Two duplicated gsdf homeologs cooperatively regulate male differentiation by inhibiting cyp19a1a transcription in a hexaploid fish.

Although evolutionary fates and expression patterns of duplicated genes have been extensively investigated, how duplicated genes co-regulate a biological process in polyploids remains largely unknown. Here, we identified two gsdf (gonadal somatic cell-derived factor) homeologous genes (gsdf-A and gsdf-B) in hexaploid gibel carp (Carassius gibelio), wherein each homeolog contained three highly conserved alleles. Interestingly, gsdf-A and gsdf-B transcription were mainly activated by dmrt1-A (dsx- and mab-3-related transcription factor 1) and dmrt1-B, respectively. Loss of either gsdf-A or gsdf-B alone resulted in partial male-to-female sex reversal and loss of both caused complete sex reversal, which could be rescued by a nonsteroidal aromatase inhibitor. Compensatory expression of gsdf-A and gsdf-B was observed in gsdf-B and gsdf-A mutants, respectively. Subsequently, we determined that in tissue culture cells, Gsdf-A and Gsdf-B both interacted with Ncoa5 (nuclear receptor coactivator 5) and blocked Ncoa5 interaction with Rora (retinoic acid-related orphan receptor-alpha) to repress Rora/Ncoa5-induced activation of cyp19a1a (cytochrome P450, family 19, subfamily A, polypeptide 1a). These findings illustrate that Gsdf-A and Gsdf-B can regulate male differentiation by inhibiting cyp19a1a transcription in hexaploid gibel carp and also reveal that Gsdf-A and Gsdf-B can interact with Ncoa5 to suppress cyp19a1a transcription in vitro. This study provides a typical case of cooperative mechanism of duplicated genes in polyploids and also sheds light on the conserved evolution of sex differentiation.

Introduction

Polyploidy or whole-genome duplication (WGD) provides extra substrates for genomic evolution and is thus considered to be an important driving force for genetic diversity, trait innovation, and ecological adaption [1-5]. The majority of plants and vertebrates have evolved from polyploid ancestors [6]. Recent polyploidy is also widespread in fishes and amphibians, though it is apparently less frequent than in plants [5,7]. Polyploidy may inherit additional set/sets of chromosomes from the same species (autopolyploidy) or from interspecific hybridization (allopolyploidy) [6]. Homologous chromosomes (and the genes they carry) resulting from allopolyploidy are commonly referred to as homeologs (also homoeologs) [3]. During the initial polyploidization, the neopolyploids are thought to experience genomic chaos resulting from the emergence of duplicated genomes [8-11]. During the subsequent diploidization processes, duplicated genes generated from polyploidy will be eliminated or will evolve divergent functions, and their evolutionary fates are closely associated with the interplay of structural and functional entanglement [12,13]. Recent studies in goldfish and common carp suggest that the subgenomes resulting from the allotetraploidy have continuously rediploidized in a manner from asymmetrical evolution to diverse stabilization [14-16]. The homeologs from subgenomes A and B are co-expressed in most pathways, and their expression dominance shifts temporally during embryogenesis [14]. However, the molecular mechanisms underlying how the duplicated genes co-regulate a biological process in polyploids remains largely unknown.

Except for the two rounds (1R and 2R) of WGD that the common vertebrate ancestor has undergone [17], a fish-specific WGD (3R) is believed to result in the dramatic radiation of teleosts [18, 19]. Intriguingly, the hexaploid gibel carp (Carassius gibelio), a cyprinid fish with a wide distribution across Eurasia [20,21], have undergone two extra rounds of polyploidy [22]. An early allotetraploidy about 10−15 Mya resulted in the formation of tetraploid C. auratus (AABB, 4n = 100) [14, 23], and a late extra autotriploidy from an ancestral tetraploid approximately 0.5 Mya led to the occurrence of hexaploid gibel carp (C. gibelio) (AAABBB, 6n≈150) [22,24]. The hexaploid gibel carp is actually an amphitriploid with two triploid sets of chromosomes derived from both ancestors [22,25,26]. Thus, in hexaploid gibel carp, most genes usually have a total of two homeologs, one from subgenome A and the other from subgenome B, and each homeolog commonly has three alleles, thereby providing an ideal model to investigate cooperative mechanisms of duplicated genes.

The amphitriploid gibel carp has overcome the meiotic obstacle caused by three homologous chromosomes via unisexual gynogenesis, in which the eggs are activated by the sperm of sympatric sexual species (kleptospermy) to initiate embryogenesis using only maternal genetic information [27,28]. In contrast to other unisexual vertebrates, variable male proportions ranging from 1.2% to 26.5% have been discovered in wild populations [29,30]. In our previous studies, we identified a genetic male-specific marker (MSM) in gibel carp [31] and revealed that male-specific supernumerary microchromosomes are closely associated with the occurrence of genotypic males in a dose-dependent relationship [32,33]. When a female gibel carp is mated with a male from other species, a typical gynogenesis is initiated that all the offspring have the same genetic information as the maternal individual. When a female gibel carp is mated with a genotypic amphitriploid male, a variant of gynogenesis is initiated, during which the sperm nuclei are also extruded but some supernumerary microchromosomes of sperm nuclei occasionally leak into the unreduced eggs. This variant of gynogenesis can accumulate microchromosomes, generate males, and create genetic diversity in the offspring [32-35]. However, details concerning the molecular mechanism of male determination and differentiation in this gynogenetic hexaploid fish are limited.

In sharp contrast to the remarkable diversity of sex-determining switches [36-38], the downstream genetic cascades of sex differentiation are relatively conserved [37,39-41]. Members of the transforming growth factor-β (TGF-β) signaling pathway have been identified as being vastly involved in sex determination and differentiation in vertebrates [36,42]. Gonadal somatic cell-derived factor (gsdf), a member of the TGF-β superfamily [43], commonly acts as a male gonad factor during sex determination/differentiation in fish species [36,37,44-49], but the molecular details underlying gsdf-mediated male determination/differentiation remain elusive. In this study, we choose the hexaploid gibel carp and the gsdf gene, as a unique system to analyze the cooperative mechanisms of duplicated genes on polyploid sex differentiation. We identified two divergent gsdf homeologous genes and revealed that the gsdf homeologs cooperatively regulated male differentiation by inhibiting cyp19a1a transcription. And interactive mechanism analyses illustrated that Gsdf-A and Gsdf-B could interact with Ncoa5 to suppress cyp19a1a transcription in vitro.

Results

Characterization of gsdf homeologs and alleles

In the hexaploid gibel carp (C. gibelio), we identified two divergent gsdf homeologs derived from subgenome A (gsdf-A) and subgenome B (gsdf-B), which were localized to chromosomes A21 and B21, respectively (Fig 1A). As gibel carp reproduce via unisexual gynogenesis without meiotic recombination [26,27], single nucleotide polymorphisms (SNPs) specific to each allele were stable through generations [25]. According to the SNPs, the sequenced fragments of each homeolog could be clearly divided into three types, indicating that gsdf-A and gsdf-B had three alleles each (Fig 1B). Subsequently, we cloned the coding sequences of three gsdf-A alleles and three gsdf-B alleles, and found that the average identity between gsdf-A and gsdf-B was 85.36 ± 0.34%, while the average identities among three alleles of gsdf-A and three alleles of gsdf-B were 99.53 ± 0.27% and 99.29 ± 0.36%, respectively (S1A Fig). Interestingly, the deduced amino acid sequences of gsdf-A and gsdf-B were less conserved (average identity = 77.66% ± 0.37%) than their coding sequences (S1B Fig), as most of the differing nucleotides (68.70 ± 1.33%) caused amino acid changes (S1 Table).

Fig 1

Characterization of gsdf-A and gsdf-B in hexaploid gibel carp.

(A) Genomic sequence diagram of two divergent gsdf homeologs including gsdf-A and gsdf-B. The black boxes indicate exons while the white boxes represent introns. Initiation codon and termination codon are marked by green and red arrows, respectively. The primer pairs used to distinguish different alleles and to examine mutant genotypes are indicated by black arrows. Red arrow heads indicate the target sites of guide RNAs (gRNA) used for knockout experiment with CRISPR/Cas9. (B) Fragments of three gsdf-A alleles amplified by primer pair of Detect-F-A and Detect-R-A, and fragments of three gsdf-B alleles amplified by primer pair of Detect-F-B and Detect-R-B. The asterisks indicate SNPs used for distinguishing different alleles. The red sequences in black box display the target sequences of guide RNAs. (C) Genomic structure of gsdf genes among different fish species. Exons and introns are depicted by rectangle boxes and thick lines, respectively. Lengths are exhibited by base pairs (bp). (D) Gene synteny of chromosomal fragments containing gsdf genes. Chromosome numbers are displayed at the left side. Conserved gene blocks are represented in matching colors. Transcription orientations are indicated by arrows. The genes marked in red color are conserved genes across all the analyzed species.

Both gsdf-A and gsdf-B contain five exons and four introns, which is similar to the gsdf genes of C. auratus, Danio rerio, Ictalurus punctatus, Oryzias latipes, and Oreochromis niloticus (Fig 1C). In addition, most genes in the neighborhood around gsdf showed conserved synteny in genomic blocks among these fish species (Fig 1D). Subsequently, we performed sequence alignment of the deduced amino acids and ascertained that the Gsdf-A and Gsdf-B of hexaploid C. gibelio had conserved TGF-β domains as in other fish species, particularly the seven or eight cysteines in this domain (S1B Fig). Phylogenetic reconstruction showed that Gsdf-A of C. gibelio and C. auratus were clustered in one clade, while Gsdf-B of C. gibelio and C. auratus were clustered into another clade (S1C Fig), and these patterns were in accordance with the common allopolyploidy origin shared by these two fish species [14,22,26].

Dynamic transcription of gsdf-A and gsdf-B are mainly activated by dmrt1-A and dmrt1-B, respectively

We first examined gsdf transcription in eight adult organs via relative real-time quantitative polymerase chain reaction (qPCR) and found out that both gsdf-A and gsdf-B mRNAs were distributed exclusively in the gonads, with much higher expression in the testis than in the ovary (Fig 2A). Then, we analyzed the dynamic expression profiles of gsdf-A and gsdf-B in male gonads during developmental stages at 17, 21, 30, 45, 60, 90, 120, 150, 210, 250, 300, and 360 days post hatching (dph). In gibel carp, gonadal morphological differentiation between females and males commonly occurs around 40 dph and gonad mature at about 1 year post hatching [33,50]. The expression of gsdf-A steadily increased with a very slow growth rate before 120 dph, displayed a sharp increase from 150 to 250 dph, and then peaked at 250 dph. The expression of gsdf-B rapidly increased at 30 dph and peaked at 120 dph. After reaching the peak value, the expression levels of both gsdf-A and gsdf-B decreased in the mature testis and remained at a certain level (Fig 2B). Subsequently, we produced a polyclonal antibody against Gsdf that could recognize both Gsdf-A and Gsdf-B (S2 Fig), as the deduced amino acid sequences of Gsdf-A and Gsdf-B were relatively conserved (S1B Fig). Immunofluorescence analysis was performed to assess the cellular distribution of Gsdf proteins (Gsdf-A and Gsdf-B) in male gonads at 30, 90, and 360 dph. According to the fluorescence intensity of the anti-Vasa antibody and nuclear morphology, we could easily distinguish germ cells from somatic cells [50,51]. Along with spermatogenesis, the green signal derived from Gsdf was mostly distributed in the cytoplasm of somatic cells surrounding the germ cells (Fig 2C), which was consistent with the main distribution in Sertoli cells of other fishes [46,47,52,53]. Gsdf, as a member of the TGF-β superfamily, is usually considered to be a secreted ligand that will bind to its cell surface receptors [54,55]. However, Gsdf proteins might also accumulate in cytoplasm before being excreted.

Fig 2

Expression characterization of gsdf-A and gsdf-B.

(A-B) Relative real-time quantitative PCR (qPCR) of gsdf-A/gsdf-B transcripts in adult organs (A) and male gonads at different developmental stages (B). (C) Immunofluorescence co-localization of Gsdf and Vasa in male gonads at 30, 90, and 360 dph (days post hatching). Green and red fluorescence were immunostained by anti-Gsdf antibody and anti-Vasa antibody, respectively, and blue fluorescence was stained by DAPI. PGC: primordial germ cell; SG: spermatogonium; PSP: primary spermatocyte; SSP: secondary spermatocyte; SPZ: spermatozoa. Arrows indicate somatic cells.

By in silico analysis, three predicted Dmrt1-binding sites (S3 Fig) and two predicted Sf1(also named as Nr5a1)-binding sites (S4 Fig) [56] were identified in the upstream sequences of both gsdf-A (from -2080 to +50) and gsdf-B (from -2150 to +50), which were defined as potential promoter of gsdf-A (2130 bp) and gsdf-B (2200 bp), respectively (Fig 3A). Subsequently, we cloned these two potential promoters into a pGL3-Basic luciferase reporter vector to analyze Dmrt1’s capability for activating gsdf promoters. Renilla luciferase plasmid pRL-TK was used as an internal reference. Expression plasmids of Sf1-A/Sf1-B, Dmrt1-A, and Dmrt1-B were constructed, and an empty expression plasmid was used as control. Similar to Nile tilapia [57], gibel carp gsdf transcription was also activated by Dmrt1 in a dose-dependent manner in the presence of Sf1 in Carassius auratus L. blastulae embryonic (CAB) cells (Fig 3B). Intriguingly, for the gsdf-A potential promoter, the transcriptional regulation ability of Dmrt1-A was much stronger than that of Dmrt1-B. For the gsdf-B potential promoter, the transcriptional regulation ability of Dmrt1-B was much stronger than that of Dmrt1-A (Fig 3B). Mutation of the Dmrt1-binding site 1 or 3 on gsdf-A potential promoter led to a decrease in both Dmrt1-A-induced and Dmrt1-B-induced transcriptional activation of gsdf-A. Meanwhile, mutation of the Dmrt1-binding site 1 or 3 on gsdf-B potential promoter resulted in a decrease in both Dmrt1-A-induced and Dmrt1-B-induced transcriptional activation of gsdf-B. However, mutation of Dmrt1-binding site 2 on gsdf-A and gsdf-B did not significantly affect Dmrt1-A/Dmrt1-B-induced transcriptional activation (Fig 3C and 3D). Thus, Dmrt1-binding site 1 and 3 of both gsdf-A and gsdf-B were important for gsdf activation.

Fig 3

Dmrt1 regulates gsdf transcription in the presence of Sf-1.

(A) Putative Dmrt1 and Sf1 cis-regulatory elements in the potential promoters of gsdf-A and gsdf-B. (B) In the presence of Sf1 (Sf1-A 25 ng + Sf1-B 25 ng), Dmrt1-A/Dmrt1-B overexpression activates the promoter activity of gsdf-A/gsdf-B in a dosage-dependent manner (50, 100, 150 ng) in CAB cells. For gsdf-A potential promoter, the transcriptional regulation ability of Dmrt1-A was significantly stronger than that of Dmrt1-B. For gsdf-B potential promoter, the transcriptional regulation ability of Dmrt1-B was significantly stronger than that of Dmrt1-A. The asterisks indicate the significant differences (*P<0.05, **P<0.01). (C-D) 5′-mutation mapping of Dmrt1-binding region on the gsdf-A (C) and gsdf-B (D) potential promoter. Blue boxes indicate predicted Dmrt1-binding sites. Red ‘×’ indicates the mutated binding site. The asterisks indicate the significant differences (*P<0.05, **P<0.01, ***P<0.001, ****P<0.0001, *****P<0.00001). Different letters represent statistical differences (P<0.01). (E-F) qPCR of dmrt1-A/dmrt1-B transcripts in adult organs (E) and male gonads at different developmental stages (F). The highest expression level in each qPCR analysis was used as control and defined as 1. (G) FISH analysis of the dmrt1(Red) and sf-1 (Pink) mRNA, and immunofluorescence analysis of the Gsdf protein (Green) in mature testis. Arrowhead indicates the somatic cells with expression of sf-1, dmrt1, and Gsdf. Scale bars: 25 μm. (H-I) Phylogenetic analysis of dmrt1 (H) and gsdf (I) coding sequences from different fish species. Phylogenetic analysis was performed using the Neighbor-Joining method. The percentage of replicate trees in which the associated taxa clustered together in the bootstrap test (2000 replicates) are shown next to the branches. The tree is drawn to scale, with branch lengths in the same units as those of the evolutionary distances used to infer the phylogenetic tree. The evolutionary distances were computed using the Nei-Gojobori method and are in the units of the number of synonymous differences per sequence. The analysis involved 7 nucleotide sequences. All positions containing gaps and missing data were eliminated. (J-K) Information of dN/dS analysis of dmrt1A/B (J) or gsdfA/B (K). The mode of each gene pairs is selected according to the P value (χ2 test), which is marked in bold. The dmrt1 or gsdf of D. rerio were served as references.

Subsequently, we found out that the expression patterns of dmrt1-A and dmrt1-B in both adult organs and in gonads at different developmental stages (Fig 3E and 3F) were closely associated with the expression patterns of gsdf-A and gsdf-B, respectively (Fig 2A and 2B). Fluorescence in situ hybridization (FISH) analysis showed that dmrt1 and sf1 mRNA were co-expressed in some Gsdf positive somatic cells in testis (Fig 3G). In addition, we performed dN/dS analyses of dmrt1 and gsdf genes to check whether one or the other homeologs are under selection. Using D. rerio as reference, the dN/dS value of dmrt1-A was significant lower than that of dmrt1-B (χ2 test p value: 7.93×10−4) under the two-ratio model (Fig 3H and 3J). On the other side, the dN/dS analysis of gsdf homeologs met one-ratio model and display the same value (Fig 3I and 3K). These results indicate that the dmrt1 and gsdf homeologs are under asymmetric and symmetric purifying selection, respectively. Thus, the differential expression between gsdf-A and gsdf-B might be resulted from the divergent evolution of dmrt1-A and dmrt1-B.

Deficiency of gsdf-A or/and gsdf-B leads to partial/complete male-to-female sex reversal

To uncover the function of gsdf in male development, we performed loss-of-function analysis using CRISPR/Cas9 in the hexaploid C. gibelio with three alleles of gsdf-A and three alleles of gsdf-B. First, the gsdf-A gRNA/Cas9 protein (S5A Fig) or gsdf-B gRNA/Cas9 protein (S5B Fig) were injected into the fertilized eggs between a WT female gibel carp without MSM (MSM−) (P: parental generation) and a male common carp to initiate typical gynogenesis [32-35]. All the individuals of the G0 generation were females (MSM−) with gsdf-A chimeric mutations (S5A Fig) or gsdf-B chimeric mutations (S5B Fig). Secondly, some of the G0 individuals (MSM−) were mated with a WT male gibel carp with MSM (MSM+) to initiate a variant of gynogenesis, in which some supernumerary microchromosomes of sperm nuclei could occasionally leak into eggs and lead to male occurrence in the offspring [32-35]. The individuals of the G1 generation had various gsdf-A genotypes (S5A Fig) or various gsdf-B genotypes (S5B Fig). Thirdly, to obtain high proportions of genotypic males in the G2 generation, we chose the sex-reversed phenotypic females (MSM+) from the G1 population as maternal fish (S5 Fig). Two gsdf-A mutants with MSM (gsdf-A+1/Δ7/Δ4 + gsdf-B+/+/+ MSM+, gsdf-AΔ4, +9/Δ4/Δ7 + gsdf-B+/+/+ MSM+) (S5A Fig) and two gsdf-B mutants with MSM (gsdf-A+/+/+ + gsdf-BΔ2/Δ1/Δ2 MSM+, gsdf-A+/+/+ + gsdf-BΔ1/Δ4/Δ5,+1 MSM+) (S5B Fig) were selected from the G1 population as maternal fish and were separately mated with a WT male gibel carp (MSM+) to construct G2 mutant lines and families. Each symbol separated by a “/” represents one allele of the hexaploid genome. Finally, we established two mutant lines of gsdf-A (gsdf-A+1/Δ7/Δ4 + gsdf-B+/+/+ and gsdf-AΔ4,+9/Δ4/Δ7+ gsdf-B+/+/+) (Figs S5A, 4A and 4B) and two mutant lines of gsdf-B (gsdf-A+/+/+ + gsdf-BΔ2/Δ1/Δ2 and gsdf-A+/+/+ + gsdf-BΔ1/Δ4/Δ5,+1) (Figs S5B, 4C and 4D).

Fig 4

Genotypes and phenotypes of gsdf mutants.

(A-F) Genotypes of gsdf mutants: gsdf-A mutant line 1 (gsdf-A+1/Δ7/Δ4 + gsdf-B+/+/+) (A), gsdf-A mutant line 2 (gsdf-AΔ4,+9/Δ4/Δ7 + gsdf-B+/+/+) (B), gsdf-B mutant line 1 (gsdf-A+/+/+ + gsdf-BΔ2/Δ1/Δ2) (C), gsdf-B mutant line 2 (gsdf-A+/+/+ + gsdf-BΔ1/Δ4/Δ5,+1) (D), gsdf-A/gsdf-B double mutant family 1 (gsdf-AΔ4,+9/Δ4/Δ7 + gsdf-B chimeric mutations) (E), and gsdf-A/gsdf-B double mutant family 2 (gsdf-A chimeric mutations + gsdf-BΔ2/Δ1/Δ2) (F). (G) Gonadal morphology and histology of adult gonads. #, sex reversal; I, primary oocyte; II, growth stage oocyte; III, vitellogenic oocyte; IV, maturing oocyte. Bars are shown at bottom-right of the images. MSM+, with MSM; MSM−, without MSM; WT, wild type.

In addition, two families of gsdf-A/gsdf-B double mutants were also established by injecting gsdf-B gRNA/Cas9 protein into the fertilized eggs of the gsdf-A mutant line (gsdf-AΔ4,+9/Δ4/Δ7 + gsdf-B chimeric mutations) (Figs S5A and 4E) and injecting gsdf-A gRNA/Cas9 protein into the fertilized eggs of the gsdf-B mutant line (gsdf-A chimeric mutations + gsdf-BΔ2/Δ1/Δ2) (Figs S5B and 4F). The genotypes of all mutant lines and families were shown in the Fig 4A–4F. As gibel carp reproduce via unisexual gynogenesis without meiotic recombination [26,27], different alleles may have different mutations in one mutant individual [25]. For instance, all the individuals in the gsdf-A mutant line (gsdf-A+1/Δ7/Δ4 + gsdf-B+/+/+ MSM+) had the same genotype where the first allele of gsdf-A had a 1-bp insertion, the second allele of gsdf-A had a 7-bp deletion, the third allele of gsdf-A had a 4-bp deletion, and three alleles of gsdf-B were all wild type genotype without mutations (Fig 4A).

Knockout of gsdf-A or gsdf-B led to partial male-to-female sex reversal (80.6% in line gsdf-A+1/Δ7/Δ4 + gsdf-B+/+/+, 85.3% in line gsdf-AΔ4,+9/Δ4/Δ7 + gsdf-B+/+/+, 83.6% in line gsdf-A+/+/+ + gsdf-BΔ2/Δ1/Δ2, and 80.0% in line gsdf-A+/+/+ + gsdf-BΔ1/Δ4/Δ5,+1), while disruption of both gsdf-A and gsdf-B resulted in complete male-to-female sex reversal (Figs S5 and 4G). These results indicate that gsdf-A and gsdf-B both have male differentiation functions.

Disruption of gsdf-A/gsdf-B results in cyp19a1a upregulation and aromatase inhibitor (AI) treatment rescues the male-to-female sex reversal

Gonadal morphological differentiation of gibel carp commonly occurs around 40 dph, during which the ovarian cavity is present in female gonads but absent in male gonads [33,50]. We performed analyses of gonadal histology and sex-related gene expression at developmental stages of 25 dph and 55 dph, respectively. At 25 dph, histological examinations in one type of gsdf-A mutants (gsdf-AΔ4,+9/Δ4/Δ7+ gsdf-B+/+/+ MSM+), one type of gsdf-B mutants (gsdf-A+/+/+ + gsdf-BΔ2/Δ1/Δ2 MSM+), and two gsdf-A/gsdf-B double mutants (gsdf-AΔ4,+9/Δ4/Δ7 + gsdf-B chimeric mutations MSM+ and gsdf-A chimeric mutations + gsdf-BΔ2/Δ1/Δ2 MSM+) showed that these gonads were similar to WT females (MSM−) and males (MSM+), all of which were at the undifferentiated developmental stage (Fig 5A). Compared with WT male gonads (MSM+), the female differentiation markers cyp19a1a and foxl2b (forkhead box L2b) were clearly upregulated in the gonads of gsdf-A mutants (gsdf-AΔ4,+9/Δ4/Δ7+ gsdf-B+/+/+ MSM+), gsdf-B mutants (gsdf-A+/+/+ + gsdf-BΔ2/Δ1/Δ2 MSM+), and gsdf-A/gsdf-B double mutants (gsdf-AΔ4,+9/Δ4/Δ7 + gsdf-B chimeric mutations MSM+ and gsdf-A chimeric mutations + gsdf-BΔ2/Δ1/Δ2) (Fig 5C). However, the male differentiation markers dmrt1 and amh (anti-Müllerian hormone) were also highly expressed in gsdf-A or gsdf-B single mutants (Fig 5C). Furthermore, the expression levels of primordial germ cell markers piwil1 (piwi-like RNA-mediated gene silencing 1) and dnd (dead end) in these four kinds of gsdf mutants (MSM+) were between those in WT males (MSM+) and WT females (MSM−) (Fig 5C). Intriguingly, compensatory high expression of gsdf-B was observed in gonads of gsdf-A mutants (gsdf-AΔ4,+9/Δ4/Δ7+ gsdf-B+/+/+ MSM+), and compensatory high expression of gsdf-A was detected in gonads of gsdf-B mutants (gsdf-A+/+/+ + gsdf-BΔ2/Δ1/Δ2 MSM+) at 25 dph (Fig 5E).

Fig 5

Aromatase inhibitor treatment rescues the male-to-female sex reversal in gsdf mutants.

(A-B) Gonadal histology of different gsdf mutants and wild type individuals at 25 (A) and 55 dph (B). dph, days post hatching; #, sex reversal; star symbol, ovarian cavity; MSM+, with MSM; MSM−, without MSM; WT, wild type. (C-D) Gonadal gene expression of different gsdf mutants and wild type individuals at 25 (C) and 55 dph (D), including female marker genes cyp19a1a and foxl2b, male marker genes dmrt1 and amh, and primordial germ cell marker genes piwil1 and dnd. (E) Gsdf-A compensatory expression in the gonads of gsdf-B mutants and gsdf-B compensatory expression in the gonads of gsdf-A mutants at 25 dph. (F-G) Gonadal histology (F) and gene expression (G) of gsdf-A/gsdf-B double mutant individuals (gsdf-AΔ4,+9/Δ4/Δ7 + gsdf-B chimeric mutations, MSM+), gsdf-A/gsdf-B double mutant individuals (gsdf-AΔ4,+9/Δ4/Δ7 + gsdf-B chimeric mutations, MSM+) with aromatase inhibitor (Letrozole) treatment, and wild type individuals at 100 dph. Different letters represent statistical differences (P<0.05). The highest expression level in each qPCR analysis was used as control and defined as 1.

At 55 dph, the gonads of WT females (MSM−) and males (MSM+) differentiated into ovaries and testes, respectively (Fig 5B). Via histological examination, the gonads of gsdf-A mutants (gsdf-AΔ4,+9/Δ4/Δ7+ gsdf-B+/+/+ MSM+) and gsdf-B mutants (gsdf-A+/+/+ + gsdf-BΔ2/Δ1/Δ2 MSM+) could be clearly distinguished into ovaries and testes, whereas the gonads of gsdf-A/gsdf-B double mutants (gsdf-AΔ4,+9/Δ4/Δ7 + gsdf-B chimeric mutations MSM+ and gsdf-A chimeric mutations + gsdf-BΔ2/Δ1/Δ2 MSM+) all developed into ovaries (Fig 5B). Compared with the WT males (MSM+), the expression levels of cyp19a1a and foxl2b were highly elevated, while the expression levels of dmrt1 and amh were repressed in all the four kinds of gsdf mutants (MSM+) with ovaries (Fig 5D). In addition, in the gsdf-A mutants (gsdf-AΔ4,+9/Δ4/Δ7 + gsdf-B+/+/+ MSM+) and gsdf-B mutants (gsdf-A+/+/+ + gsdf-BΔ2/Δ1/Δ2 MSM+) with testes, the expression levels of all the examined genes were similar to those in WT males (MSM+) (Fig 5D).

Although gonads at 25 dph could not be distinguished as ovaries or testes by morphological analysis, the expression levels of marker genes in the gsdf mutants, especially the gsdf-A/gsdf-B double mutants, could provide clues to sexual fate (Fig 5C and 5D). The gonadal aromatase gene cyp19a1a is a conserved factor of ovarian differentiation in fish [58,59] and also has been demonstrated to play a role in female differentiation in hexaploid C. gibelio (S6 Fig). In the undifferentiated gonads of all gsdf mutants (MSM+), the ovarian aromatase gene cyp19a1a was dramatically upregulated (Fig 5C), indicating that deficiency of gsdf might abolish its repression of cyp19a1a. Thus, we used nonsteroidal aromatase inhibitor letrozole to analyze whether blockage of Cyp19a1a enzyme activity could rescue the male-to-female sex reversal caused by gsdf dysfunction. As expected, administration of letrozole from 15 to 55 dph prevented male-to-female sex reversal in 84.4% of gsdf-A/gsdf-B double mutants (MSM+) (Fig 5F). As the period before 15 dph was key developmental stages of sex determination/differentiation, letrozole treatment after 15 dph might be the reason that 15.6% gsdf-A/gsdf-B double mutants still developed into phenotypic females. And the gene expression patterns of these letrozole treated testes were similar to that in WT males (MSM+), except for cyp19a1a and foxl2b (Fig 5G). Upregulation of cyp19a1a and its active transcriptional factor foxl2b [25,60] in the testis of gsdf-A/gsdf-B double mutants (MSM+) subjected to letrozole treatment may have been caused by inhibition of Cyp19a1a enzyme activity. In addition, cyp19a1a mRNA was co-expressed with Gsdf in some somatic cells of mature testis (S7 Fig). These results indicate that gsdf-A and gsdf-B co-inhibit cyp19a1a expression, resulting in male differentiation in WT males (MSM+).

Identification of Gsdf-Ncoa5 interaction via yeast two-hybrid assay and co-immunoprecipitation

As a member of TGF-β superfamily, Gsdf is commonly considered to be a ligand to bind to its cell surface receptors [54]. To identify potential interaction membrane proteins of secreted Gsdf, the coding sequence of Gsdf-A mature peptide was cloned into pBT3-SUC (pBT3-SUC-Gsdf-A mature peptide) as a bait to perform yeast two hybrid assay via DUAL membrane system (Dualsystems Biotech). However, screens did not yield any interactors (S8 Fig). Subsequently, the open reading frame of gsdf-A was cloned into pGBKT7 (pGBTKT7-Gsdf-A) as bait to perform yeast two hybrid assay via GAL4 system (Clontech). A total of 239 positive transformants were selected, and their plasmids were isolated for sequencing. These obtained coding sequences belonged to 34 genes according to the genome and transcription data of gibel carp [26,33]. To exclude false positive results, the full-length coding sequences of the 34 genes were cloned into vector pGADT7-AD and co-transformed with gsdf-A-pGBTKT7 separately. Finally, a total of 27 proteins were confirmed to be the potential interaction partners of Gsdf-A (Fig 6A and 6B).

Fig 6

Identification of Gsdf-Ncoa5 interaction via yeast two-hybrid assay and co-immunoprecipitation.

(A) Confirmation of 34 candidate interacting proteins of Gsdf-A via yeast two-hybrid assay. +: pGBKT7–53 and pGADT7-T co-transformed as positive control; −: pGBKT7-Lam and pGADT7-T co-transformed as negative control. (B) List of the 27 potential interaction partners of Gsdf-A. (C-D) qPCR analysis of cyp19a1a expression in the CAB cells transfected with different plasmids. Different letters represent statistical differences (P<0.01). The highest expression level in each qPCR analysis was used as control and defined as 1. (E) Co-IP of Gsdf-A-Flag and Gsdf-B-Flag with Ncoa5-HA in CAB cells transfected with the indicated plasmids. Anti-Flag Ab was used for Co-IP. Sf1: Sf1-A and Sf1-B; Foxl2b: Foxl2b-A and Foxl2b-B; Ncoa5: Ncoa5-B.

We overexpressed these 27 isolated genes in order to identify whether these genes were involved in sex differentiation pathway, by analyzing transcription of sex differentiation genes such as cyp19a1a, foxl2b, dmrt1, and amh. In CAB cells, amh had no constitutive expression, so we obtained the data of the rest three sex differentiation genes (S9 Fig). Among the 27 proteins, a total of 6 proteins could activate cyp19a1a transcription significantly, however, only Ncoa5 activated cyp19a1a transcription but did not change expression levels of foxl2b and dmrt1 (S9 Fig). Besides, Ncoa5 was revealed to be involved in the regulating pathway of sex hormones [61,62] and cyp19a1a was one of the most important downstream genes of gsdf (Fig 5), so we selected Ncoa5 for subsequent analyses. There were two homeologs of ncoa5 (ncoa5-A and ncoa5-B) in the gibel carp genome (S10A and S10C Fig) and the protein isolated from yeast two-hybrid assay was Ncoa5-B (Fig 6B). As the protein sequences between Ncoa5-A and Ncoa5-B was highly conserved (identity = 91.53%) (S10C Fig), we only used Ncoa5-B for subsequent in vitro experiments.

Subsequently, we found that the presence of Gsdf-A/Gsdf-B could eliminate Ncoa5-induced upregulation of cyp19a1a expression (Fig 6C), but could not eliminate Foxl2b/Sf1-induced upregulation of cyp19a1a (Fig 6D). In addition, the interactions of Gsdf-A/Gsdf-B and Ncoa5 were confirmed by co-immunoprecipitation, in which the anti-Flag Ab-immunoprecipitated protein Ncoa5 was recognized by the anti-HA Ab (Fig 6E). These findings indicate that Gsdf-A and Gsdf-B both interact with Ncoa5 to regulate cyp19a1a transcription in vitro.

Ncoa5 participates in cyp19a1a regulation via interaction with Rora

In humans, Rora is known to interact with Ncoa5 to enhance cyp19a1a transcription [61,62]. It would be interesting to know whether gibel carp Rora and Ncoa5 are involved in the expression modulation of cyp19a1a. We identified two homeologs of roraα (roraα-A and roraα-B) and two homeologs of roraβ (roraβ-A and roraβ-B) in the gibel carp genome (S10B and S10D Fig). The sequence identities between Roraα-A/Roraα-B and human RORA was much higher than those between Roraβ-A/Roraβ-B and human RORA (S10D Fig). As the protein sequences between Roraα-A and Roraα-B (identity = 91.88%) was highly conserved (S10D Fig) and Roraα-B had higher identity than Roraα-A compared with human ortholog, we used Roraα-B for subsequent in vitro experiments.

In CAB cells, co-immunoprecipitation showed that Rora could interact with Ncoa5 (Fig 7A). In addition, Rora activated cyp19a1a transcription in a dose-dependent manner in the presence of Ncoa5 (Fig 7B). Potential Rora-binding sites were predicted in the promoter of cyp19a1a (Figs 7C and S11) [63]. Mutation of the potential Rora-binding sites resulted in a decrease in the Rora/Ncoa5-induced transcriptional activation of cyp19a1a but did not affect Foxl2b/Sf1-induced upregulation of cyp19a1a, indicating that these binding sites were specific to Rora/Ncoa5 (Fig 7D). Subsequently, the Rora-binding site 1 was further confirmed via chromatin immunoprecipitation (ChIP) in CAB cells transfected with Rora-Myc vector. The PCR band containing the binding site 1 of Rora was detected in the chromatin precipitated with the Myc antibody, while no band was observed in the negative control chromatin that was precipitated with nonspecific IgG (Fig 7E). Besides, FISH analysis showed that rora and ncoa5 mRNA were co-expressed in Cyp19a1a positive somatic cells of mature testis (Fig 7F). Thus, these findings indicate that Rora positively regulates transcription of cyp19a1a by binding to the promoter in the presence of Ncoa5.

Fig 7

Rora interacts with Ncoa5 to regulate the transcriptional activity of cyp19a1a.

(A) Co-IP of Rora-Myc with Ncoa5-HA in CAB cells transfected with the indicated plasmids. Anti-HA Ab was used for Co-IP. (B) In the presence of Ncoa5 (50 ng), Rora overexpression activates cyp19a1a promoter in a dosage-dependent manner in EPC cells. Different letters represent statistical differences (P<0.01). (C) Putative Rora cis-regulatory elements in the potential promoter of cyp19a1a. (D) 5′-mutation mapping of Rora-binding region on the cyp19a1a potential promoter. Red boxes indicate predicted Rora binding sites. Black ‘X’ indicates the mutated binding site. Different letters represent statistical differences (P<0.01). (E) ChIP-PCR assay in CAB cells transfected with Rora-Myc vector. Specific primers were used to amplify the fragment spanning the Rora binding site on the cyp19a1a promoter. The PCR products were detected by 1.5% agarose gel electrophoresis. IgG antibody-based ChIP assay was used as a negative control. (F) FISH analysis of the rora (Pink) and ncoa5 (Red) mRNA, and immunofluorescence analysis of the Cyp19a1a protein (Green) in mature testis. Arrowhead indicates the somatic cells with expression of rora, ncoa5, and Cyp19a1a. Scale bars: 25 μm.

Gsdf-A and Gsdf-B both inhibit cyp19a1a transcription via competitive interaction with Ncoa5

During sex differentiation, expression of gsdf was closely negatively associated with expression of cyp19a1a in female and male gonads (Fig 8A). The expression of ncoa5 had no significant difference between females and males in gonads at early developmental stages, while the gonadal expression of rora has no difference between females and males before 45 dph but display female-biased expression at 60 dph (Fig 8A). FISH analysis showed that rora and ncoa5 mRNA were co-expressed in some Gsdf positive somatic cells of mature testis (Fig 8B). To elucidate how gsdf-A or gsdf-B regulates cyp19a1a expression, we performed an overexpression analysis, and the overexpression of Gsdf-A or Gsdf-B significantly repressed cyp19a1a transcription in vitro accompanied by downregulation of rora (Fig 8C and 8D). However, the expression of ncoa5 was not affected by Gsdf-A or Gsdf-B overexpression in tissue culture cells (Fig 8C and 8D), consistent with the in vivo result. Meanwhile, the cyp19a1a transcription induced by Rora and Ncoa5 was repressed by Gsdf-A and Gsdf-B in a dose-dependent manner (Fig 8E). Furthermore, siRNAs against both ncoa5-A and ncoa5-B were designed, and the repression of Gsdf-A or Gsdf-B on cyp19a1a transcription was abolished by knockdown of both ncoa5-A and ncoa5-B (Fig 8F). The competitive interaction analysis revealed that Rora recruitment by Ncoa5 decreased upon the participation of Gsdf-A or Gsdf-B but was not affected by the participation of other proteins such as female differentiation factor Foxl2b and immune-related factor Sting (Fig 8G). Thus, these results indicate that the upregulation of Gsdf-A and Gsdf-B increases the interaction of Ncoa5 with both homeologs and reduces the interaction of Ncoa5 with Rora, leading to the downregulation of cyp19a1a and subsequent male differentiation.

Fig 8

Gsdf inhibits cyp19a1a transcription via competitive interaction with Ncoa5.

(A) Gene expression detected by qPCR in gonads during different developmental stages. MSM+, wild type males with MSM; MSM−, wild type females without MSM; (B) FISH analysis of the rora (Pink) and ncoa5 (Red) mRNA, and immunofluorescence analysis of the Gsdf protein (Green) in mature testis. Arrowhead indicates the somatic cells with expression of rora, ncoa5, and Gsdf. Scale bars: 25 μm. (C-D) Gene expression detected by qPCR after overexpression of Gsdf-A (C) and Gsdf-B (D). The asterisks indicate the significant differences (*P<0.05, **P<0.01). (E) In the presence of Ncoa5 and Rora, Gsdf-A/Gsdf-B overexpression inhibits the promoter activity of cyp19a1a in a dosage-dependent manner in EPC cells. Different letters represent statistical differences (P<0.05). (F) Effect of ncoa5 (ncoa5-A and ncoa5-B) RNAi on the Gsdf-A/Gsdf-B-induced repression of cyp19a1a. qPCR analysis of ncoa5 and cyp19a1a in the EPC cells after transfection with different plasmids and ncoa5-A/B siRNA for 24 h (*P<0.05). NC, negative control; Si, ncoa5-A/B siRNA. (G) Co-IP of Ncoa5-Flag and Rora-HA with Gsdf-A-Myc, Gsdf-B-Myc, Foxl2b-Myc, and Sting-Myc in EPC cells transfected with the indicated plasmids. Anti-Flag Ab was used for Co-IP. Black arrow, gray arrow, star, and arrow head indicate bands of Gsdf-A-Myc, Gsdf-B-Myc, Foxl2b-Myc, and Sting-Myc, respectively. The asterisks indicate the decreased Rora-HA. The highest expression level in each qPCR analysis was used as control and defined as 1.

Discussion

Polyploidy provides a source of new genes and these duplicated genes will be eliminated/pseudogenized or evolve a sub/neo function during the evolutionary trajectory [2,12,25]. The hexaploid gibel carp (AAABBB) with extra two rounds of polyploidy origins has retained most of the duplicates, where most genes usually have two homeologs, and each homeolog commonly has three alleles [22,25,26,64]. Recently, asymmetrical evolution, homoeologous exchanges, and expression divergence of subgenomes A and B have been observed in allotetroploid goldfish, common carp, and hexaploid gibel carp [14,65]. And we also have presented functional divergence of foxl2 and viperin homeologs in hexaploid gibel carp [25,64]. Here, we found expression divergence between gsdf-A and gsdf-B (Fig 2A and 2B), but knockout gsdf-A or gsdf-B displayed similar sex-reversal rates (S5 Fig), indicating the contribution of each homeolog is similar in male differentiation. In addition, disruption of gsdf-A or gsdf-B triggers highly compensatory expression of gsdf-B or gsdf-A during the critical period of sex differentiation (Fig 5E), and missing either gsdf-A or gsdf-B does not give complete sex reversal but missing both does, suggesting that gsdf-A and gsdf-B cooperatively regulate male differentiation in gibel carp.

An intriguing finding of this study is the revelation of potential molecular rationales underlying male differentiation mediated by gsdf homeologs. In male gibel carp with MSM (MSM+), high expression of gsdf-A and gsdf-B in somatic cells suppresses cyp19a1a to induce Sertoli cell development and male differentiation. In female individuals without MSM (MSM−), the low levels of Gsdf-A and Gsdf-B cannot inhibit cyp19a1a transcription, leading to estrogen production, granulosa cell development, and female differentiation (Fig 9). Besides, in vitro analyses revealed that Gsdf-A and Gsdf-B can interact with Ncoa5 to block Ncoa5 interaction with Rora, inhibiting Rora/Ncoa5-induced activation of cyp19a1a (Fig 9). Commonly, as a member of TGF-β superfamily, Gsdf is considered to be a secreted ligand to bind to its cell surface receptors [54]. Here we demonstrated that Gsdf might also have functions in cells. However, we still do not known whether other factors are involved in the interaction between Gsdf and Ncoa5.

Fig 9

Hypothetical molecular mechanism underlying gsdf-A/gsdf-B-mediated male differentiation in gibel carp.

In MSM+ individuals, highly expressed Gsdf-A and Gsdf-B can inhibit cyp19a1a to induce Sertoli cell development and male development. In MSM− individuals, lowly expressed Gsdf-A and Gsdf-B cannot inhibit cyp19a1a, leading to estrogen production, granulosa cell development, and female development. In vitro, Gsdf-A and Gsdf-B interact with Ncoa5 and blocks Ncoa5 interaction with Rora, resulting in the reduction of Rora/Ncoa5-induced activation on cyp19a1a. The expressions of gsdf and cyp19a1a are mainly in the somatic cells around germ cells.

As we known, Foxl2/Sf1-cyp19a1a pathway is an important pathway of female sex differentiation and knockout of foxl2 results in female to male sex reversal in fish [25,66]. We suppose that the pathway of Rora/ Ncoa5 induced cyp19a1a activation is not independent of Foxl2/Sf1-cyp19a1a pathway. For instance, the cyp19a1a expression change induced by Foxl2/Sf1 can affect estradiol synthesis, which may lead to expression changes of er (estrogen receptor) and rora in the presence of Ncoa5 and then affect cyp19a1a transcription via Rora/Ncoa5-cyp19a1a pathway [61,62]. Thus, these two pathways of cyp19a1a regulation may interact with each other.

The sexual phenotype is the result of antagonism between the female and male pathways, with multiple feedback loops that are influenced by genotypic and/or environmental factors [37]. In hexaploid gibel carp, we have found that gsdf and cyp19a1a play antagonistic roles in sex differentiation. Gsdf represses cyp19a1a by blocking Ncoa5’s availability for activation of cyp19a1a transcription. Meanwhile, cyp19a1a also has the ability to downregulate gsdf by suppressing gsdf’s transcription factor dmrt1 [59]. Antagonistic actions of dmrt1 and foxl2 have been found in many other vertebrates [67-70] and these two genes also display conserved expression patterns during sex differentiation in gibel carp (Fig 5) [25,71]. In addition, Foxl2 also has been demonstrated to be a positive transcriptional factor of cyp19a1a as previously reported [60], indicating that Dmrt1 and Foxl2 may also play conserved antagonistic roles in gibel carp. As shown in previous studies, on one hand upregulation of cyp19a1a would lead to dmrt1 inhibition while on the other it would upregulate foxl2.

In this study, we have identified two duplicated gsdf homeologous genes, gsdf-A and gsdf-B, and each homeolog has three alleles in the gynogenetic hexaploid gibel carp. The transcription of gsdf-A and gsdf-B is mainly activated by dmrt1-A and dmrt1-B, respectively. Moreover, loss-of-function experiments reveal the cooperative ability of two gsdf homeologs to regulate male differentiation by interacting cyp19a1a transcription. And the interactive mechanism analyses demonstrate that Gsdf interacts with Ncoa5 to suppress cyp19a1a transcription in vitro. This study provides a typical case of cooperative mechanism of duplicated genes in polyploids and also sheds light on the conserved evolution of sex differentiation.

Materials and methods

Ethics statement

Animal experiments and treatments were performed according to the Guidelines for Animal Care and Use Committee of Institute of Hydrobiology, Chinese Academy of Sciences (IHB, CAS, Protocol No. 2016–018).

Fishes and cells

Experimental fish species including hexaploid gibel carp (C. gibelio) and red common carp (C. carpio) were provided and raised by the National Aquatic Biological Resource Center (NABRC), Institute of Hydrobiology, Chinese Academy of Sciences, Wuhan, China. Fish cell line Carassius auratus L. blastulae embryonic (CAB) cells and epithelioma papulosum cyprini (EPC) cells were maintained at 28°C in 5% CO2 in medium 199 (Invitrogen) supplemented with 10% fetal bovine serum (FBS) (Invitrogen).

Cloning and sequence analysis

The divergent gsdf homeologs including gsdf-A and gsdf-B were identified according to the assembled genome of hexaploid gibel carp (C. gibelio) (Genbank accession numbers: PRJNA546443). Full-length cDNAs of gsdf-A and gsdf-B were obtained by 5’ and 3’ rapid amplification of cDNA ends (RACE) (SMARTer RACE 5’/3’ Kit, Clontech) using testicular cDNA library. Specific primers for RACE amplification (S2 Table) were designed according to the genome sequences of gsdf-A and gsdf-B. Multiple alignments of gsdf genomic and cDNA sequences was performed by DNAMAN 8.0 software.

The deduced amino acid sequences were predicted by DNAMAN 8.0 software. Multiple alignments of deduced amino acid sequences was performed by ClustalX program and exhibited by Bioedit program. Phylogenetic construction was adjusted by bootstrap analysis (1000 replicates) using the neighbor-joining method (NJ) in MEGA version 7.0 [72]. All the amino acid sequences of Gsdf in other fishes used in this analysis were obtained from Genebank. The accession number are as follows: C. auratus Gsdf-A, XP_026110442.1; C. auratus Gsdf-B, XP_026111149.1; D. rerio Gsdf, XP_017208308.1; I. punctatus Gsdf, XP_017347335.1; O. latipes Gsdf, BAJ05045.1 and O. niloticus Gsdf, BAJ78985.1. Syntenic analyses were conducted by comparing the chromosomic regions around gsdf genes in different fish species, including C. gibelio chromosomes (CgA21 and CgB21), C. auratus chromosomes (CaA21 and CaB21), D. rerio chromosome 21, I. punctatus chromosome 18, O. latipes chromosome 12, and O. niloticus chromosome 7. These information was obtained from GenBank (http://www.ncbi.nlm.nih.gov/, last accessed August 12, 2021).

RNA extraction and qPCR

Adult organs, including heart, liver, hypothalamus, pituitary, kidney, spleen, ovary and testis, were isolated from three mature WT females (MSM-) and three mature WT males (MSM+), respectively. Male individuals were obtained from the offspring of a WT female mating with a WT male and all the female offspring were excluded by PCR detection using the male-specific marker. WT male gonads at different developmental stages were carefully dissected, and a total of 25, 25, 20, 10, 5, 5, 5, 5, 5, 5, 5, and 5 gonads were pooled for RNA extraction at the developmental stages of 17, 21, 30, 45, 60, 90, 120, 150, 210, 250, 300, and 360 dph, respectively. Besides, a total of 25, 5, 5 gonads from gsdf mutants and corresponding WT individuals were pooled for RNA extraction at the stages of 25, 55, 100 dph, respectively.

Total RNA isolation was performed using SV Total RNA isolation System (Promega), and the isolated RNAs were reverse-transcribed by the PrimeScript RT Reagent Kit (Takara). qPCR was performed on S1000 Thermal Cycler (BioRad), using iQSYBR Green Supermix (BioRad) as described previously [73]. β-actin was used as internal reference. All samples were analyzed in triplicates, and relative expression level of target gene was calculated with 2-ΔΔCT method. The highest expression level in each qPCR analysis was used as control and defined as 1 separately. Data were displayed as mean ± standard deviation. Significant differences were calculated by one-way ANOVA followed by Tukey test.

Polyclonal anti-Gsdf antibody preparation and western blot

The gsdf-A cDNA sequence coding for 170 amino acids (S1 Fig) was cloned into Frd-GST vector (Friendbio Science and Technology) and the prokaryotic fusion protein was used as antigen to immunize a rabbit. Anti-Gsdf polyclonal antibody was produced by Friendbio Science and Technology Company Limited (Wuhan). Sample protein was extracted from cells using RIPA Lysis Buffer (Beyotime). Western blot detection was performed according to the previous reports and β-actin was used as internal control [74]. The images were obtained by ImageQuant LAS 4000mini (GE).

Histological analysis and immunofluorescence

The gonads of gibel carp were fixed with 4% paraformaldehyde in PBS at 4°C over night. After washing with PBS, the samples were immersed in 30% saccharose-PBS buffer for 5 h at 4°C, embedded in paraffin, and then were cut into 4μm sections. Hematoxylin-eosin staining was performed as described previously [50]. Immunofluorescence co-localization of Gsdf and Vasa was performed as described previously [71]. The images were obtained by upright fluorescence microscope Axio Imager M2 (Carl Zeiss).

Fluorescence in situ hybridization (FISH)

Probes for ncoa5 and dmrt1 antisense/sense digoxigenin-labeled RNA strands were transcribed in vitro using the DIG RNA labeling kit (Roche). Probes for roar, cyp19a1a, and sf-1 antisense/sense fluorescein-labeled RNA strands were transcribed in vitro using the Fluorescein RNA labeling kit (Roche). Specific primers with a T7 RNA polymerase promoter were designed to amplify complementary DNA (cDNA) fragment of each gene (S2 Table). Each probe was used at a final concentration of 0.5ng/μL. For more sensitive fluorescence in situ hybridization detection, the tyramide signal amplification TSA Plus Cyanine 3/Cyanine 5 System (PerkinElmer Life Science) was used according to the manufacturer’s instructions. Digoxigenin-labeled RNA was stained with cy5, fluorescein -labeled RNA was stained with cy3. FISH analyses using sense RNA strands were shown in S12 Fig.

Evolutionary analysis of gsdf or dmrt1 homeologs

To investigate the potential role of selection on the evolution of gsdf or dmrt1 homeologs, the gsdf or dmrt1 gene dataset was assessed by branch model tests [75]. Alternative branch models, which allow foreground and background lineages to evolve differently (with different dN/dS), were compared to null models that assume the same ratio for all branches. Tree with divergence time was taken from MEGA version 7.0 [76]. The alternative models were evaluated for statistically significance (P < 0.05) by likelihood ratio tests (LRTs), with the null model using a χ2 distribution [77].

Generation of gsdf mutants by CRISPR/Cas9

Mutant line of gsdf-A and gsdf-B were generated by CRISPR/Cas9 as described previously [25] and the process was shown in S5 Fig. The sgRNA target sites of gsdf-A and gsdf-B were designed on the first exon and the second exon, respectively (Fig 1A and 1B). gRNAs were transcribed with the TranscriptAid T7 High-Yield Transcription Kit (Thermo Fisher Scientific). The gRNA and Cas9 protein (Invitrogen) were co-injected into one-cell-stage embryos at a concentration of 200 ng/μL and 100 ng/μL, respectively.

DNA extraction and PCR detection of MSM

A small piece of fin was used to extract genomic DNA for each sampled fish, using DNA extraction kit (Promega) according to the manufacturer’s instructions. The MSM was detected by PCR using the primer pair Cg-MSM-F and Cg-MSM-R [32]. PCR analysis was performed as previously described [35].

Aromatase inhibitor treatment

Individuals from a gynogenetic family (WT, MSM−) and individuals from a gsdf-A/gsdf-B double mutant family (gsdf-AΔ4,+9/Δ4/Δ7 + gsdf-B chimeric mutations, MSM+) were divided into two groups respectively, including a control group and a treatment group. The treated fish fry were fed with fairy shrimp that had been placed in 95% ethanol containing Letrozole (MCE) at a final concentration of 150 μg/L for 0.5 hr, whereas the control fish were fed with fairy shrimp that had been socked with 95% ethanol only. The treatment lasted for 40 days from 15 to 55 dph, and then all groups were fed with normal diet and maintained in outdoor tanks as described previously [32,78].

Plasmid construction

According to genome database of gibel carp, the upstream sequences of gsdf–A (from -2080 to +50), gsdf-B (from -2150 to +50), and cyp19a1a-B (from -2066 to +50) were amplified from the genomic DNA and cloned into pGL3-Basic luciferase reporter vector (Promega). The open reading frame (ORF) of gsdf-A, gsdf-B, dmrt1-A, dmrt1-B, sf1-A, sf1-B, ncoa5-B, roraα-B, foxl2b-A, and foxl2b-B were amplified from testicular cDNAs of mature gibel carp by PCR and cloned into pcDNA3.1(+) vector, separately. Roraα-B and ncoa5-B ORF with N-terminal hemagglutinin (HA)-tag were cloned into pCGN-ham vector, while gsdf-A, gsdf-B, and ncoa5-B ORF with N-terminal Flag-tag were cloned into pCMV-Tag 2 vector (Agilent Technologies). Gsdf-A, gsdf-B, ncoa5-B, roraα-B, foxl2b-A, and sting ORF with C-terminal Myc-tag were cloned into pcDNA3.1/myc-His(-) A Vector (Invitrogen). The full-length coding sequences of 27 genes confirmed by yeast two-hybrid assay were cloned into pcDNA3.1(+) vector, separately. All constructs were confirmed by sequence analysis.

Transient transfection

CAB cells were cultured in 6-well plates of phenol red-free M199 media (Gibco) supplemented with 10% charcoal dextran-treated serum (BI) until the cultures became approximately 75% confluent. Confluent cells were transfected using FuGENE HD Transfection Reagent (Promega) with 2 μg expression vectors. To analyze how Gsdf inhibits cyp19a1a transcription, we added 17β-estradiol (E2; Sigma-Aldrich) to the cells at a final concentration of 10 nM at 4 h post-transfection to elevate cyp19a1a transcription. The cells were harvested for RNA extraction and subsequent qPCR analysis. All the experiments were performed in triplicates.

Luciferase activity assays

CAB cells (or EPC cells) were seeded in 24-well plates of phenol red M199 medium and co-transfected with various plasmid constructs at a ratio of 10:10:1 (250 ng luciferase reporter gene plasmid: 250 ng expression plasmid: 25 ng Renilla luciferase plasmid pRL-TK) using FuGENE HD Transfection Reagent (Promega). Then, transfected cells were harvested at 24 h post-transfection and measured by the Dual-Luciferase Reporter Assay System (Promega). Luciferase activities were measured by a Junior LB9509 luminometer (Berthold, Pforzheim, Germany) and normalized to the amounts of Renilla luciferase activities. All experiments were performed at least 3 times and the significant differences were calculated by SPSS soft-ware (SPSS Inc.).

Yeast two-hybrid assay using DUAL membrane system

RNAs from female (MSM−) and male (MSM+) gonads at 15, 18, 22, 26, 30, 35, 40, 47, 58, 70, 110, 200, and 360 dph were pooled and reverse-transcribed. The purified double strand cDNAs were cloned into pDONR222 (Invitrogen) by BP Clonase II enzyme mix (Invitrogen) and the library titre was 1.12 × 107 cfu (colony-forming units) (S13A, S13B and S13E Fig). Then, these cDNAs were transferred from plasmid pDONR222 to pPR3-N-DEST (Dualsystems Biotech) by BP Clonase II enzyme mix (Invitrogen) and the library titre was 3.40 × 107 cfu (colony-forming units) (S13A, S13C and S13F Fig). The mature peptide sequence of gsdf-A (from 95 to 186 amino acids) was cloned into pBT3-SUC vector (Dualsystems Biotech) as bait (pBT3-SUC -Gsdf-A) and yeast library screening was performed according to protocol of the DUAL membrane starter kits User Manual (Dualsystems Biotech).

Yeast two-hybrid assay using GAL4 system

cDNAs were transferred from plasmid pDONR222 to pGADT7-DEST via homologous recombination by LR Clonase II Mix (Invitrogen) (S13A, S13D and S13G Fig). The extracted pGADT7-DEST-cDNA plasmids were transfected into yeast competent cell Y187, the library titre was 1.60 × 107 cfu. The library titer was calculated as described previously. Insert size was identify by PCR using primer pair pGADT7-F (T7) and pGADT7-R (ADR) (S2 Table). Subsequently, the well-distributed yeast cells were cultured on 100 plates (150 mm) with dropout medium (SD/-Leu) at 30°C for 5 days. All the appeared colonies were collected into freezing medium (YPDA medium with 25% glycerol) and stored at −80°C for yeast two-hybrid screening. The above construction of cDNA libraries were performed by OE BioTech (Shanghai, China).

The coding sequence of gsdf-A was cloned into pGBKT7 vector (Clontech) as bait (pGBTKT7-Gsdf-A), and yeast library screening was performed according to protocol of the Matchmaker Gold Yeast Two-Hybrid System (Clontech). All positive colonies were collected from quadruple dropout medium (QDO supplemented with X-alpha-Gal and Aureobasidin A, lack of Ade, His, Leu, and Trp) separately and used for plasmid extraction. Each plasmid was transformed into TOP10 chemically competent cell for subsequent sequencing. To exclude false positive results, the full-length coding sequences of all candidate genes were constructed into pGADT7 (Clontech) and then co-transformed to Y2H competent cell with pGBKT7-Gsdf-A on QDO/X-alpha-Gal/AbA plates and DDO (lack of Leu and Trp) plates, respectively.

RNA interference

EPC cells were cultured in 12-well plates overnight, and then transfected with 50 nM small interfering RNAs (siRNA) of ncoa5 (ncoa5-A and ncoa5-B) and the negative control (si-Nc) by using FishTrans (MeiSenTe Biotechnology). 17β-estradiol was added to the cells at a final concentration of 10 nM at 4 h post-transfection to elevate cyp19a1a transcription. siRNA of ncoa5 (ncoa5-A and ncoa5-B) and si-Nc were synthesized by Sangon Biotech (Shanghai). The following sequences were targeted for ncoa5 (ncoa5-A and ncoa5-B): si-ncoa5: CCGUCAUAGUCGUCAACAATT.

Co-immunoprecipitation assay

CAB cells (or EPC cells) were seeded in 10 cm2 dishes overnight and then transfected with a total of 10 μg of various plasmid combinations. The transfected cells were washed twice with 10 mL ice-cold PBS and then lysed by radioimmunoprecipitation (RIPA) lysis buffer with protease inhibitor cocktail (Sigma-Aldrich). After removing cellular debris, the supernatant was transferred to a 1.5 mL clean tube and incubated with 25 μL anti-Flag Affinity gel (Sigma-Aldrich) or anti-HA Magnetic Beads (Thermo Fisher) overnight at 4°C with constant rotating incubation. Immunoprecipitated proteins were collected by Magnetic Stand (Promega), washed five times with lysis buffer, and resuspended in 100 μL SDS-PAGE protein loading buffer (Beyotime). The immunoprecipitates and whole cell lysates (WCLs) were separated by 10–12% SDS-PAGE and then transferred to polyvinylidene fluoride membranes (Millipore) for subsequent western blot analysis. Antibodies were diluted as follows: anti-β-actin (Cell Signaling Technology) at 1:3,000, anti-Flag/HA antibody (Cell Signaling Technology) at 1:3,000, anti-Myc antibody (Abcam) at 1:2,000, and HRP-conjugated anti-rabbit IgG (Thermo Scientific) at 1:5,000. Images were captured by ImageQuant LAS 4000mini (GE). Results were representative of three independent experiments.

Chromatin immunoprecipitation (ChIP)

CAB cells were cultured in 15 cm2 plate overnight and then transfected with 20 μg expression vector pCS2+-Rora-Myc. The transfected cells were used for ChIP analysis by ChIP-IT Express Chromatin Immunoprecipitation Kits (Active Motif). The protein and chromatin complexes were immunoprecipitated by 3 μg Anti-Myc antibody (Abcam) or anti-IgG antibody (Dia-An Biotec), respectively. The purified DNA was used for PCR analysis after Immunoprecipitation purification. The PCR products were electrophoresed in 2% agarose gels. The primer pair Chip-rora-F and Chip-rora-R (S2 Table) was used to amplify specific region spanning the potential binding site for Rora.

Sequence alignments and phylogenetic construction.

(A) Coding sequence alignment of three gsdf-A alleles and three gsdf-B alleles. Sequence lengths and identities are exhibited at the end of sequences. (B) Multiple amino acid sequence alignment of Gsdf proteins from different fish species. Star marks the conserved cysteine. TGF-β superfamily domain is highlighted by black box. Sequences highlighted by red box were used as antigen for anti-Gsdf antibody preparation. (C) Phylogenetic tree of Gsdf proteins from different fish species.

(TIF)

Click here for additional data file.

Specificity of polyclonal anti-Gsdf antibody confirmed by western blot.

(TIF)

Click here for additional data file.

Sequence preference of Dmrt1 recognition motif and putative Dmrt1-binding sites.

(A) Sequence preference of Dmrt1 recognition motif (MA1603.1) from JASPAR database. (B) Information about putative Dmrt1-binding sites of gsdf promoter.

(TIF)

Click here for additional data file.

Sequence preference of Sf1 recognition motif and putative Sf1-binding sites.

(A) Sequence preference of Sf1 recognition motif (MA1540.1) from JASPAR database. (B) Information about putative Sf1-binding sites of gsdf promoter.

(TIF)

Click here for additional data file.

Establishment of gsdf mutant families.

(A-B) Construction of different gsdf-A mutant lines (A), gsdf-B mutant lines (B), and gsdf-A/gsdf-B double mutant families (A, B). Sex reversed individuals are marked in red color and sex reversal rate was shown at the bottom of each line or family. ♀, phenotypic female; ♂, phenotypic male.

(TIF)

Click here for additional data file.

Aromatase inhibitor treatment on gynogenetic offspring.

(A) Gonadal histology of gynogenetic offspring without letrozole treatment at 100 dph. (B) Gonadal histology of gynogenetic offspring with letrozole treatment at 100 dph. Bar: 20 μm. I, primary oocyte; II, growth stage oocyte; SG: spermatogonium; PSP: primary spermatocyte; SSP: secondary spermatocyte; SC: somatic cell. MSM−, without MSM; WT, wild type.

(TIF)

Click here for additional data file.

FISH analysis of the cyp19a1a (Red) mRNA and immunofluorescence analysis of the Gsdf protein (Green) in mature testis.

Arrowhead indicates the somatic cells with expression of cyp19a1a and Gsdf. Scale bars: 25 μm.

(TIF)

Click here for additional data file.

Yeast two-hybrid assay using DUAL membrane system.

(A) Plate counting of 1,000-fold diluted yeast cells co-transformed with pBT3-SUC-Gsdf-A mature peptide and pPR3-N-library on DDO plate. The number of clones is displayed at the right bottom. (B) Undiluted yeast cells co-transformed with pBT3-SUC-Gsdf-A mature peptide and pPR3-N-library on QDO plate. No positive cells.

(TIF)

Click here for additional data file.

Transcription of sex differentiation genes in response to overexpression of Gsdf-A’s potential interaction partners.

qPCR analysis of cyp19a1a, foxl2b, and dmrt1 expression in the CAB cells transfected with different plasmids. Candidate genes represented by different numbers are given in Fig 6B. Different letters represent statistical differences (*P<0.05, **P<0.01, ***P<0.001). Ncoa5 activated cyp19a1a transcription but could not change expression levels of foxl2b and dmrt1.

(TIF)

Click here for additional data file.

Identification of ncoa5 and rora homeologs in hexaploid gibel carp.

(A-B) Chromosomal localization of ncoa5 (A) and rora (B). Chromosome numbers are displayed at the left side. Conserved gene blocks are represented in matching colors. Transcription orientations are indicated by arrows. (C-D) Deduced amino acid sequence alignment of Ncoa5 (C) and Rora (D). The identities relative to human orthologs are exhibited at the end of each sequence. Ncoa5-B and Roraα-B were selected for subsequent in vitro analyses.

(TIF)

Click here for additional data file.

Sequence preference of Rora recognition motif and putative Rora-binding sites.

(A) Sequence preference of Rora recognition motif (MA0071.1) from JASPAR database. (B) Information about putative Rora-binding sites of cyp19a1a promoter.

(TIF)

Click here for additional data file.

FISH analyses using sense RNA strands.

Sections of mature testis were subjected for FISH with sense riboprobes of dmrt1 (Red), ncoa5 (Red), cyp19a1a (Pink), roar (Pink), and sf-1 (Pink), and analyzed by fluorescence microscopy. Scale bars: 25 μm.

(TIF)

Click here for additional data file.

Evaluation of cDNA libraries.

(A) Summary of three cDNA libraries using different plasmids including pDNOR222, pPR3-N-DEST, and pGADT7-DEST. (B-D) Plate counting of 200-fold diluted E. coli cells from the libraries of pDNOR222 (B), pPR3-N-DEST (C), and pGADT7-DEST (D). (E-G) Agarose gel electrophoresis of PCR products from randomly selected 24 colonies from the library of pDNOR222 (E), pPR3-N-DEST (F), and pGADT7-DEST (G). Marker is DL2000 DNA marker.

(TIF)

Click here for additional data file.

Comparison of coding sequences and protein sequences between gsdf-A and gsdf-B alleles.

(DOCX)

Click here for additional data file.

Primers used in this study.

(DOCX)

Click here for additional data file.

Transfer Alert

This paper was transferred from another journal. As a result, its full editorial history (including decision letters, peer reviews and author responses) may not be present.

13 Apr 2022

Dear Dr Gui,

Thank you very much for submitting your Research Article entitled 'Two duplicated gsdf homeologs cooperatively regulate male differentiation by inhibiting cyp19a1a transcription in a hexaploid fish' to PLOS Genetics.

The manuscript was fully evaluated at the editorial level and by independent peer reviewers. The reviewers appreciated the attention to an important problem, but raised some substantial concerns about the current manuscript.Based on the reviews, we will not be able to accept this version of the manuscript, but we would be willing to review a much-revised version. We cannot, of course, promise publication at that time.

Should you decide to revise the manuscript for further consideration here, your revisions should address the specific points made by each reviewer, in particular those concerning the subgenome-specific regulation of gsdf.  We will also require a detailed list of your responses to the review comments and a description of the changes you have made in the manuscript.

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Reviewer's Responses to Questions

Comments to the Authors:

Please note here if the review is uploaded as an attachment.

Reviewer #1: Review about Wang et al.; PLoS Genetics

Two duplicated gsdf homeologs cooperatively regulate male differentiation by inhibiting cyp19a1a transcription in a hexaploid fish.

In their study, Wang et al. have identified two gsdf homeologous genes in the hexaploid gibel carp. They further propose to better characterize their regulation and function regarding to gonad development.

First, having in mind to decipher how duplicated genes co-regulate a biological process (gonadal development for instance) in a polyploid species, they show that the transcription of gsdfA and gsdfB is mainly activated by dmrt1a and dmrt1b respectively, suggesting asymmetrical evolution to explain the expression divergence in the context of the two sub-genomes. Then, functional characterization is carried on through generation of gsdf mutants, leading to partial or total sex reversion. The cooperative ability of the two gsdf homeologs to regulate male differentiation is then shown. Using cell culture system, gsdf interactions with Ncoa5 and subsequent transcriptional modulation of Cyp19a1a is further demonstrated.

All in one, although the whole story presented in this paper draft is interesting in terms of (i) evolutionary biology (how duplicated genes co-regulate a biological process, and how asymmetrical evolution rule expression divergence), and (ii) mechanistic/physiology (how gsdfs exert their function(s)), I think some major issues need to be addressed before this draft is ready for publication in PLoS Genetics.

Major Issues:

-Characterization of gsdf homeologs and alleles/dynamic transcription of gsdfA/Bare mainly activated by dmrt1A/B respectively.

Co-evolution of the two subgenomes (regarding to gsdfs regulation by Dmrt1) is indeed an interesting aspect of that paper.

-The analysis might benefit from a dN/dS analysis (gsdf and dmrt1) to check whether one or the other homeologs are under selection. This might tell us more about the co-evolution. It should be then discussed if necessary.

-In silico analysis: authors need to tell us more about the predicted dmrt1/sf1-binding sites (which matrix was employed, sequence, homology, divergence to the canonical sequence…)

-At the end, we need to know if this differential regulation is due to the evolution (divergence) of the Dmrt1s only, or of the targets only, or co-evolution of both.

-Cytoplasmic localization of the Gsdf proteins is OK for me, although we know it is a secreted protein that will diffuse from one cell to others, and bind at cell membranes. It might accumulate in some cells before being excreted; this should be told and discussed.

-Disruption of gsdfA/B results in cyp19a1a upregulation and aromatase inhibitor treatment rescues the male to female sex reversal.

That part is OK for me.

-Figure 3A/3B is very complex and I am not sure it helps much here. I would move it to supplemental data.

-Figure 4E: it is difficult to compare that figure 4E with what has been shown in figure 2B: in 2B, basal relative expression of Gsdfs is nearly not detectable (at 25 dph), while that basal expression is quite high in 4E (same stage). Reference to (E) is actually not well written in the figure legend.

So, please unify the relative expressions (2B/4E) or explain that discrepancy.

The rest is OK for me, although at one point you will need RNA in situs to show that Gsdf, Ncoa5 and rora are indeed co-expressed to validate the physiological relevance of the inferred interactions.

-Well, although I could be at first convinced by the yeast two hybrid assay, I am nevertheless very concerned about the fact that the full open reading frame of Gsdf was used as a bait. Indeed, if I am right the whole ORF usually encodes for the pro-mature peptide. Then to be active the pro-domain has to be cleaved to release the mature gsdf active peptide. In these conditions, using the un-cleaved Gsdf, I am not certain that the found interactions are relevant. This is an issue.

-The same question now stands for the antibody that was prepared from (most of) the whole sequence; and that might also recognize the pro-domain instead of the mature gsdf peptide.

-NCOa5 participates in cyp19a1a regulation via interaction with Rora.

-Like for Sf1 and Dmrt1 predicted binding sites, potential Rora binding sites should be described with more details: statistics concerning the homology to the matrix and so on.

The rest of that part seems OK for me.

-Ncoa5 participates in cyp19a1a regulation via interaction with Rora.

-Regulations and interactions were established in cells lines after transfection experiments. While the experiments performed for that part are quite OK, we should not forget the physiology behind, and how to interpret these results “physiologically”.

Indeed, while Figure 8 compile the whole story, it is difficult for me to understand how a TGF-beta ligand, that is an excreted molecule that usually binds at the cell surface, would here have an activity inside of the nucleus together with Ncoa5 and Rora. To my knowledge this is impossible.

Then to make the necessary connexion between the results in cell lines together with what the physiology would allow, it is to my opinion necessary to show co-localization of the Gsdf-Ncoa5-Rora complexes inside of the nucleus.

Reviewer #2: Gsdf is a member of transforming growth factor β (TGF-β) superfamily which has been implicated in male sex differentiation of multiple teleost species. Despite of its critical role in testis differentiation, the specific molecular pathway of Gsdf-mediated sex differentiation remains elusive. In this MS, the authors identified two duplicated gsdf homeologs genes (gsdf-A and gsdf-B) in the gynogenetic hexaploid gibel carp, and found that the transcription of gsdf-A and gsdf-B is activated by dmrt1-A and dmrt1-B, respectively. By loss-of-function experiments, the authors demonstrated that two gsdf homeologs cooperatively regulate male differentiation by inhibiting cyp19a1a transcription. By in vitro analyses, the authors demonstrated that Gsdf-A and Gsdf-B interact with Ncoa5 to block Ncoa5 interaction with Rora, inhibiting Rora/Ncoa5-induced activation of cyp19a1a.

Generally, this study is interesting and provides a special mechanism of gsdf in regulating male sex determination and differentiation. However, the following issues should be addressed before the article is accepted.

Major concerns:

1) As we know, Gsdf is a secretory protein which belongs to the transforming growth factor β family, and it is supposed to function with its receptor. In this MS, the author demonstrated a direct interaction between Gsdf-A/B and Ncoa5. Is there a TGF- β type II receptor involved in this regulatory process by binding gsdf?

2) Usually, the male and female pathway genes display obvious sexually dimorphic expression. If both Ncoa5 and Rora are involved in sex differentiation, why there is no difference in the expression of ncoa5 and rora between females and males in the Figure 7A. Further, in Figure 7B, overexpression of gsdf led to downregulation of roar, why there was no difference in the expression of rora between females and males in the Figure 7A?

3) An important finding of this work is that in females Ncoa5/Rora induced activation of cyp19a1a independent of Foxl2/Sf1-cyp19a1a pathway. We all know that mutation of Foxl2 lead to female to male sex reversal. The specific function of Ncoa5 in fish and whether it is involved in fish sex differentiation has not been explored. For this reviewer, it is hard to believe Ncoa5/Rora-cyp19a1a and Foxl2/Sf1-cyp19a1a pathway is independent in female sex differentiation.

4) In Figure 2D and 2E, the authors demonstrated that Dmrt1 and Sf1 activate gsdf expression through binding to their potential binding sites in the gsdf promoter, it is better to determine which site is important for gsdf activation. It is also better to demonstrate that Dmrt1 and Sf1 are co-expressed in gsdf expressing cells.

Miner concerns:

Line 172：“Expression plasmids of Sf1-A/Sf1-B, Dmrt1-A, Dmrt1-B, and an empty expression plasmid were used as controls” should be “Expression plasmids of Sf1-A/Sf1-B, Dmrt1-A, Dmrt1-B were constructed, and an empty expression plasmid was used as controls”.

Line 555: “mediaand” should be “medium”.

Line 556: “luc” should be “luciferase reporter gene”.

Line 633: A repeated copy of “under the accession number of” should be deleted.

Line 904: “exam” should be “examine”.

Line 908: “indicates” should be “indicate”.

Line 921: “transcriptions” should be “transcripts” or “mRNA expression”, and the same for line 934.

Line 958: The letter “e” in the Figure legend should be “(E)”.

Line 980: “activates the promoter activity of gsdf-A/gsdf-B”, the promoter described here should be the “cyp19a1a promoter”.

Line 1071：“noca5” should be “ncoa5”.

Reviewer #3: This is an interesting and well-conducted study showing the role of a member of the transforming growth factor beta (TGF-ß) signaling pathway, gonadal somatic cell-derived factor (gsdf) to be involved in male differentiation in the hexaploid gibel carp (Carassius gibelio). This species was used as a system to investigate the mechanisms that duplicate genes use in a developmental process such as sex differentiation. Authors demonstrate the presence of two homeologues of gsdf, termed gsdf-A and gsdf-B that cooperatively regulate male sex differentiation by interacting with the nuclear co-activator factor 5 (Ncoa5) to suppress gonadal aromatase (cyp19a1a) gene expression. In lower vertebrates, it is well established that aromatase activity is necessary for estrogen production and female development. The study is very complete and includes the molecular characterization of gsdf-A and gsdf-B genes, expression during gonad development, transcriptional regulation, and functional analysis by wisely combining induced gynogenesis using males with or without a male determinant contained in supernumerary microchromosomes and loss-of-function analysis by the production of single or double mutants by means of CRISPR technology. Authors convincingly show that disruption of either gsdf-A or gsdf-B results in partial sex reversal while disruption of both homeologs results in complete male-to-female sex reversal. Furthermore, male-to-female sex reversal in double mutants is due to stimulation of cyp19a1a, the effects of which can be inhibited by treatment with a non-steroidal aromatase inhibitor. Authors identify Ncoa5 as interaction partner of Gsdf and show that gsdf binding of Ncoa5 prevents the latter to interact with Ncoa to upregulate cyp19a1 transcription. The study nicely shows how both forms of gsdf cooperate towards male sex differentiation. I have some comments that may help to improve the ms.

L27 and Throughout the ms. The term “homeologous”, used in polyploidy plant research, may not be familiar with some readers, who may confuse it with “homologous”. Please explain that homologous genes resulting from allopolyploidy are commonly referred to as “homoeologs”. Here you use the term “homeologs”, which I am not sure how common it is. All this may create confusion. Please provide a clear definition of the term and its possible variants right from the start.

L. 91-93. “a variant of gynogenesis”. This needs to be better explained to readers. Please indicate ploidy level of each progenitor in the initial cross. Also explain whether there is a dose-dependent relationship between the microchromosomes and the percent of males.

L109. I am not sure if it would be more appropriate to speak about “sex determination” rather than to “sex differentiation”. Duplicates of gsdf has been implicated in sex determination. In fact, in lines 101-102 authors write “Members of the transforming growth factor-β (TGF-β) signaling pathway have been identified as being vastly involved in sex determination”.

L151. Gsdf-A expression during sex differentiation peaked at 250 dph while the expression of Gsdf-B peaked at 120 dph. However, gonadal morphological differentiation between males and females occurs around 40 dpf. How then it is possible that Gsdf-A and B are drivers of sex determination/differentiation?

L166-168. Consider rephrasing the sentence to clearly indicate that the identified upstream sequences of gsdf-A and gsdf-B were considered the potential promoter sequences; otherwise it seems that the binding sites per se are the promoter regions.

L191. Indicate how did you ensure that the female gibel carp was devoid of MSM.

L263. Delete “obviously”.

L276. Add “nonsteroidal” before “aromatase inhibitor”.

L277. Add “enzyme activity” after “Cyp19a1a”.

L279. Explain why there were approximately 15% of the fish in which male-to-female sex reversal could not be prevented.

L282. Upregulation of foxl2b in the testis of gsdf-A/gsdf-B double mutants (MSM+) subjected to letrozole treatment comes as a bit of surprise since usually foxl2a and Cyp19a1a expression goes hand in hand due to the regulatory positive feedback loop involving these two genes. Please clarify.

L317. How can you be sure that Ncoa5-A had no role in sex differentiation

L394. It comes as a bit of surprise that no further explanation of the different functions of gsdf-A and gsdf-B are discussed. This is relevant to the study but these functions are not properly explained. This is relevant in view of the overall aim of the paper. It should be stated whether the contribution of each homeologue is similar, if there is any indication that one has more relevance than the other in male differentiation.

L421. “Thus, dysfunction of gsdf triggers upregulation of cyp19a1a and subsequently leads to the inhibition of dmrt1 and activation of foxl2”. I would rephrase a bit this sentence because in this study the whole chain of events is not shown. So I suggest indicating that, as shown in previous studies, on one hand upregulation of cyp19a1a would lead to dmrt1 inhibition while on the other it would upregulate foxl2.

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Have all data underlying the figures and results presented in the manuscript been provided?

Large-scale datasets should be made available via a public repository as described in the PLOS Genetics data availability policy, and numerical data that underlies graphs or summary statistics should be provided in spreadsheet form as supporting information.

Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: Yes

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Reviewer #1: No

Reviewer #2: Yes: Deshou Wang

Reviewer #3: No

25 May 2022

Submitted filename: Response to Reviewers.docx

Click here for additional data file.

8 Jun 2022

Dear Dr Gui,

We are pleased to inform you that your manuscript entitled "Two duplicated gsdf homeologs cooperatively regulate male differentiation by inhibiting cyp19a1a transcription in a hexaploid fish" has been editorially accepted for publication in PLOS Genetics. Congratulations!

Before your submission can be formally accepted and sent to production you will need to correct the minor mistakes identified by reviewer #2 and complete our formatting changes, which you will receive in a follow up email. Please be aware that it may take several days for you to receive this email; during this time no action is required by you. Please note: the accept date on your published article will reflect the date of this provisional acceptance, but your manuscript will not be scheduled for publication until the required changes have been made.

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----------------------------------------------------

Comments from the reviewers (if applicable):

Reviewer's Responses to Questions

Comments to the Authors:

Please note here if the review is uploaded as an attachment.

Reviewer #1: Well, all my concerns have been addressed and discussed, although it was requiring quite a bit of additional work.

I do think this paper draft has been much improved, and that it is now ready for publication.

I have to add that it is a nice piece of work, and that is was real pleasure to review it.

Congratulation!

Reviewer #2: In this revised manuscript, the authors have carefully considered the reviewer’s comments and provided new evidences. Co-localization results of dmrt1/sf-1 with Gsdf along with mutation of Dmrt1 binding sites on gsdf promoter further supported the regulation of Dmrt1 on gsdf promoter in vitro. Additionally, new in situ expression data of rora/ncoa5 showed their co-expression with Gsdf and Cyp19a1a, respectively. These results strongly supported the regulation of Rora/Ncoa5 on cyp19a1a and validated the physical interaction between Gsdf and Ncoa5. Together the additional experiments and careful editing have significantly improved the quality of the manuscript. Overall, the authors have taken all the suggestions in consideration and have satisfactorily addressed all of my concerns. In one word, the authors have done a commendable job at revising the manuscript and have successfully improved it.

Attached below were a few minor mistakes:

Line 276: “are shown” should be “were shown”.

Line 611: The process concerning generation of gsdf mutants has been moved into revised Figure S5, it is not in Figure 3 now. Please revise “shown in Fig 3.” as “shown in S5 Fig.”

Line 613: “Fig 1A and 3B” here should be “Fig 1A and 1B”.

Line 644: “roarα-B” should be “roraα-B”.

Reviewer #3: The authors have successfully addresed all comments and clarified the doubts. This is a good paper suitable for publication.

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Have all data underlying the figures and results presented in the manuscript been provided?

Large-scale datasets should be made available via a public repository as described in the PLOS Genetics data availability policy, and numerical data that underlies graphs or summary statistics should be provided in spreadsheet form as supporting information.

Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: Yes

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Reviewer #1: Yes: Amaury HERPIN

Reviewer #2: Yes: Deshou Wang

Reviewer #3: No

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Data Deposition

If you have submitted a Research Article or Front Matter that has associated data that are not suitable for deposition in a subject-specific public repository (such as GenBank or ArrayExpress), one way to make that data available is to deposit it in the Dryad Digital Repository. As you may recall, we ask all authors to agree to make data available; this is one way to achieve that. A full list of recommended repositories can be found on our website.

The following link will take you to the Dryad record for your article, so you won't have to re‐enter its bibliographic information, and can upload your files directly:

http://datadryad.org/submit?journalID=pgenetics&manu=PGENETICS-D-22-00317R1

More information about depositing data in Dryad is available at http://www.datadryad.org/depositing. If you experience any difficulties in submitting your data, please contact help@datadryad.org for support.

Additionally, please be aware that our data availability policy requires that all numerical data underlying display items are included with the submission, and you will need to provide this before we can formally accept your manuscript, if not already present.

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Press Queries

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25 Jun 2022

PGENETICS-D-22-00317R1

Two duplicated gsdf homeologs cooperatively regulate male differentiation by inhibiting cyp19a1atranscription in a hexaploid fish

Dear Dr Gui,

We are pleased to inform you that your manuscript entitled "Two duplicated gsdf homeologs cooperatively regulate male differentiation by inhibiting cyp19a1atranscription in a hexaploid fish" has been formally accepted for publication in PLOS Genetics! Your manuscript is now with our production department and you will be notified of the publication date in due course.

The corresponding author will soon be receiving a typeset proof for review, to ensure errors have not been introduced during production. Please review the PDF proof of your manuscript carefully, as this is the last chance to correct any errors. Please note that major changes, or those which affect the scientific understanding of the work, will likely cause delays to the publication date of your manuscript.

Soon after your final files are uploaded, unless you have opted out or your manuscript is a front-matter piece, the early version of your manuscript will be published online. The date of the early version will be your article's publication date. The final article will be published to the same URL, and all versions of the paper will be accessible to readers.

Thank you again for supporting PLOS Genetics and open-access publishing. We are looking forward to publishing your work!

With kind regards,

Zsofia Freund

PLOS Genetics

On behalf of:

The PLOS Genetics Team

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