The channel catfish fries used in the present study were produced at the National Genetic Breeding Center of Channel Catfish (Yangzhong, Jiangsu, China). Artificial compound feed containing 17β-estradiol (17β-E₂, 60 μg/g) was selected as a vector to induce the sexual reversal of male fish by feeding catfish larvae continuously for 27 days. The dosage of 17β-E₂ in the compound feed was 60 μg/g. The gonads of sex-reversed and normal female catfish at 60 dahs were identified by histological section and sex-linkage molecular marker (Zhang et al., 2019; Pan et al., 2022), respectively.

Furthermore, the ovary tissues of the two catfish types were dissected from three individuals based on the identification results. At the same time, five tissues, namely, liver, brain, kidney, head kidney, and testes/ovaries, were also dissected from three male and three female individuals. All tissue samples were preserved in the RNA Keeper Tissue Stabilizer (Vazyme, China) and were stored at -20°C until RNA extraction. During sampling, the experimental fish were anesthetized using 0.1% tricaine methanesulfonate (MS-222) (Merck, Germany). All animal handling was carried out in accordance with the ethical guidelines and protocols of the Freshwater Fisheries Research Institute of Jiangsu Province.

Candidate IpDmrt family genes were identified on the channel catfish genome using the BLAST search program based on the conserved DM domain (Pfam 00,751) with amino acids. The identified candidates and their amino acid sequences were downloaded from the channel catfish genome database stored in the NCBI database (https://www.ncbi.nlm.nih.gov/). The genes were further confirmed to contain the conserved DM domains by scanning their sequences through the Pfam (http://pfam.sanger.ac.uk/search), SMART (http://smart.embl-heidelberg.de/), and InterPro (http://www.ebi.ac.uk/interpro/) online databases.

The total RNA of sampled tissues was isolated using the RNeasy Mini Kit (Qiagen, Germany). The cDNA was subjected to reverse transcription using the PrimeScript™ first Strand cDNA Synthesis Kit (TaKaRa, Dalian, China) according to the manufacturer’s instructions. According to the DNA sequences of channel catfish Dmrt genes, we designed seven pairs of primers (Supplementary Table S1) to clone their full-length cDNA sequences. The PCR conditions were as follows: 94°C for 30 s, gene-specific annealing for 30 s and elongation stage at 72°C for 60 s, a total of 34 cycles. The target products were purified using the universal DNA Purification Kit (Tiangen, Beijing, China), and then sequenced by the Sangon Biotech Co., Ltd. (Shanghai, China).

Basic information related to the IpDmrt genes, including the number of introns and exons, coding sequence length, and chromosome location, were extracted from the channel catfish genome database. The physicochemical characteristics of IpDmrt proteins, including the theoretical molecular weight (kDa), lengths, isoelectric points (pI), and grand average of hydropathicity (GRAVY), were evaluated using the online ExPASY tool (https://web.expasy.org/protparam/). The intron and exon positions, and untranslated regions were visualized using the Gene Structure Display Server 2.0 (http://gsds.gao-lab.org/).

Multiple sequence alignments of the IpDmrt proteins were performed and visualized using the T-COFFEE (tcoffee.crg.cat/apps/tcoffee/index.html) and EsPript 3.0 (https://espript.ibcp.fr/ESPript/ESPript) online software, respectively. The amino acid sequences of Dmrt proteins from human (Homo sapiens), chicken (Gallus), African clawed frog (Xenopus laevis), zebrafish (Danio rerio), large yellow croaker (Larimichthys crocea), Nile tilapia (Oreochromis niloticus), Japanese medaka, Japanese pufferfish, and channel catfish were selected to construct the phylogenetic tree in MEGA7 using the neighbor-joining method. The Jones-Taylor-Thornton (JTT) algorithm + Nearest-Neighbor-Interchange (NNI) distance were selected to automatically generate the initial tree (Default - NJ/BioNJ) model and evaluate the phylogenetic tree. The Dmrt genes were named using an “Ip” prefix and were numbered in ascending order based on the results of the phylogenetic analysis. The name of Dmrt protein sequences showed in Supplementary Table S2.

In order to examine the extent of preservation of genomic neighborhoods between IpDmrt genes and the potential counterparts of other species, such as electric eel (Electrophorus electricus), Nile tilapia, zebrafish, and Atlantic cod (Gadus morhua), the Synteny Database (http://teleost.cs.uoregon.edu/synteny_db/) and Genomicus Browser (http://www.dyogen.ens.fr/genomicus-63.01/cgi-bin/search.pl) were used as the main visualization tools. To perform the synteny analysis, the channel catfish orthologs of the Dmrt genes neighboring other teleosts’ genes were firstly examined at the chromosome level through the Synteny Database, and at the local neighborhood level using the Genomicus Browser, which provides detailed gene information related to each teleost gene’s orthologs on an individual chromosome. In the synteny analyses of Dmrt genes, the channel catfish neighboring genes as follows: Fbrsl1, Ap1b1, Fbxo21, Fbxw8, Abca2, Adamts3, Dmrt2a, Dmrt3, Dmrt1, and Adgrv1 on Chr22; Abl2, Bend5, Dmrt5, Calr, Abhd17 ab, Tmem47, Tab3, Nexn, Armh1, Multh, and Acbd6 on Chr5; Irrc40, Irrc7, Insl5a, Ak4, Jak1, Foxd3, Kank4, Cyldl, Adcyaplr1b, and Dmrt2b on Chr11; and Ndrg2, Arhgef40, Casq1b, Ipcat4, Khnyn, Drap1, Rela, Elavl2, Dmrt4, Rbpms, and Alpk1 on Chr29.

The purpose of protein interaction network analysis was to examine the relationship between Dmrt and other genes and explore their potential functions. The protein interaction networks of the IpDmrt gene family were constructed using the STRING online software (https://www.string-db.org).

Gene-specific primers (listed in Supplementary Table S3) were designed using Primer Premier 5.0 software and were synthesized by Sangon Biotech Co., Ltd (Shanghai, China). The α-tubulin (Zhang et al., 2020) gene was employed as an internal reference gene. The qRT-PCR reaction system contained 12.5 μL of SYBR green (TaKaRa, Dalian, China), 1 μL of gene-specific primers (1.0 μM), 1 μL of cDNA, and 8.5 μL of DEPC water, reaching a total reaction volume of 25 μL. The PCR conditions were as follows: 95°C for 30 s, followed by 40 cycles of 15 s at 95°C and 30 s at 57°C; 95°C for 15 s, 60°C for 15 s, and 95°C for 15 s. Relative mRNA expression levels were determined using the 2^{−△△ CT} method (Livak and Schmittgen, 2001). Finally, the statistical significance was calculated in R v. 4.0.5. Statistically significant differences were set at p < 0.05.

A total of seven Dmrt genes (IpDmrt1, IpDmrt2a, IpDmrt2b, IpDmrt3, IpDmrt4, IpDmrt5, and IpDmrt6) were identified in the channel catfish genome, and were named based on the nomenclature of teleost Dmrt genes and the results of phylogenetic analyses. These gene numbers were relatively consistent compared to that previously reported in other fish species, such as large yellow croaker (Wan et al., 2020), while it was less than that reported for grass carp (Ctenopharyngodon idella) (Chen et al., 2019) and rainbow trout (Oncorhynchus mykiss) (Wang et al., 2014), which experienced four rounds of whole genome duplication during evolution. The exon-intron structure analysis revealed that the IpDmrt gene family contained two to five exons; in particularly, IpDmrt1 had five exons; IpDmrt2a and IpDmrt6 had four; IpDmrt2b had three; and IpDmrt3, IpDmrt4, and IpDmrt5 had two. The length of IpDmrt proteins ranged from 282 to 507 amino acids, with an average molecular weight of 42.30062 kDa. The average pI of the IpDmrt protein family was 8.13; with the highest and lowest points reaching 9.36 and 5.91, respectively. Only one basic protein, IpDmrt3, was detected, while the others were all acidic proteins. The calculated grand average of the GRAVY values of all IpDmrt genes was -0.2835 to -0.928, indicating that they were hydrophobic in nature (Table1).

Each IpDmrt protein had a characteristic conserved DM domain (Pfam 00,751), and only protein IpDmrt1 had an additional Dmrt1 domain, while a DMA domain was detected in IpDmrt3, IpDmrt4, and IpDmrt5 (Figure 1).

The multiple alignment of IpDmrt protein sequences revealed the presence of a conserved DM domain at the N-terminal (Figure 2), while outside the DM domain there was an extremely lower conservation. Sequence identity analysis of DM domain showed that IpDmrt1 shared a 75% identity with its homologs from IpDmrt2a (74.58%) and IpDmrt3 (66.10%), which were located on the same chromosome (Chr 22). However, IpDmrt1 shared a higher identity with its counterparts from IpDmrt5 (82.76%). Although IpDmrt4 and IpDmrt5 were on the different chromosome, they shared the highest percentage of identity (89.66%) compared to all chromosomes (Table 2).

To analyze the phylogenetic relationships among the IpDmrt gene family members, a phylogenetic tree was generated using 57 Dmrt protein sequences from various vertebrate species, ranging from fish to mammals, based on the maximum likelihood method. As shown in Figure 3, Dmrt1, Dmrt2a, Dmrt3, and Dmrt5 were found in all five teleost species. As expected, Dmrt genes were clustered into six subfamilies, which is consistent with previous studies (Dong et al., 2020; Kikkawa and Osumi, 2021), and each Dmrt gene branched along with its counterparts from different vertebrate species. Within each subfamily, the phylogenetic topology could be further divided into two subgroups: tetrapods and teleosts. Dmrt2 experienced teleost-specific whole genome duplication during evolution, and at least two copies (Dmrt2a and Dmrt2b) were observed in teleosts. However, Dmrt7 and Dmrt8 were lost in teleosts, and are only present in the mammal genome (Veith et al., 2006; Date et al., 2012).

Gene duplication is an important process in species evolution in terms of gene expansion and functional diversity. The aim of synteny analysis is to show whether genes are conserved during evolution in teleosts. Conservative regions can be clearly identified by observing the distribution and permutation of genes on chromosomes. In this study, in order to investigate the preservation extent of the Dmrt genes among teleosts, the distribution and permutation on the corresponding chromosomes of these genes were compared, as well as their neighboring genes from channel catfish, zebrafish, electric eel, Nile tilapia, and Atlantic cod. Seven IpDmrt genes were unevenly distributed on five chromosomes by chromosomal location analysis. Specifically, IpDmrt2b, IpDmrt4, IpDmrt5, and IpDmrt6 were located on Chr11, Chr29, Chr5, and Chr7, respectively; while IpDmrt1, IpDmrt2a, and IpDmrt3 were located on the same chromosome (Chr22).

Syntenic analyses revealed that the Dmrt1-Dmrt2a-Dmrt3 syntenic block was highly conserved in teleosts (Figure 4A). Interestingly, Dmrt5 and neighboring genes were not completely conserved, showing differences in replication direction and location (Figure 4B), which indicated that this region experienced genome arrangement during evolution. On the contrary, the most conservative syntenic block, Irrc40-Irrc7-Insl5a-Ak4-Jak1-Foxd3-Kank4-Cyldl-Adcyaplr1b-Dmrt2b, was detected in channel catfish, electric eel, and zebrafish, despite the location and copy direction of these genes being different in Atlantic cod (Figure 4C). It was also found that Dmrt4 is preserved only in channel catfish, electric eel, and Atlantic cod, while it was lost in zebrafish and Nile tilapia (Figure 4D).

The interaction network of channel catfish Dmrt proteins was constructed using the STRING database. Dmrt1 closely interacted not only with the Amh protein, which is thought to be associated with male sex determination, but also with Cyp19a1, a key downstream gene of female sex regulation (Figure 5A). Both Dmrt2a and Dmrt2b were related to Pxdc1 in the protein interaction network; in addition, Dmrt2a was also associated with Wnt4 (Figures 5B, C); Figures 5D–F showed that there was protein interaction among Dmrt3, Dmrt4, and Dmrt5. Moreover, Dmrt3 was demonstrated to interact with Foxl1, which was also compactly linked with Dmrt6 in the protein interaction network (Figure 5G). This analysis revealed that most genes interacting with Dmrt genes have functions in sex differentiation and gonad development, contributing to understand the mechanism/dynamics of more genes related to sex determination and differentiation in channel catfish and even in teleosts.

To study the spatiotemporal specific expression of IpDmrt genes, their expression profiles were investigated in five tissues obtained from female and male channel catfish, namely the brain, head kidney, kidney, liver, and ovaries or testes using qRT-PCR. Subsequently, the mRNA expression of IpDmrt genes in the ovary tissues of sex-reversed individuals were also explored. The results indicated that IpDmrt1 and IpDmrt6 exhibited a sexually dimorphic expression pattern with higher expression levels in the testes than in the ovaries (p < 0.01, Figures 6A, G). IpDmrt2a, IpDmrt3, and IpDmrt4 were highly expressed in the kidney, head kidney, and liver of male channel catfish (p < 0.05, Figures 6B, D, E). In addition, a sexually dimorphic expression pattern was also detected in IpDmrt2b and IpDmrt5, which presented more significant expression levels in the ovaries than in other tissues (p < 0.05, Figures 6C, F). Also, a relatively high expression level of Dmrt5 was also detected in the kidney, head kidney, and liver of male channel catfish (p < 0.05, Figure 6F).

The qRT-PCR assay was performed to verify the response expression of IpDmrt genes during sex reversal. The expression of IpDmrt2a, IpDmrt3, and IpDmrt4 was not carried out because a sexually dimorphic expression pattern was not observed. Compared with XX channel catfish, IpDmrt2b and IpDmrt5 were significantly up-regulated in the ovaries of XY individuals, while IpDmrt1 and IpDmrt6 were significantly down-regulated. The results indicate that these Dmrt gene family members may play an important role during sex reversal in XY channel catfish.