K, Nagahama Y, Abe S-I. Gonadal sex differentiation in japanese flounder (Paralichthys olivaceus). Fish Sci. 2002;68:679–80.
Article Google Scholar
Sun C, Huang C, Su X, Zhao X, Dong Q. Optimization of handling and refrigerated storage of guppy Poecilia reticulata sperm. J Fish Biol. 2010;77(1):54–66.
Fan Z, Zou Y, Liang D, Tan X, Jiao S, Wu Z, et al. Roles of forkhead box protein L2 (foxl2) during gonad differentiation and maintenance in a fish, the olive flounder (Paralichthys olivaceus). Reprod Fertil Dev. 2019;31(11):1742–52.
Article CAS PubMed Google Scholar
Hulata G, Moav R, Wohlfarth G. Relationship of gonad and egg size to weight and age in european and chinese races of common carp Cyprinus carpiol. J Fish Biol. 1974;6(6):745–58.
Article Google Scholar
Chen J-J, Xia X-H, Wang L-F, Jia Y-f, Nan P, Li L, et al. Identification and comparison of gonadal transcripts of testis and ovary of adult common carp Cyprinus carpio using suppression subtractive hybridization. Theriogenology. 2015;83(9):1416–27.
Article CAS PubMed Google Scholar
Feng-ping Z, Min-hai LIU, Zhi-lan P, Jian Z, Rong-bing FU, Hai-zhong LUO. Histology of ovary development in striped knifejaw Oplegnathus fasciatus. J Dalian Fish Coll. 2010;25(2):102–6.
Google Scholar
Liu HY, Xia WW, Li BJ, Liu L, Wang YK, Luo Q, et al. Sex-specific markers developed by 2b-RAD and genome sequencing reveal an XX/XY sex-determination system in mud carp (Cirrhinus molitorella). Aquaculture. 2023;565:8.
Article Google Scholar
Lubzens E, Young G, Bobe J, Cerda J. Oogenesis in teleosts: How fish eggs are formed. Gen Comp Endocrinol. 2010;165(3):367–89.
Article CAS PubMed Google Scholar
Schulz RW, de Franca LR, Lareyre J-J, LeGac F, Chiarini-Garcia H, Nobrega RH, et al. Spermatogenesis in fish. Gen Comp Endocrinol. 2010;165(3):390–411.
Article CAS PubMed Google Scholar
Metcalfe CD, Metcalfe TL, Kiparissis Y, Koenig BG, Khan C, Hughes RJ, et al. Estrogenic potency of chemicals detected in sewage treatment plant effluents as determined by in vivo assays with Japanese medaka (Oryzias latipes). Environ Toxicol Chem. 2001;20(2):297–308.
Article CAS PubMed Google Scholar
Piferrer F, Guiguen Y. Fish Gonadogenesis. Part II: Molecular biology and genomics of sex differentiation. Rev Fisheries Sci. 2008;16:35–55.
Article CAS Google Scholar
Yano A, Nicol B, Jouanno E, Quillet E, Fostier A, Guyomard R, et al. The sexually dimorphic on the Y-chromosome gene (sdY) is a conserved male-specific Y-chromosome sequence in many salmonids. Evol Appl. 2013;6(3):486–96.
Article CAS PubMed Google Scholar
Pan QW, Kay T, Depincé A, Adolfi M, Schartl M, Guiguen Y, et al. Evolution of master sex determiners: TGF-β signalling pathways at regulatory crossroads. Philos Trans R Soc B Biol Sci. 2021;376(1832):20200091.
Qu M, Liu Y, Zhang Y, Wan S, Ravi V, Qin G, et al. Seadragon genome analysis provides insights into its phenotype and sex determination locus. Sci Adv. 2021;7(34):eabg5196.
Shaw DE, White MA. The evolution of gene regulation on sex chromosomes. Trends Genet. 2022;38(8):844–55.
Article CAS PubMed Google Scholar
Liew WC, Orban L. Zebrafish sex: a complicated affair. Brief Funct Genomics. 2014;13(2):172–87.
Article PubMed Google Scholar
Wen M, Pan QW, Jouanno E, Montfort J, Zahm M, Cabau C, et al. An ancient truncated duplication of the anti-Mullerian hormone receptor type 2 gene is a potential conserved master sex determinant in the Pangasiidae catfish family. Mol Ecol Resour. 2022;22(6):2411–28.
Edgecombe J, Urban L, Todd EV, Gemmell NJ. Might Gene Duplication and neofunctionalization contribute to the sexual lability observed in fish? Sexual Development. 2021;15(1–3):122–33.
Article CAS PubMed Google Scholar
Hart KN, Stocker WA, Nagykery NG, Walton KL, Harrison CA, Donahoe PK, et al. Structure of AMH bound to AMHR2 provides insight into a unique signaling pair in the TGF-β family. P Natl Acad Sci USA. 2021;118(26):e2104809118.
Zheng SQ, Tao WJ, Yang HW, Kocher TD, Wang ZJ, Peng ZG, et al. Identification of sex chromosome and sex-determining gene of southern catfish (Silurus meridionalis) based on XX, XY and YY genome sequencing. Proc Biol Sci. 2022;289(1971):20212645.
Adolfi MC, Nakajima RT, Nóbrega RH, Schartl M. Intersex, hermaphroditism, and gonadal plasticity in vertebrates: evolution of the mullerian duct and amh/amhr2 signaling. Annu Rev Anim Biosci. 2019;7:149–72.

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Acknowledgements

Thanks to all laboratory members for their suggestions, support, and encouragement. We also thank State Key Laboratory of Breeding Biotechnology and Sustainable Aquaculture (CAS) for financial support.

Funding

This work was supported by grants from the National Natural Science Foundation of China (No. 42276107), Key Deployment Projects of Center for Ocean Mega-Science, Chinese Academy of Sciences (Frontier Cross-category, COMS2020Q05), Natural Science Foundation of Shandong Province (ZR2024MC071), Shandong Province Key R&D Program (2023LZGCQY001), National Key Research and Development Program (2022YFC3103600).

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Haixia Zhao and Jun Li contributed equally to this work.

Authors and Affiliations

State Key Laboratory of Breeding Biotechnology and Sustainable Aquaculture, Chinese Academy of Sciences (CAS), Qingdao, China
Haixia Zhao, Jun Li, Zhizhong Xiao & Yongshuang Xiao
Laboratory for Marine Biology and Biotechnology, Qingdao Marine Science and Technology Center, Qingdao, China
Haixia Zhao, Jun Li, Zhizhong Xiao & Yongshuang Xiao
Shandong Province Key Laboratory of Experimental Marine Biology, Institute of Oceanology, Chinese Academy of Sciences, Qingdao, China
Haixia Zhao, Jun Li, Zhizhong Xiao & Yongshuang Xiao
Marine Biology Institute of Shandong Province, Qingdao, Shandong, China
Haixia Zhao

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Haixia Zhao
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Jun Li
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Zhizhong Xiao
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Yongshuang Xiao
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Contributions

J.L. and Y.S.X. planned and managed the project, H.X. Z. and Y.S.X. wrote the main manuscript and edit the manuscript, H.X. Z. and X.Y.S. assembled the genome, X.Z.Z. collected samples, J. L. and Y.S. X. contributed to the funding acquisition. All authors reviewed the manuscript.

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Correspondence to Jun Li or Yongshuang Xiao.

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Zhao, H., Li, J., Xiao, Z. et al. Dose-dependent role of AMH and AMHR2 signaling in male differentiation and regulation of sex determination in Spotted knifejaw (Oplegnathus punctatus) with X₁X₁X₂X₂/X₁X₂Y chromosome system. Cell Commun Signal 23, 59 (2025). https://doiorg.publicaciones.saludcastillayleon.es/10.1186/s12964-025-02038-w

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Received: 14 September 2024
Accepted: 10 January 2025
Published: 01 February 2025
DOI: https://doiorg.publicaciones.saludcastillayleon.es/10.1186/s12964-025-02038-w