Integrated analysis of mRNA-seq and miRNA-seq reveal the dynamics of the sexual differentiation of Procypris mera

Integrated analysis of mRNA-seq and miRNA-seq reveal the dynamics of the sexual differentiation of Procypris mera | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Integrated analysis of mRNA-seq and miRNA-seq reveal the dynamics of the sexual differentiation of Procypris mera Zhenlin Ke, Weijun Wu, Zhe Li, Yusen Li, Yaoquan Han, Jun Shi, and 6 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-8288461/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 13 Mar, 2026 Read the published version in Marine Biotechnology → Version 1 posted 10 You are reading this latest preprint version Abstract The Chinese ink carp ( Procypris mera ), a primitive species within the Cyprinidae family, exhibits sexual dimorphism in growth, with females growing significantly faster than males. Due to the depletion of wild resources and limitations in breeding technology, its commercial viability has become concerning. Research into all-female breeding and germplasm resource recovery is crucial for addressing these challenges, with sex control intervention playing a pivotal role. In this study, we conducted histological observations of P. mera gonads using Hematoxylin-Eosin (HE) staining and identified four key stages of gonadal differentiation (60 dph, 95 dph, 110 dph, and 150 dph,) for transcriptome sequencing. Additionally, miRNA sequencing was performed at the late differentiation stage (150 dph). Our results indicate that gonadal differentiation in P. mera is largely completed by 150 dph. During ovarian differentiation, the estrogen synthesis rate-limiting gene cyp19a1a is promoted by foxl2 , facilitating estrogen production to drive ovarian differentiation while antagonizing male pathway genes. In contrast, during testicular differentiation, genes such as dmrt1 , sox9-b , and fgf1 are upregulated across all four stages, inhibiting the expression of female pathway genes dominated by foxl2 and cyp19a1a to ensure commitment to testis differentiation. Furthermore, miRNAs also play a critical role in gonadal differentiation and maturation during the later stages. The miR-200 family regulates multiple genes associated with gonadal differentiation, potentially serving as core regulators. Additionally, some newly identified miRNAs (novell-m0207, novell-m0123-5P, and novell-m0122-5P) may also contribute to the regulation of gonadal differentiation processes. Overall, this study may provide valuable insights into the gonadal differentiation process and its underlying molecular regulatory network in P. mera . These findings offer a solid foundation for improving production performance and advancing aquaculture practices for this species. Procypris mera Gonadal differentiation Transcriptome miRNA Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 1. Introduction Sexual reproduction, as the primary mode of multiplication in life, represents the most common breeding method in the vast majority of vertebrates[ 1 ]. Serves as the foundation for species continuation, the normal development and differentiation of gonads are essential prerequisites for reproduction. Sex determination occurs after primordial germ cells (PGCs) migrate to the genital ridge and form the primitive gonad. This process initiates the expression of genes associated with gonadal differentiation, guiding the undifferentiated gonads to develop into either testes or ovaries. Ultimately, the gonads developed into mature with the emergence of secondary sexual characteristics[ 2 ]. As the largest group of vertebrates, fish exhibit remarkable diversity and plasticity in gonadal differentiation, which provides an excellent model for analyzing the genetic mechanism and gonadal differentiation[ 3 ]. To data, numerous key genes involved in gonadal differentiation have been identified in fish, such as gsdf , cyp19a1a , hsd11b2 , foxl2 and cyp17a1 , these genes are regulated by sex-determining genes and are specifically expressed in testis or ovary during the gonadal differentiation. The knock-out of these genes will cause sexual reversal[ 4 – 7 ]. In addition, microRNA (miRNA), a type of small non-coding RNA, as the second network for regulating gene expression and protein translation, it can target multiple genes and regulate their expression[ 8 ], which also plays an important role in the early gonadal differentiation of fish[ 9 ], miRNA have been proven that it can specifically regulate the differentiation of testis or ovary[ 10 – 13 ]. Consequently, integrated analysis of miRNA and mRNA transcriptomes can provide deeper insights into post-transcriptional regulatory networks. In summary, while many genetic factors contributing to gonadal differentiation have been identified, significant gaps remain in our understanding of how these factors interact, whether at the level of mRNA or miRNA regulation. Therefore, elucidating the mechanisms of fish gonadal differentiation and uncovering the associated regulatory networks, especially the processes governing early sex differentiation, is of great importance[ 14 ]. Such research could offer valuable references for developing effective fish breeding strategies. Chinese ink carp ( Procypris mera ), indigenous to the Pearl river basin, is one of the most primitive fishes within the subfamily Cyprininae[ 15 , 16 ]. Similar to the common carp ( Cyprinus carpio ), P. mera showed an obvious sexual dimorphism in growth and females possessed a faster growth rate, which was once an economic fish in Guangxi, therefore, all-female breeding can effectively improve its breeding benefit[ 17 ]. However, the wild resources of P mera have experienced an amazing decline due to overfishing, dam construction, and destruction of feeding and spawning grounds. At present, it has been classified as endangered species (VU) in the China Red and listed as a national second-class protected animal[ 15 , 18 – 20 ]. Therefore, it is important to study gonadal determination and differentiation of P mera , which is significant for P. mera in both resource restoration and aquaculture production. Currently, there were few studies investigated the ecological habits[ 21 ], breeding[ 22 ], reproduction[ 23 , 24 ] and seedling cultivation[ 25 – 27 ], while the detailed and complex process of gonadal differentiation and development haven't been reported. In order to define the gonadal differentiation and development process of P mera , and further dissect the mechanisms of its differentiation. In this study, the histological changes of the formation and differentiation of primitive gonads in P. mera were explored by paraffin tissue sections and HE staining, and comparative transcriptome analysis was performed for four key periods of males and females early gonadal differentiation, including: gonadal differentiation for females (60 and 110 dph), gonadal differentiation for males (90 and 150 dph). Additionally, to further understand the molecular regulatory network of late differentiation of P. mera , which is the key stage to control the maturation of early gonads, miRNA sequencing was performed in the critical period of gametogenesis. In general, this study has determined the early gonadal development process of P. mera , and the sequencing results showed that there were many gender-biased genes and miRNAs, which participated in the gonadal differentiation of males and females. Moreover, cyp19a1a has significant differential expression between males and females in four critical stages of differentiation, which is a critical gene for estrogen synthesis. And many miRNAs also play a regulatory role in gonadal differentiation. This study may provide further insight into the early gonadal differentiation in P. mera and assist to explain its molecule mechanisms in this species, which will offer reference for future sex-controlled breeding. 2. Materials and Methods 2.1. Ethics statement This study was conducted in accordance with the Declaration of Helsinki and approved by the Animal Care and Use Committee of Southwest University, Chongqing, China. 2.2. sample collection The P. mera used in the experiment were derived from the rare and endangered fish breeding base of Guangxi Fisheries Research Institute, all the larvae were reproduced by the same parents (Breeding certificate number: 2023-0108004). During the whole breeding process, the water quality conditions are as follows: temperature: 25 ± 1℃, DOI ≥ 5 mg/L, pH: 7–8, NH3-N: 0.1–0.2 mg/L, NIT: 0.01–0.02 mg/L. Gonad were collected at 5, 10, 15, 20, 25, 30, 35 …… dph until gonadal differentiation is completed. The one-half of the gonad are used to identify the sex and development situation, which were stored in 4% paraformaldehyde, the other half were stored in the 4℃ RNA later and transferred to -80℃ after 24 hours. Only gonads during the critical stage of differentiation were used for subsequent sequencing. 2.3. RNA extraction, library construction, transcriptome sequencing and data analysis. According to the histological section results, testis and ovary in the critical period of gonadal differentiation (60, 95, 110, 150dph) were used to extract RNA, there were three samples of testis and ovary in each period, and each sample is mixed by two samples. Total RNA of each samples were extracted using TRIzol Reagent Kit (Invitrogen, Carlsbad, CA, USA) with the manufacturer's program. The quality and integrity of RNA samples were assessed using an Agilent 2100 Bioanalyzer (Agilent Technologies, Palo Alto, CA, USA). Sequencing library was constructed using Illumina TruSeq RNA Sample Preparation Kit (Illumina, San Diego, CA, USA) following the manufacturer's protocol. The libraries were sequenced on the Illumina Novaseq 6000 sequencing platform. Low quality data of raw data were eliminated by Fastp[ 28 ]( https://github.com/OpenGene/fastp ) and the clean data were compared with reference genome by hisat2[ 29 ](v.4.8.5) ( https://daehwankimlab.github.io/hisat2/ ), then the transcript were assembled by stringtie[ 30 ], the differential expression genes(DEGs) analysis were performed using edgeR[ 31 ] with FDR 1, the corresponding biological functionand related pathways of DEGs were performed using OmicShare online tools ( www.omicshare.com/tools ). 2.4. miRNA extraction, library construction, transcriptome sequencing and data analysis The testis and ovary in the critical period of primordial germ cells (150dph) were used to sequencing. The miRNA libraries were constructed using the NEBNext® Multiplex Small RNA Library Prep Set for Illumina® kit strictly following the operation instructions. After the quality inspection, the qualified libraries were sequenced on the Illumina Hiseq 2000 (Illumina, San Diego, CA, USA). The raw sequences were processed through the Illumina pipeline program. After eliminating contaminated reads, such as adapter dimers, junk sequences, those with low complexity, members of common RNA families (rRNA, tRNA, snRNA, snoRNA), and repeats, the clean reads were further filtered by the software package ACGT101 - miR - v4.2 (LC Sciences, Houston, Texas, USA). The clean sequence reads were aligned with miRBase 21.0[ 29 ], permitting a mismatch of one or two nucleotide bases, to identify both known miRNAs and novel 3p - and 5p - derived miRNAs. EdgeR[ 31 ] was used to analyze the differential expression of the identified miRNA (dispersion = 0.1, and the rest were the default parameters). The screening criteria are that the difference multiple is greater than 2 and P < 0.05. 2.5. miRNA target predictions and integrate analysis of miRNA and mRNA Miranda[ 32 ] (parameter: set score threshold to 140, set energy threshold to -10 kcal/mol, demand strict 5' seed pairing, set gap-open penalty to -4.0 and set gap-extend penalty to -9.0) (v3.3a) and TargetScan[ 33 ] (V 7.0) were used to predict target genes, and the prediction result shared by the two softwires as the final results. In addition, the differential expression mRNAs of the concurrent period were used to integrate analysis in omicshare tools ( www.omicshare.com/tools ) with two demands; 1: There is a targeted relationship between miRNA and mRNA; 2: miRNA was negatively correlated with mRNA expression. 2.6. The verification of miRNA and mRNA with qRT-PCR There were 7 genes and 9 miRNAs were selected to verify the sequencing data. For mRNA, the quantitative real-time PCR (qRT-PCR) primers were designed in primer 5.0, and β-actin was used as reference gene (The primers were shown in Table S1 ). The cDNA of mRNA was synthesized with the PrimeScript™ FAST RT reagent Kit and the express level of mRNAs were determined using TB Green® Fast qPCR Mix (TaKaRa, Beijing, China). All primers used five serially diluted cDNA as templates to construct standard curves, and determine the amplification efficiency of the primer. For miRNA, the Mir-XTM miRNA qRT-PCR TB GreenTM Kit and TB Green® Premix Ex Taq™ II (Tli RNaseH Plus) (TaKaRa, Beijing, China) were used to complete real-time fluorescence quantitative analysis, and the reverse primer is the universal primer included in the kit, the forward primer is the full length of miRNA sequence (U base instead T base substitution sequence), and the reference U6 are included in the kit (The primers were shown in Table S2 ). The relative expression level of mRNA and miRNA were calculated by the 2^- ΔΔCt method. 3. Results 3.1. The gonadal differentiation process of P. mera The histological results showed that the gonadal differentiation of P. mera was completed at 150 dph. At 15 dph, the single PGC was observed above the intestine, it has arrived the genital crest (Fig. 1 A). With the development of gonad, the primordial germ was surrounded by somatic cell and formed primitive undifferentiated gonads (Fig. 1 B, C). At 55 dph, there are two sections of undifferentiated gonad, one of them was long shape which will develop into ovaries, another one still was water drops shape which will develop into testis. At 60 dph, the long gonads begin to have a small cavity between the back of gonad and mesentery, suggesting the initiation of the gonad histological differentiation, and the cavity gradually develop into ovarian cavity and enlarge. At 110 dph, the oogoniums have presented the morphology of primary oocyte, it marked the beginning of ovarian cytological differentiation (Fig. 1 D, E, F). The sperm primordium appeared at the base of gonad at 95 dph in the gonads of water drops shape, which suggested the initiation of the testis histological differentiation, and spermatogonia cells presented the morphology of primary spermatocytes at 150 dph, indicating the initiation of testicular cytological differentiation (Fig. 1 G, H, I). A: Single primordial germ cell in genital crest at 15 dph; B: Primitive gonadal were formed at 20 dph; C: The primordial germ cell numbers increased at 25 dph; D: Ovary histological differentiation with ovarian cavity appeared at 60 dph; E, F: Ovary development with enlargement of ovarian cavity, the primary oocytes appeared at 110dph, and ovarian cytological differentiation began; G: Sperm primordium appears at the base of gonad, and histological differentiation of testis begins at 95 dph; H, I: Testis developed, and the spermatogonia appeared spermatocyte-like at 150 dph, indicating that the cytological differentiation of testis began; PGC: Primordial germ cells; AB: air bladder; G: gut; PG: primary gonad; OC: Ovarian cavity; BV: blood vessel; PO: primary oocyte; SA: Sperm anlage; PS: Primary spermatocyte. 3.2. Overview of transcriptome sequencing data A total of 24 samples from four stages of gonadal differentiation of P. mera were sequenced using Illumina NovaSeq sequencing platform, and 1149852254 raw reads were obtained. After quality control, 1137396262 clean reads were obtained, the average Q30 ratio of clean reads is 93.46%, which indicates that the clean reads can be used for the subsequent analysis. The data were compared to the reference genome of P. mera , and the average total comparison rate was 93.26%, the average unique comparison rate was 88.74%. A total of 47541 genes were detected in the sequencing data (Table 1 ). Table 1 Summary statistics of sequencing data Sample Raw reads Clean reads Q30 (%) Total mapping ratio (%) Uniquely mapping ratio (%) m60 dph-1 48554896 47732030 92.42% 91.73% 88.01% m60 dph-2 47324756 45990558 91.72% 87.45% 83.56% m60 dph-3 47717928 46622366 91.93% 89.56% 84.65% m95 dph-1 57509484 56890792 91.47% 93.70% 88.54% m95 dph-2 43823372 43340520 89.35% 92.63% 88.75% m95 dph-3 57012282 56545560 94.05% 94.75% 90.35% m110 dph-1 40379064 40164198 95.39% 95.21% 91.30% m110 dph-2 39704502 39500594 95.38% 95.53% 91.62% m110 dph-3 43282212 42970878 94.02% 95.05% 91.17% m150 dph-1 57994336 57584718 94.18% 95.07% 91.13% m150 dph-2 69825146 69298336 94.10% 94.95% 91.06% m150 dph-3 37826662 37617134 94.97% 94.59% 88.86% f60 dph-1 45399048 44105590 91.48% 83.10% 79.01% f60 dph-2 45941564 45070634 91.62% 89.79% 84.87% f60 dph-3 47918168 47259338 90.38% 90.14% 86.21% f95 dph-1 38107292 37861510 95.02% 94.80% 90.88% f95 dph-2 42335224 42034914 94.33% 95.15% 91.31% f95 dph-3 37670940 37444940 94.95% 95.14% 86.81% f110 dph-1 46973400 46530270 92.93% 94.33% 89.98% f110 dph-2 50275914 49950856 94.79% 95.02% 90.67% f110 dph-3 45963804 45783608 95.67% 95.44% 91.13% f150 dph-1 42003708 41703404 94.27% 94.99% 91.05% f150 dph-2 59059068 58658882 94.68% 94.91% 91.37% f150 dph-3 57249484 56734632 93.82% 95.22% 87.40% 3.3. Identification, annotation and expression analysis of DEGs between testis and ovary at four differentiation stages The edgeR was used to identify the DEGs of four stages, a total of 8755 DEGs were identified, of which 2372 were up-regulated and 6383 were down-regulated. The specific number of DEGs of different stages of differentiation were displayed in Fig. 2 A and B. DEGs between testis and ovary gradually increased following the development of gonad, indicating that more and more gender-related genes were involved in the differentiation process. Additionally, DEGs of each stage were used to KEGG enrichment analyse to explore its potential function. Interestingly, the steroid hormone synthesis relate pathway was significantly enriched in four periods, it suggested steroid hormone synthesis pathway probably was the key pathway to lead the gonadal differentiation of P. mera (Fig. 2 C, D, E, F). In order to further understand the function of this pathway, the genes related to steroid synthesis with differential expression were screened to applied in the expression profile analysis and protein-protein interaction(PPI) network analysis, the results showed that there were different dominant genes in different stages of differentiation, such as: cyp19a1a , ldlr and cyp27a1 of 60 dph; hsd3b , pla2g4d and cyp11a1 of 95 dph; foxl2 , star and cyp11b of 110 dph; esr1 , dmrt1 and cyp17a1 of 150 dph. There were 8 genes including cyp17a1 , hsd3b1 , cyp11b , cyp3a27 , hsd3b2 , cyp2c8 and cyp19a1a were identified as hub genes in PPI network, which may play a key role in gonadal differentiation of P. mera (Fig. 2 G, H). 3.4. Identification, spatiotemporal and trend analysis of DEGs in testis and ovary differentiation In order to further explore the molecular regulatory network of gonadal differentiation in P. mera , neighbor periods in testis or ovary were compared and analyzed respectively. In testis, a total of 13466 DEGs were identified, in which 107 DEGs were overlapped. As the differentiation of gonads, the number of DEGs between adjacent days decreased gradually, which may result from the gradual completion of gonadal differentiation and the steady expression of differentiation-related genes, it is helpful to maintain the development of testis (Fig. 3 C, D). Additionally, the expression thermogram showed that there were significantly different expression profiles at different stages, which further indicated that gonadal differentiation was regulated by a complex network (Fig. 3 A). Further trend analysis of DEGs shows that these DEGs are significantly enriched in 9 plates (Fig. 3 B). The DEGs including dmrtb1 , dmrt2 , sox9-b , zar and cyp26a1 etc in profile17 was stably up-regulated after testis differentiation, and maintained at a certain level in the later stage, indicating that they may be closely related to testis differentiation, whereas the expression level of dmrt1 , hsd11b2 , and dmrt3a etc in profile 10 were low in the early stage of differentiation and started to rise after 110 dph, which demonstrated that these genes may play a key role in the development and maintenance of testis. In ovary, there were 14198 DEGs between ovaries in several stages, and 273 DEGs were overlap (Fig. 4C, D). Consistent with the testis, the number of differential genes between adjacent ovarian ages decreased gradually, and the expression thermogram of each period was significantly different. The trend analysis showed that these DEGs significantly enriched in 6 plates(Fig. 4A, B). The expression level of foxl2 , kdm2b , wnt4a , map2k6 and tdrd9 in profile 18 increased gradually and reached the highest at 110 dph, it is proved that these genes mainly play a role in the late stage of ovarian differentiation. In addition, sox11 , wt1 , fgf1, fgf13, fgf2, smad4 and smad6 etc were enriched in profile 17, which began to up-regulated from 95 dph and keep highly expression, indicating that these genes were important for the later development and maintenance of the ovary. Figure 4 Heatmap of all DEGs between each ovary differentiation stages. B: Trend diagram of different expressed genes. C, D: UpSet and Venn plots showing the distribution of DEGs at 4 differentiation periods. The bar chart above represents the number of genes contained in each type of group. The bar chart at the bottom left represents the number of DEGs included in each stage of gonadal differentiation. 3.5. Combination analysis of miRNA and mRNA Furthermore, we selected the 150 dph gonads for small RNA sequencing, which is helpful to further explore the regulatory network in the later stage of gonadal differentiation, because this is the key period to control gonadal maturation. The details of the original sequencing data and clean tags of each sample were displayed in Table S3, the clean tags of each sample were compared with the genome of P.mera , and the comparison rate was between 73.71% and 78.11%, and the gap rate between samples was small, indicating that the samples were not polluted (Table S4). The aligned sequences were utilized for miRNA identification, resulting in the discovery of 1561 miRNAs, among which 924 were novel miRNAs (Figure S1 ). These identified miRNAs were subsequently subjected to differential expression analysis, revealing a total of 224 miRNAs that exhibited significant differential expression between the testis and ovary. To further investigate the regulatory network during the late differentiation stage, differentially expressed mRNAs from the same stage were employed for target gene prediction. The analysis demonstrated a notable negative regulatory relationship between 189 miRNAs and 3,813 mRNAs (Figure S2 ). Furthermore, given the pivotal role of steroid hormone synthesis-related pathways, core genes involved in steroid hormone synthesis and significantly different expression of gender-biased miRNA were selected to construct the mRNA-miRNA regulatory axis (Fig. 5 ), thereby providing insights into the intricate regulatory mechanisms underlying late meiosis. The results showed that miRNA-200-y was regulated by several genes related to sex differentiation and gametogenesis, it was highly expressed in testis, while its corresponding target genes including sox9-b , zp3 , spdya , ago3 and tyro3 were down-regulated in males. Concordantly, miRA-144-y was highly expressed in ovary and its target genes of camk2a, ppp2r5b and hsd11b2 were down-regulated expression in ovary. Single miRNA could be regulated by multiple mRNA and single mRNA also could be regulated by multiple miRNAs, which emphasized the complexity of gonadal differentiation. represent miRNA, and blue indicate their target genes) 3.6 Validation of differential expression mRNA and miRNA Differentially expressed genes and miRNAs in gonads were randomly selected for verification. The results showed that the expression levels of mRNA and miRNAs were consistent with RNA-seq data, which ensured the reliability of the above sequencing results. 4. Discussion Sex determination and sex differentiation were an intricate and highly regulated processes. Upon initiation of the sexual developmental pathway, a network of genes involved in sex differentiation emerges, which was two mutually antagonistic regulatory systems[ 3 , 34 ]. These opposing networks govern the fate of bipotential gonadal primordium, which possess the potential for either testicular or ovarian differentiation, ultimately driving their commitment to one of the two reproductive organ destinies. This process ensures the precise establishment of male or female gonadal identity, a critical step in sexual development[ 35 ]. Monitoring this complex developmental process could provide invaluable data support for subsequent breeding efforts. To elucidate the entire process of gonadal differentiation and the sex-specific molecular regulatory mechanisms of P. mera , we collected gonads from 5 dph, from the appearance of the ovarian cavity at 60 dph to the presence of spermatocytes at 150 dph. The early sex differentiation of P. mera was essentially complete by this stage. Based on the differentiation time, the gonad differentiation of P. mera occurs later compared to other carp species within the same family, such as common carp and rare gudgeon ( Gobiocypris rarus )[ 35 – 37 ]. This delay may be attributed to the extended early development and life cycles of P. mera , thus resulting in a relatively later differentiation time. Additionally, we utilized the testis and ovary at a critical stage of sex differentiation to sequence the transcriptome and systematically analyzed these dynamic transcriptional data. Among the findings, we observed that genes associated with steroid hormones synthesis played a significant role in the gonadal differentiation of P. mera . These genes exhibited sexual dimorphism across four distinct periods. Steroids, being the most crucial precursors of synthetic sex hormones, are essential for the development of male or female characteristics. Previous studies have indicated that the balance of sex hormones can determine the direction of sex differentiation[ 38 ]. Fish, in particular, were highly sensitive to the perception of sex hormones, while this balance was regulated by aromatase[ 39 ]. In this study, the expression of cyp19a1a during four differentiation periods was significantly higher in ovary than in testis at all stages of differentiation, and it reached its peak at 110 dph. Early studies have shown that aromatase was mainly used to hydrolyze conjugated steroids and promote the synthesis of synthetic estrogens[ 40 ]. Thus, vitellogenesis and steroid hormone synthesis were the reasons of the up-regulation of cyp19a1a expression. In this study, the oocyte of P. mera began meiosis at 110 dph, and with the maturation of oocytes, vitellogenesis was gradually completed, accompanied by the completion of gonadal differentiation. Therefore, the expression of cyp19a1a in the gonads of P. mera reached the highest at 110 dph and decreased at 150 dph. These results emphasize the importance of cyp19a1a in ovarian differentiation of P. mera . Accumulating evidence of previous investigations had unequivocally demonstrated the pivotal role of cyp19a1a in the differentiation and maintenance of fish ovaries. Such as Danio rerio [ 41 ], Oreochromis nilotica [ 42 ], and Scatophagus argus [ 43 ], genetic mutations in cyp19a1a consistently result in ovarian-to-testicular sex reversal. This conserved phenomenon across diverse fish species highlights the evolutionary significance of cyp19a1a in regulating gonadal development, underscoring its indispensable function in maintaining the ovarian fate of germ cells. Interestingly, foxl2 , one of the ovary makers, has showed great similarity to the expression trend of cyp19a1a (Fig. 6 ). Previous research has demonstrated that foxl2 played a crucial role too in the differentiation and maintenance of ovarian, with mutations in foxl2 leading to ovarian-to-testicular sex reversal[ 42 , 44 ]. Notably, substantial evidence indicates that foxl2 binds to the promoter region of cyp19a1a , thereby promoting its transcription[ 45 – 47 ]. Consequently, we propose that during the differentiation of P. mera , foxl2 may similarly enhance the transcription of cyp19a1a , facilitating the production of estrogen to finish the commitment to ovarian differentiation. In addition to the classical sex differentiation genes, we found some genes that exhibited significantly up-regulated expression during the late stage of ovarian differentiation. Including kdm2b , wnt4a , map2k6 , and tdrd9 , as well as s x11 , wt1 , smad6 and smad4 . Notably, both wt1 and wnt4a are components of the WNT signaling pathway. Researches in mammals has demonstrated that the WNT signaling pathway plays a pivotal role in ovarian determination and differentiation through the stable activity of WNT ligand on β-catenin, which regulates downstream target genes essential for ovarian development[ 3 , 48 ]. Furthermore, the WNT signaling pathway could suppress the expression of sox9 , a key transcription factor involved in male differentiation[ 49 ]. These findings highlight the central role of the WNT signaling pathway in regulating ovarian fate and inhibiting male-specific pathways during sex differentiation. In comparison to females, males exhibited a variety of genes with specific high-expression patterns. Among these, dmrt1 , a member of the dmrt gene family, was recognized as a conserved male differentiation gene that plays an essential role in gonadal differentiation and maintenance across fish and even many vertebrates[ 35 , 50 , 51 ]. In our study, dmrt1 was found to be virtually non-expressed in females, which contrasts sharply with the expression patterns of foxl2 and cyp19a1a in the gonads. This suggested that dmrt1 may serve as a key gene for testis differentiation in P. mera . Furthermore, drawing from studies in other species[ 52 ], it is speculated that dmrt1 might inhibit the expression of foxl2 and cyp19a1a in the testes of P. mera while promoting testis differentiation. Additionally, sox9-b demonstrated male-biased expression at all developmental stages examined in this study, aligning with the male-positive feedback regulatory pathway ( dmrt1 - sox9-b - fgf ) reported in numerous studies[ 35 , 53 ]. This indicates that sox9-b plays a crucial role in the testis differentiation of P. mera . Although no significant difference was observed in the expression of fgf9 between male and female individuals in this study, genes such as fgf1 , fgf13 , and fgfbp3 were up-regulated in males during all four periods analyzed. It is possible that other members of the fgf family also contribute to this regulation through specific expression patterns. These findings collectively underscore the complex interplay of genetic factors in sex determination and differentiation pathways, highlighting the conservation and divergence of mechanisms across different species. To further elucidate the regulatory network underlying gonadal differentiation and maturation in P. mera , we conducted miRNA sequencing of gonads at 150 dph and integrated these data with existing transcriptomic data. The results revealed a total of 224 differentially expressed miRNAs between testes and ovaries at this stage, among these, several miRNAs known to be involved in gonadal differentiation regulation were identified, such as lett-7, miR-21-x and miR-184[ 54 – 56 ]. Notably, miR-21-x, is essential for porcine oocytes entering meiosis, showed upregulated expression from the onset of oocyte entry into meiosis until metaphase II of the second meiotic division[ 54 ]. Target gene prediction analysis indicated a significant negative correlation between the expression of 189 miRNAs and 3,813 genes, underscoring the critical role of miRNAs during late-stage gonadal differentiation in P. mera . The intricate regulatory network formed by miRNA-mRNA interactions reveals that individual miRNAs can regulate multiple target genes simultaneously, while multiple miRNAs may converge on a single target gene. This suggests an exceedingly complex regulatory framework within the gonadal development process. Among the identified miRNAs, miR-200-y, miR-144-y and miR-141-x emerged as key regulators, potentially targeting several genes associated with gonadal differentiation. Specifically, miR-200-y and miR-141, members of the miR-200 family, exhibited ovary-specific upregulation. Importantly, their predicted targets include male pathway genes such as sox9 , tyro3 , dmrt2 and dmrt2a . This implies that they may suppress male-related pathways in the ovary, thus promoting ovarian differentiation. Supporting evidence comes from studies in zebrafish, where knockout of the miR-200 family impaired oocyte maturation[ 57 ], and research in sheep indicating that miR-200b regulated follicular granulosa cell development[ 58 ]. Collectively, these findings suggest that the miR-200 family may represent a pivotal factor in oocyte maturation in P. mera . In addition to known miRNAs, our study identified several novel gender biased miRNAs, including novel-m0207, novel-m0123-5p, novel-m0123-5p and novol-m0122-5p. Their predicted target genes encompass key regulators of gonadal differentiation, such as sox9-b , cyp19a1a , and wnt4a . While these newly discovered miRNAs are speculated to play potential roles in testicular or ovarian differentiation, further functional validation experiments are warranted to confirm their specific roles. In summary, our study provides valuable insights into the regulatory mechanisms of gonadal differentiation and maturation in P. mera , highlighting the importance of both conserved and novel miRNAs in this process. Future investigations should focus on unraveling the precise regulatory mechanisms of these miRNAs and their downstream target genes to deepen our understanding of sex determination and gonadal development in fish. 5. Conclusions In this study, we conducted a comprehensive analysis of the gonadal differentiation process in P. mera using histology, transcriptomics, and miRNA sequencing. Our findings provide a detailed description of the genes and miRNAs expressed during sexual dimorphism in P. mera differentiation. We identified that the genes involved in sex steroid synthesis play a crucial role in the sex differentiation of P. mera . Notably, the key rate-limiting gene for estrogen synthesis, cyp19a1a , exhibited significant differential expression between testes and ovaries across four differentiation stages. This suggests that cyp19a1a is critical for ovarian differentiation in P. mera . Additionally, other potential female candidate genes, including foxl2 , esr1 , wnt4a , wt1 , smad6 , and smad4 , may also play essential roles in ovarian differentiation. In testis, potential male candidate genes such as dmrt1 , sox9-b , dmrt2a , fgf1 , fgf13 , and cyp17a1 , are likely to be important for testis differentiation. Furthermore, we discovered numerous miRNAs implicated in gonadal maturation and differentiation in the later stages in P. mera . These include members of the well-known miR-200 family, as well as newly identified miRNAs such as novel-m0207, novel-m0123-5p, and novel-m0122-5p. The dynamic changes in these genetic factors may provide valuable insights into the regulatory network underlying gonadal differentiation in P. mera . Understanding these mechanisms will aid in improving all-female culture technologies for P. mera in aquaculture, thereby enhancing breeding efficiency and productivity. This study contributes significantly to deciphering the complex regulatory network governing gonadal differentiation in P. mera , offering promising prospects for advancing aquaculture practices. Declarations Funding This research was supported by the Investigation of Fishery Resources in Guangxi (GXZC2022-G3-001062-ZHZB) and Shuangcheng Cooperative Agreement Research Grant of Yibin, China (XNDX2022020004). Author Contribution Zhenlin Ke: Conceptualization, Methodology, Formal analysis, Visualization, Investigation, Writing - Original Draft, Writing - Review & EditinWeijun Wu: Methodology, Visualization, Investigation, Writing - Review & EditingZhe Li: Formal analysis, InvestigationYusen Li: Formal analysis, Writing - Review & EditingYaoquan Han: Formal analysis, Writing - Review & EditingJun Shi: Formal analysis, Writing - Review & EditingLilong Chen: Methodology, InvestigationDapeng Wang: Methodology, Writing—Review & EditingYong Lin: Conceptualization, methodologyMin Li: Conceptualization, , Writing—Review & EditingHua Ye: Conceptualization, Methodology, Writing - Review & Editing References Kitano J, Ansai S, Takehana Y, Yamamoto Y (2024) Diversity and Convergence of Sex-Determination Mechanisms in Teleost Fish. Annu Rev Anim Biosci 12:233–259 Devlin RH, Nagahama Y (2002) Sex determination and sex differentiation in fish: an overview of genetic, physiological, and environmental influences. Aquaculture 208:191–364 Lobo IKC, Nascimento ÁR, do, Yamagishi MEB, Guiguen Y, Silva GF da, Severac D et al (2020) Transcriptome of tambaqui Colossoma macropomum during gonad differentiation: Different molecular signals leading to sex identity. Genomics. ; 112:2478–88 Nakamoto M, Shibata Y, Ohno K, Usami T, Kamei Y, Taniguchi Y et al (2018) Ovarian aromatase loss-of-function mutant medaka undergo ovary degeneration and partial female-to-male sex reversal after puberty. Mol Cell Endocrinol 460:104–122 Jiang D-N, Yang H-H, Li M-H, Shi H-J, Zhang X-B, Wang D-S (2016) gsdf is a downstream gene of dmrt1 that functions in the male sex determination pathway of the Nile tilapia. Mol Reprod Dev 83:497–508 Hayashida T, Soma S, Nakamura Y, Higuchi K, Kazeto Y, Gen K (2023) Transcriptome characterization of gonadal sex differentiation in Pacific bluefin tuna, Thunnus orientalis (Temminck et Schlegel). Sci Rep 13:13867 Sun D, Yu H, Li Q (2022) Examination of the roles of Foxl2 and Dmrt1 in sex differentiation and gonadal development of oysters by using RNA interference. Aquaculture 548:737732 Bartel DP, MicroRNAs (2004) Genomics, Biogenesis, Mechanism, and Function. Cell 116:281–297 Tao W, Sun L, Shi H, Cheng Y, Jiang D, Fu B et al (2016) Integrated analysis of miRNA and mRNA expression profiles in tilapia gonads at an early stage of sex differentiation. BMC Genomics 17:328 Juanchich A, Le Cam A, Montfort J, Guiguen Y, Bobe J (2013) Identification of differentially expressed miRNAs and their potential targets during fish ovarian development. Biol Reprod 88(128):1–11 Sex-Biased (2014) miRNAs in gonad and their potential roles for testis development in Yellow Catfish. PLoS ONE Zhao H, Zhang L, Li Q, Zhao Z, Duan Y, Huang Z et al (2022) Integrated analysis of the miRNA and mRNA expression profiles in Leiocassis longirostris at gonadal maturation. Funct Integr Genomics 22:655–667 Gao Y, Wu Q, Wang G, Zhang S, Ma W, Shi X et al (2024) Histomorphic analysis and expression of mRNA and miRNA in embryonic gonadal differentiation in Chinese soft-shelled turtle ( Pelodiscus sinensis ). Gene 893:147913 Jiang M, Jia S, Chen J, Chen K, Ma W, Wu X et al (2020) Timing of gonadal development and dimorphic expression of sex-related genes in gonads during early sex differentiation in the Yellow River carp. Aquaculture 518:734825 Wang Y (1979) On the classification, distribution, origin and evolution of the fishes referred to the subfamily cyprininae of China, with description of a new species. Acta Hydrobiol Sin 3:419–438 Zhu Y (2006) Cyprinidae subfamily Abainae (except Haliotis), Cynanchinae and Cyprininae. Guangxi people’s publishing house, NanNing Zhai G, Shu T, Chen K, Lou Q, Jia J, Huang J et al (2022) Successful Production of an All-Female Common Carp ( Cyprinus carpio L.) Population Using cyp17a1 -Deficient Neomale Carp. Engineering 8:181–189 Wang S, Le P, Chen Y (1988) China Red Data Book of Endangered Animals-Fish. Science, Beijing, China Wu W, Tan H, Han Y, Yao M, Deng D, Chen J (2024) Identification and virulence gene analysis of pathogenic Aeromonas veronii isolated from Procypris merus . 55(5):1520–1529 Han Y, He A, Lan J, Wu W, Li Y (2018) Individual fecundity and phenotypic traits of Procypris merus . Jiangsu Agricultural Sci 46:134–137 Li Y, Lei J, Han Y, Shi J, Wu W, Wang D et al (2019) Effect of temperature and light intensity on oxygen consumption rate and critical asphyxiation point of Chinese ink carp ( Procypris merus ). J South Agric 50:418–423 Xu H, Lu Z, Lin B, Liu Q, Tan D, Wei X et al (2021) Domestication and artificial reproduction of Chinese ink carp ( Procypris mera ). Hubei Agricultural Sci 60:118–120 Wu Q, Sun H, Li Y (2011) Effects of Different K Sperm Motility in Chinese Ink Carp, Ca and Glucose Concentrations on. Fish Sci 30:202–205 Han Y, He A, Lan J, Wu W, Li Y, Wang D et al (2018) Embryonic Development of Chinese Ink Carp. Fish Sci 37:368–373 Chen L, Wu W, Ye H, Li Z, Ke Z, Zhou K et al (2024) Changes in growth, the activities of major digestive enzymes and intestinal tissues during the early development of Procypris merus . Freshw Fisheries 54:85–92 Li J, Que Y, Xu L, Zhu B, Lian H, Wang L et al (2023) Effects of initial diet and weaning strategies on the growth and survival of Percocypris merus . Freshwtaer Fisheries 53:55–60 Li H, Yang B, Dong W, Que Y, Zhu B, Qiao Y (2023) Feeding and Starvation Tolerance of Yolk-sac Larvae of Percocypris mera . J Hydroecology 44:89–95 Chen S, Zhou Y, Chen Y, Jia G (2018) fastp: an ultra-fast all-in-one FASTQ preprocessor. Bioinformatics 34(17):i884–i890 Kim D, Paggi JM, Park C, Bennett C, Salzberg SL (2019) Graph-based genome alignment and genotyping with HISAT2 and HISAT-genotype. Nat Biotechnol 37(8):907–915 Pertea M, Pertea GM, Antonescu CM, Chang T-C, Mendell JT, Salzberg SL (2015) StringTie enables improved reconstruction of a transcriptome from RNA-seq reads. Nat Biotechnol 33:290–295 Robinson MD, McCarthy DJ, Smyth GK (2010) edgeR: a Bioconductor package for differential expression analysis of digital gene expression data. Bioinformatics 26:139–140 Griffiths-Jones S, Saini HK, Van DS, Enright AJ (2008) Mirbase: tools for microRNA genomics. Nucleic Acids Res 36:154–158 Edris Badreddin (2011) A comparison of the Oligomap and TargetScan algorithms for miRNA target analysis. bmi231.stanford.edu Ungewitter EK, Yao HH-C (2012) How to Make a Gonad: Cellular Mechanisms Governing Formation of the Testes and Ovaries. Sex Dev 7:7–20 Wang M, Chen L, Zhou Z, Xiao J, Chen B, Huang P et al (2023) Comparative transcriptome analysis of early sexual differentiation in the male and female gonads of common carp ( Cyprinus carpio ). Aquaculture 563:738984 Jia Y, Nan P, Zhang W, Wang F, Zhang R, Liang T et al (2018) Transcriptome analysis of three critical periods of ovarian development in Yellow River carp ( Cyprinus carpio ). Theriogenology 105:15–26 Dai L, Liu B, Wang Z (2013) Histological observation of gonadal differentiation of Gobiocypris rarus . J Southwest China Normal Univ Nat Sci Ed. ;38 Bogart MH (1987) Sex determination: A hypothesis based on steroid ratios. J Theor Biol 128:349–357 Simpson ER, Clyne C, Rubin G, Boon WC, Robertson K, Britt K et al (2002) Aromatase—A Brief Overv Annual Rev Physiol 64:93–127 Guiguen Y, Fostier A, Piferrer F, Chang C-F (2010) Ovarian aromatase and estrogens: A pivotal role for gonadal sex differentiation and sex change in fish. Gen Comp Endocrinol 165:352–366 Chen W, Liu L, Ge W (2023) Expression analysis of growth differentiation factor 9 ( Gdf9 / gdf9 ), anti-müllerian hormone ( Amh / amh ) and aromatase ( Cyp19a1a / cyp19a1a ) during gonadal differentiation of the zebrafish, Danio rerio . Freshwtaer Fisheries 53:55–60 Zhang X, Li M, Ma H, Liu X, Shi H, Li M et al (2017) Mutation of foxl2 or cyp19a1a Results in Female to Male Sex Reversal in XX Nile Tilapia. Endocrinology 158:2634–2647 Mustapha UF, Peng Y-X, Huang Y-Q, Assan D, Zhi F, Shi G et al (2022) Comparative transcriptome analysis of the differentiating gonads in Scatophagus argus . Front Mar Sci. ;9 Ren Z, Ye D, Su N, Wang C, He L, Wang H et al (2024) foxl2l is a germ cell-intrinsic gatekeeper of oogenesis in zebrafish. Zoological Res 45:1116–1130 Zhang C, He Q, Cheng H, Li L, Ruan X, Duan X et al (2022) Transcription factors foxl2 and foxl3 regulate cyp19a1a and cyp11b in orange-spotted grouper ( Epinephelus coioides ). Aquaculture Reports. ; 25:101243 Lee KH, Yamaguchi A, Rashid H, Kadomura K, Yasumoto S, Matsuyama M (2009) Germ cell degeneration in high-temperature treated pufferfish, Takifugu rubripes . Sex Dev 3:225–232 Pannetier M, Fabre S, Batista F, Kocer A, Renault L, Jolivet G et al (2006) FOXL2 activates P450 aromatase gene transcription: towards a better characterization of the early steps of mammalian ovarian development. 36(3):399–413 Kim K-A, Wagle M, Tran K, Zhan X, Dixon MA, Liu S et al (2008) R-Spondin Family Members Regulate the Wnt Pathway by a Common Mechanism. Mol Biol Cell 19:2588–2596 Tevosian SG (2012) Genetic Control of Ovarian Development. Sex Dev 7:33–45 Mustapha UF, Peng Y-X, Huang Y-Q, Assan D, Zhi F, Shi G et al (2022) Comparative transcriptome analysis of the differentiating gonads in Scatophagus argus . Front Mar Sci. ;9 Dai S, Qi S, Wei X, Liu X, Li Y, Zhou X et al (2021) Germline sexual fate is determined by the antagonistic action of dmrt1 and foxl3 / foxl2 in tilapia. Development 48:8 Augstenová B, Ma W-J (2025) Decoding Dmrt1 : insights into vertebrate sex determination and gonadal sex differentiation Lavery R, Chassot A-A, Pauper E, Gregoire EP, Klopfenstein M, de Rooij DG et al (2012) Testicular differentiation occurs in absence of R-spondin1 and Sox9 in mouse sex reversals. Plos Genetic 8:1003170 Song C, Yao J, Cao C, Liang X, Huang J, Han Z et al (2016) PPARγ is regulated by miR-27b-3p negatively and plays an important role in porcine oocyte maturation. Biochem Biophys Res Commun 479:224–230 Wu Y, Wang Y, Xue Y, Wu N, Wu J, Zhang J (2023) cbx2 is a functional target of the let-7 family in the gonad of Japanese flounder ( Paralichthys olivaceus ). Comparative Biochemistry and Physiology Part B: Biochemistry and Molecular Biology. ; 263:110799 Wu J, Bao J, Wang L, Hu Y, Xu C (2011) MicroRNA-184 downregulates nuclear receptor corepressor 2 in mouse spermatogenesis. BMC Dev Biol 11:64 Xiong S, Tian J, Ge S, Li Z, Long Z, Guo W et al (2020) The microRNA-200 cluster on chromosome 23 is required for oocyte maturation and ovulation in zebrafish. Biol Reprod 103:769–778 Song P, Xi J, Zhang Z, Fu Q, Yue Q, Zhang P et al Effects of Ovine miR-200b on Cell Cycle and Apoptosis of Follicular Granulosa Cells. Acta veterinaria et zootechnica sinica. 52:3471–3479 Additional Declarations No competing interests reported. Supplementary Files supplementaltables.doc supplementalfigures.zip Cite Share Download PDF Status: Published Journal Publication published 13 Mar, 2026 Read the published version in Marine Biotechnology → Version 1 posted Editorial decision: Revision requested 02 Jan, 2026 Reviews received at journal 02 Jan, 2026 Reviews received at journal 27 Dec, 2025 Reviewers agreed at journal 13 Dec, 2025 Reviewers agreed at journal 13 Dec, 2025 Reviewers agreed at journal 12 Dec, 2025 Reviewers invited by journal 12 Dec, 2025 Editor assigned by journal 11 Dec, 2025 Submission checks completed at journal 11 Dec, 2025 First submitted to journal 05 Dec, 2025 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-8288461","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":560022888,"identity":"49e91b3b-5aee-4440-9700-43203c4620a2","order_by":0,"name":"Zhenlin Ke","email":"","orcid":"","institution":"Southwest University","correspondingAuthor":false,"prefix":"","firstName":"Zhenlin","middleName":"","lastName":"Ke","suffix":""},{"id":560022892,"identity":"43d1c766-47f6-41dc-8882-f077f95af532","order_by":1,"name":"Weijun Wu","email":"","orcid":"","institution":"Guangxi Academy of Fishery Sciences","correspondingAuthor":false,"prefix":"","firstName":"Weijun","middleName":"","lastName":"Wu","suffix":""},{"id":560022895,"identity":"bb843ad2-884c-4d01-94d6-10541f756cd1","order_by":2,"name":"Zhe Li","email":"","orcid":"","institution":"Guangxi Academy of Fishery Sciences","correspondingAuthor":false,"prefix":"","firstName":"Zhe","middleName":"","lastName":"Li","suffix":""},{"id":560022896,"identity":"565b0265-91c3-4f70-94e1-457ad123f882","order_by":3,"name":"Yusen Li","email":"","orcid":"","institution":"Guangxi Academy of Fishery Sciences","correspondingAuthor":false,"prefix":"","firstName":"Yusen","middleName":"","lastName":"Li","suffix":""},{"id":560022897,"identity":"7e9373c3-4f3f-4013-9203-43bc51f48894","order_by":4,"name":"Yaoquan Han","email":"","orcid":"","institution":"Guangxi Academy of Fishery Sciences","correspondingAuthor":false,"prefix":"","firstName":"Yaoquan","middleName":"","lastName":"Han","suffix":""},{"id":560022898,"identity":"bc8759ac-08cf-41b1-8c78-30b38cbc4877","order_by":5,"name":"Jun Shi","email":"","orcid":"","institution":"Guangxi Academy of Fishery Sciences","correspondingAuthor":false,"prefix":"","firstName":"Jun","middleName":"","lastName":"Shi","suffix":""},{"id":560022899,"identity":"134fdd51-e9f5-43a7-977a-6b9094df02df","order_by":6,"name":"Lilong Chen","email":"","orcid":"","institution":"Southwest University","correspondingAuthor":false,"prefix":"","firstName":"Lilong","middleName":"","lastName":"Chen","suffix":""},{"id":560022901,"identity":"e7cb70b7-a449-488d-ab36-7e7154d1af07","order_by":7,"name":"Kangqi Zhou","email":"","orcid":"","institution":"Guangxi Academy of Fishery Sciences","correspondingAuthor":false,"prefix":"","firstName":"Kangqi","middleName":"","lastName":"Zhou","suffix":""},{"id":560022904,"identity":"ae5e795b-acf5-4977-8af3-06d9cea83a2d","order_by":8,"name":"Dapeng Wang","email":"","orcid":"","institution":"Guangxi Academy of Fishery Sciences","correspondingAuthor":false,"prefix":"","firstName":"Dapeng","middleName":"","lastName":"Wang","suffix":""},{"id":560022907,"identity":"06e24683-6989-4ce6-99f7-a979180c63a5","order_by":9,"name":"Yong Lin","email":"","orcid":"","institution":"Guangxi Academy of Fishery Sciences","correspondingAuthor":false,"prefix":"","firstName":"Yong","middleName":"","lastName":"Lin","suffix":""},{"id":560022917,"identity":"980f1806-0ec8-4307-8d73-e67faac431ee","order_by":10,"name":"Min Li","email":"","orcid":"","institution":"Guangxi Academy of Fishery Sciences","correspondingAuthor":false,"prefix":"","firstName":"Min","middleName":"","lastName":"Li","suffix":""},{"id":560022918,"identity":"399f6b09-28b7-4e19-a9f1-03d6a64efb86","order_by":11,"name":"Hua Ye","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAu0lEQVRIiWNgGAWjYDACCeY2hg8GEjz8zMwHHxCphbGNcUaFjZxkO1uyAdFamHnOpBkbnOcxEyBKh8HtxrYHvG2HEzcfZjBjYKixiSas5c7BdgNJoJZthxnSHjAcS8ttIKjlRmKbhCFEy3EDxobDRGpJBDmsmbFNgngtB0DeZ2ZmI06LJFCLZAMwkCUOszEbJBDjF74bycek/4Cisv/8xwcfamwIa1E4gMxLIKQcBOQJGjoKRsEoGAWjAACEiEQSnHeZ/AAAAABJRU5ErkJggg==","orcid":"","institution":"Southwest University","correspondingAuthor":true,"prefix":"","firstName":"Hua","middleName":"","lastName":"Ye","suffix":""}],"badges":[],"createdAt":"2025-12-05 13:53:18","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-8288461/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-8288461/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1007/s10126-026-10581-x","type":"published","date":"2026-03-13T15:58:02+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":98399465,"identity":"71019790-2a17-48f7-86a8-56274b8831be","added_by":"auto","created_at":"2025-12-17 11:20:41","extension":"docx","order_by":0,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":4052038,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.docx","url":"https://assets-eu.researchsquare.com/files/rs-8288461/v1/c57e24a33b1602f761c1ca34.docx"},{"id":98441334,"identity":"a258821d-30e1-4588-aaf7-353dea4f50ff","added_by":"auto","created_at":"2025-12-17 17:05:13","extension":"json","order_by":1,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":12501,"visible":true,"origin":"","legend":"","description":"","filename":"432b25492b1a4fdf8f2524a2dd0fe442.json","url":"https://assets-eu.researchsquare.com/files/rs-8288461/v1/fac15d512f9c3b94eac68f0e.json"},{"id":98440994,"identity":"1f8269d5-3d49-43b6-b881-057458f6fabe","added_by":"auto","created_at":"2025-12-17 17:04:43","extension":"zip","order_by":2,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":367737,"visible":true,"origin":"","legend":"","description":"","filename":"supplementalfigures.zip","url":"https://assets-eu.researchsquare.com/files/rs-8288461/v1/8d750fca496fafc6b10863cf.zip"},{"id":98399466,"identity":"c8f6ece6-d80d-49f7-8305-ca4708496253","added_by":"auto","created_at":"2025-12-17 11:20:41","extension":"doc","order_by":3,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":65024,"visible":true,"origin":"","legend":"","description":"","filename":"supplementaltables.doc","url":"https://assets-eu.researchsquare.com/files/rs-8288461/v1/9a7955f7a74ce1614360a35d.doc"},{"id":98441557,"identity":"5b7086f9-1918-46cc-bc0b-e6a05094ec74","added_by":"auto","created_at":"2025-12-17 17:05:35","extension":"xml","order_by":4,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":148212,"visible":true,"origin":"","legend":"","description":"","filename":"432b25492b1a4fdf8f2524a2dd0fe4421enriched.xml","url":"https://assets-eu.researchsquare.com/files/rs-8288461/v1/6fa9d7ba660fbcd283d2d425.xml"},{"id":98399483,"identity":"3d1368b5-b7cd-43a7-9960-1604dbc80dd1","added_by":"auto","created_at":"2025-12-17 11:20:42","extension":"jpeg","order_by":5,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":3764712,"visible":true,"origin":"","legend":"","description":"","filename":"floatimage1.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-8288461/v1/0a5944f9c09c77741e8438a7.jpeg"},{"id":98441407,"identity":"71a3d86e-17b5-40b0-b35f-620ceb8404dc","added_by":"auto","created_at":"2025-12-17 17:05:20","extension":"jpeg","order_by":6,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":9607794,"visible":true,"origin":"","legend":"","description":"","filename":"floatimage2.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-8288461/v1/38488ca1c836ce2b2f354b9f.jpeg"},{"id":98440609,"identity":"e1f1aec0-8a6f-4ba7-bc4c-7cc1a2e62a59","added_by":"auto","created_at":"2025-12-17 17:04:06","extension":"jpeg","order_by":7,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":4512028,"visible":true,"origin":"","legend":"","description":"","filename":"floatimage3.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-8288461/v1/47223288ff700ce25837e542.jpeg"},{"id":98440785,"identity":"a70c1e59-937e-4684-907e-61ec305bdda9","added_by":"auto","created_at":"2025-12-17 17:04:21","extension":"jpeg","order_by":8,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":4505314,"visible":true,"origin":"","legend":"","description":"","filename":"floatimage4.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-8288461/v1/e97cb781cd90163b36e29748.jpeg"},{"id":98399481,"identity":"1ea9682f-e3d6-4f31-85b3-606ca75766fc","added_by":"auto","created_at":"2025-12-17 11:20:42","extension":"png","order_by":9,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":487249,"visible":true,"origin":"","legend":"","description":"","filename":"floatimage5.png","url":"https://assets-eu.researchsquare.com/files/rs-8288461/v1/9679d0c932fa8cb5cc3f3c3f.png"},{"id":98399479,"identity":"f0d48093-375f-4a5b-bb4a-7f7533a19350","added_by":"auto","created_at":"2025-12-17 11:20:42","extension":"jpeg","order_by":10,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":6579902,"visible":true,"origin":"","legend":"","description":"","filename":"floatimage6.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-8288461/v1/267afdf3895c60b6a484d780.jpeg"},{"id":98441444,"identity":"0a6f30f8-6399-4fef-b156-e9eec7017597","added_by":"auto","created_at":"2025-12-17 17:05:25","extension":"png","order_by":11,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":178795,"visible":true,"origin":"","legend":"","description":"","filename":"Onlinefloatimage1.png","url":"https://assets-eu.researchsquare.com/files/rs-8288461/v1/665f71a37fb01df7d5edf56a.png"},{"id":98399484,"identity":"13d035ed-9beb-4965-9c7a-331f7a2e1968","added_by":"auto","created_at":"2025-12-17 11:20:42","extension":"png","order_by":12,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":118252,"visible":true,"origin":"","legend":"","description":"","filename":"Onlinefloatimage2.png","url":"https://assets-eu.researchsquare.com/files/rs-8288461/v1/7a1c61ec2d457767556880fd.png"},{"id":98441172,"identity":"ccb08683-ca89-46b3-926b-ec5a58c1972c","added_by":"auto","created_at":"2025-12-17 17:05:01","extension":"png","order_by":13,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":79335,"visible":true,"origin":"","legend":"","description":"","filename":"Onlinefloatimage3.png","url":"https://assets-eu.researchsquare.com/files/rs-8288461/v1/7b6022285bc41fd03f096043.png"},{"id":98399475,"identity":"6fc872c7-007a-4e17-889b-f977142b3458","added_by":"auto","created_at":"2025-12-17 11:20:42","extension":"png","order_by":14,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":69728,"visible":true,"origin":"","legend":"","description":"","filename":"Onlinefloatimage4.png","url":"https://assets-eu.researchsquare.com/files/rs-8288461/v1/c24caf97deeab951529e2121.png"},{"id":98399485,"identity":"44144a6a-a890-4eb8-8a92-6af1d885f702","added_by":"auto","created_at":"2025-12-17 11:20:42","extension":"png","order_by":15,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":106658,"visible":true,"origin":"","legend":"","description":"","filename":"Onlinefloatimage5.png","url":"https://assets-eu.researchsquare.com/files/rs-8288461/v1/3094ab96f5cd2274bf87e76d.png"},{"id":98441541,"identity":"66759194-d8bb-4372-a516-9dabc00a4135","added_by":"auto","created_at":"2025-12-17 17:05:34","extension":"png","order_by":16,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":110063,"visible":true,"origin":"","legend":"","description":"","filename":"Onlinefloatimage6.png","url":"https://assets-eu.researchsquare.com/files/rs-8288461/v1/8548d8a89fade98d77e54e88.png"},{"id":98399489,"identity":"053af465-1207-449f-bc6d-79078559d488","added_by":"auto","created_at":"2025-12-17 11:20:42","extension":"xml","order_by":17,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":146316,"visible":true,"origin":"","legend":"","description":"","filename":"432b25492b1a4fdf8f2524a2dd0fe4421structuring.xml","url":"https://assets-eu.researchsquare.com/files/rs-8288461/v1/3645a4982eb39d66c933776c.xml"},{"id":98441415,"identity":"e9436e3d-690c-4321-a54d-424c0f5e0233","added_by":"auto","created_at":"2025-12-17 17:05:21","extension":"html","order_by":18,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":164470,"visible":true,"origin":"","legend":"","description":"","filename":"earlyproof.html","url":"https://assets-eu.researchsquare.com/files/rs-8288461/v1/5bb040027b00c7663069a308.html"},{"id":98399463,"identity":"1b0eefde-5562-4389-a6b4-87670044427b","added_by":"auto","created_at":"2025-12-17 11:20:41","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":4165932,"visible":true,"origin":"","legend":"\u003cp\u003eEarly gonadal differentiation of \u003cem\u003eP. mera\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eA: Single primordial germ cell in genital crest at 15 dph; B: Primitive gonadal were formed at 20 dph; C: The primordial germ cell numbers increased at 25 dph; D: Ovary histological differentiation with ovarian cavity appeared at 60 dph; E, F: Ovary development with enlargement of ovarian cavity, the primary oocytes appeared at 110dph, and ovarian cytological differentiation began; G: Sperm primordium appears at the base of gonad, and histological differentiation of testis begins at 95 dph; H, I: Testis developed, and the spermatogonia appeared spermatocyte-like at 150 dph, indicating that the cytological differentiation of testis began;\u003c/p\u003e\n\u003cp\u003ePGC: Primordial germ cells; AB: air bladder; G: gut; PG: primary gonad; OC: Ovarian cavity; BV: blood vessel; PO: primary oocyte; SA: Sperm anlage; PS: Primary spermatocyte.\u003c/p\u003e","description":"","filename":"image1.png","url":"https://assets-eu.researchsquare.com/files/rs-8288461/v1/9335b9a63fc50869fd18e575.png"},{"id":98440301,"identity":"6cef78a9-2163-44f5-9c45-6ed980daabd7","added_by":"auto","created_at":"2025-12-17 17:03:41","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":2355656,"visible":true,"origin":"","legend":"\u003cp\u003eA: The DEGs numbers in different stages of differentiation. Upregulated: red, downregulated: green (male vs female); B: Venn plots showed the distribution of DEGs at 4 differentiation periods; C-F: The differentially expressed genes TOP20 KEGG in four stages of differentiation enriched of significantly bubble chart; G: The expression heatmap of steroid synthesis related genes during early differentiation of the ovary and testis; H: Protein-protein interaction network of steroid synthesis related genes.\u003c/p\u003e","description":"","filename":"image2.png","url":"https://assets-eu.researchsquare.com/files/rs-8288461/v1/5bb88174cf5b6cd175f6819e.png"},{"id":98441131,"identity":"93984050-a043-4ce0-9d83-8350e5f2b729","added_by":"auto","created_at":"2025-12-17 17:04:55","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":2047362,"visible":true,"origin":"","legend":"\u003cp\u003eA: Heatmap of all DEGs between each testis differentiation stages; B: Trend diagram of differentially expressed genes; C, D: UpSet and Venn plots showing the distribution of DEGs at 4 differentiation periods. The bar chart above represents the number of genes contained in each type of group. The bar chart at the bottom left represents the number of DEGs included in each stage of gonadal differentiation.\u003c/p\u003e","description":"","filename":"image3.png","url":"https://assets-eu.researchsquare.com/files/rs-8288461/v1/cfa778169d80f08bb415234e.png"},{"id":98441360,"identity":"3c71143f-ed9a-41c2-b43f-3c2f57356f57","added_by":"auto","created_at":"2025-12-17 17:05:15","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":1751885,"visible":true,"origin":"","legend":"\u003cp\u003eHeatmap of all DEGs between each ovary differentiation stages. B: Trend diagram of different expressed genes. C, D: UpSet and Venn plots showing the distribution of DEGs at 4 differentiation periods. The bar chart above represents the number of genes contained in each type of group. The bar chart at the bottom left represents the number of DEGs included in each stage of gonadal differentiation.\u003c/p\u003e","description":"","filename":"image4.png","url":"https://assets-eu.researchsquare.com/files/rs-8288461/v1/e8a54baf91e7677fcc5cb30a.png"},{"id":98399470,"identity":"1c817da3-579b-4f4f-a166-300cdc3f83a2","added_by":"auto","created_at":"2025-12-17 11:20:41","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":487249,"visible":true,"origin":"","legend":"\u003cp\u003eDiagram of regulation network of miRNA-RNA (pink\u003c/p\u003e\n\u003cp\u003erepresent miRNA, and blue indicate their target genes)\u003c/p\u003e","description":"","filename":"image5.png","url":"https://assets-eu.researchsquare.com/files/rs-8288461/v1/9c8557d3f67f8c572b51c7c5.png"},{"id":98440982,"identity":"9e3e539e-387e-4a4e-a7c2-7cc89dff3c06","added_by":"auto","created_at":"2025-12-17 17:04:43","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":1821914,"visible":true,"origin":"","legend":"\u003cp\u003eThe verification results of RNA-seq and miRNA-seq by qRT-PCR (For mRNA, developmental stages are revealed in x-axis, The relative expression levels and RNA-seq results were revealed in y-axis, \u003cem\u003eβ-actin\u003c/em\u003e was the internal reference gene. For miRNA, the core miRNAs were used for verification. F represents for ovary and M represents for testis)\u003c/p\u003e","description":"","filename":"image6.png","url":"https://assets-eu.researchsquare.com/files/rs-8288461/v1/f5525ece34807df263dd504a.png"},{"id":104740828,"identity":"d0f8aefb-a662-4d51-b6a5-c73fcfa5780b","added_by":"auto","created_at":"2026-03-16 16:19:13","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":14513993,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8288461/v1/e75ec0da-40db-406b-be38-61e2d28ed224.pdf"},{"id":98399462,"identity":"1479ccb5-4cd7-48e7-bd78-71952f50fa2a","added_by":"auto","created_at":"2025-12-17 11:20:41","extension":"doc","order_by":0,"title":"","display":"","copyAsset":false,"role":"supplement","size":65024,"visible":true,"origin":"","legend":"","description":"","filename":"supplementaltables.doc","url":"https://assets-eu.researchsquare.com/files/rs-8288461/v1/8fad2f419ffc682689ab3fce.doc"},{"id":98399472,"identity":"41f1b378-8d1f-4f63-bbb4-19e71c7d625f","added_by":"auto","created_at":"2025-12-17 11:20:41","extension":"zip","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":367737,"visible":true,"origin":"","legend":"","description":"","filename":"supplementalfigures.zip","url":"https://assets-eu.researchsquare.com/files/rs-8288461/v1/5fbfedd3e0f0899ceef7b64c.zip"}],"financialInterests":"No competing interests reported.","formattedTitle":"Integrated analysis of mRNA-seq and miRNA-seq reveal the dynamics of the sexual differentiation of Procypris mera","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003eSexual reproduction, as the primary mode of multiplication in life, represents the most common breeding method in the vast majority of vertebrates[\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. Serves as the foundation for species continuation, the normal development and differentiation of gonads are essential prerequisites for reproduction. Sex determination occurs after primordial germ cells (PGCs) migrate to the genital ridge and form the primitive gonad. This process initiates the expression of genes associated with gonadal differentiation, guiding the undifferentiated gonads to develop into either testes or ovaries. Ultimately, the gonads developed into mature with the emergence of secondary sexual characteristics[\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eAs the largest group of vertebrates, fish exhibit remarkable diversity and plasticity in gonadal differentiation, which provides an excellent model for analyzing the genetic mechanism and gonadal differentiation[\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. To data, numerous key genes involved in gonadal differentiation have been identified in fish, such as \u003cem\u003egsdf\u003c/em\u003e, \u003cem\u003ecyp19a1a\u003c/em\u003e, \u003cem\u003ehsd11b2\u003c/em\u003e, \u003cem\u003efoxl2\u003c/em\u003e and \u003cem\u003ecyp17a1\u003c/em\u003e, these genes are regulated by sex-determining genes and are specifically expressed in testis or ovary during the gonadal differentiation. The knock-out of these genes will cause sexual reversal[\u003cspan additionalcitationids=\"CR5 CR6\" citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. In addition, microRNA (miRNA), a type of small non-coding RNA, as the second network for regulating gene expression and protein translation, it can target multiple genes and regulate their expression[\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e], which also plays an important role in the early gonadal differentiation of fish[\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e], miRNA have been proven that it can specifically regulate the differentiation of testis or ovary[\u003cspan additionalcitationids=\"CR11 CR12\" citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. Consequently, integrated analysis of miRNA and mRNA transcriptomes can provide deeper insights into post-transcriptional regulatory networks. In summary, while many genetic factors contributing to gonadal differentiation have been identified, significant gaps remain in our understanding of how these factors interact, whether at the level of mRNA or miRNA regulation. Therefore, elucidating the mechanisms of fish gonadal differentiation and uncovering the associated regulatory networks, especially the processes governing early sex differentiation, is of great importance[\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. Such research could offer valuable references for developing effective fish breeding strategies.\u003c/p\u003e \u003cp\u003eChinese ink carp (\u003cem\u003eProcypris mera\u003c/em\u003e), indigenous to the Pearl river basin, is one of the most primitive fishes within the subfamily Cyprininae[\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e, \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. Similar to the common carp (\u003cem\u003eCyprinus carpio\u003c/em\u003e), \u003cem\u003eP. mera\u003c/em\u003e showed an obvious sexual dimorphism in growth and females possessed a faster growth rate, which was once an economic fish in Guangxi, therefore, all-female breeding can effectively improve its breeding benefit[\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. However, the wild resources of \u003cem\u003eP mera\u003c/em\u003e have experienced an amazing decline due to overfishing, dam construction, and destruction of feeding and spawning grounds. At present, it has been classified as endangered species (VU) in the China Red and listed as a national second-class protected animal[\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e, \u003cspan additionalcitationids=\"CR19\" citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. Therefore, it is important to study gonadal determination and differentiation of \u003cem\u003eP mera\u003c/em\u003e, which is significant for \u003cem\u003eP. mera\u003c/em\u003e in both resource restoration and aquaculture production. Currently, there were few studies investigated the ecological habits[\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e], breeding[\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e], reproduction[\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e, \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e] and seedling cultivation[\u003cspan additionalcitationids=\"CR26\" citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e], while the detailed and complex process of gonadal differentiation and development haven't been reported.\u003c/p\u003e \u003cp\u003eIn order to define the gonadal differentiation and development process of \u003cem\u003eP mera\u003c/em\u003e, and further dissect the mechanisms of its differentiation. In this study, the histological changes of the formation and differentiation of primitive gonads in \u003cem\u003eP. mera\u003c/em\u003e were explored by paraffin tissue sections and HE staining, and comparative transcriptome analysis was performed for four key periods of males and females early gonadal differentiation, including: gonadal differentiation for females (60 and 110 dph), gonadal differentiation for males (90 and 150 dph). Additionally, to further understand the molecular regulatory network of late differentiation of \u003cem\u003eP. mera\u003c/em\u003e, which is the key stage to control the maturation of early gonads, miRNA sequencing was performed in the critical period of gametogenesis. In general, this study has determined the early gonadal development process of \u003cem\u003eP. mera\u003c/em\u003e, and the sequencing results showed that there were many gender-biased genes and miRNAs, which participated in the gonadal differentiation of males and females. Moreover, \u003cem\u003ecyp19a1a\u003c/em\u003e has significant differential expression between males and females in four critical stages of differentiation, which is a critical gene for estrogen synthesis. And many miRNAs also play a regulatory role in gonadal differentiation. This study may provide further insight into the early gonadal differentiation in \u003cem\u003eP. mera\u003c/em\u003e and assist to explain its molecule mechanisms in this species, which will offer reference for future sex-controlled breeding.\u003c/p\u003e"},{"header":"2. Materials and Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003e2.1. Ethics statement\u003c/h2\u003e \u003cp\u003e This study was conducted in accordance with the Declaration of Helsinki and approved by the Animal Care and Use Committee of Southwest University, Chongqing, China.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003e2.2. sample collection\u003c/h2\u003e \u003cp\u003eThe \u003cem\u003eP. mera\u003c/em\u003e used in the experiment were derived from the rare and endangered fish breeding base of Guangxi Fisheries Research Institute, all the larvae were reproduced by the same parents (Breeding certificate number: 2023-0108004). During the whole breeding process, the water quality conditions are as follows: temperature: 25\u0026thinsp;\u0026plusmn;\u0026thinsp;1℃, DOI\u0026thinsp;\u0026ge;\u0026thinsp;5 mg/L, pH: 7\u0026ndash;8, NH3-N: 0.1\u0026ndash;0.2 mg/L, NIT: 0.01\u0026ndash;0.02 mg/L. Gonad were collected at 5, 10, 15, 20, 25, 30, 35 \u0026hellip;\u0026hellip; dph until gonadal differentiation is completed. The one-half of the gonad are used to identify the sex and development situation, which were stored in 4% paraformaldehyde, the other half were stored in the 4℃ RNA later and transferred to -80℃ after 24 hours. Only gonads during the critical stage of differentiation were used for subsequent sequencing.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003e2.3. RNA extraction, library construction, transcriptome sequencing and data analysis.\u003c/h2\u003e \u003cp\u003eAccording to the histological section results, testis and ovary in the critical period of gonadal differentiation (60, 95, 110, 150dph) were used to extract RNA, there were three samples of testis and ovary in each period, and each sample is mixed by two samples. Total RNA of each samples were extracted using TRIzol Reagent Kit (Invitrogen, Carlsbad, CA, USA) with the manufacturer's program. The quality and integrity of RNA samples were assessed using an Agilent 2100 Bioanalyzer (Agilent Technologies, Palo Alto, CA, USA).\u003c/p\u003e \u003cp\u003eSequencing library was constructed using Illumina TruSeq RNA Sample Preparation Kit (Illumina, San Diego, CA, USA) following the manufacturer's protocol. The libraries were sequenced on the Illumina Novaseq 6000 sequencing platform. Low quality data of raw data were eliminated by Fastp[\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e](\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://github.com/OpenGene/fastp\u003c/span\u003e\u003cspan address=\"https://github.com/OpenGene/fastp\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) and the clean data were compared with reference genome by hisat2[\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e](v.4.8.5) (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://daehwankimlab.github.io/hisat2/\u003c/span\u003e\u003cspan address=\"https://daehwankimlab.github.io/hisat2/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e), then the transcript were assembled by stringtie[\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e], the differential expression genes(DEGs) analysis were performed using edgeR[\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e] with FDR\u0026thinsp;\u0026lt;\u0026thinsp;0.05and |log2FC|\u0026gt;1, the corresponding biological functionand related pathways of DEGs were performed using OmicShare online tools (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e\u003ca href=\"https://github.com/OpenGene/fastp\" target=\"_blank\"\u003ewww.omicshare.com/tools\u003c/a\u003e\u003c/span\u003e\u003cspan address=\"http://www.omicshare.com/tools\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003e2.4. miRNA extraction, library construction, transcriptome sequencing and data analysis\u003c/h2\u003e \u003cp\u003eThe testis and ovary in the critical period of primordial germ cells (150dph) were used to sequencing. The miRNA libraries were constructed using the NEBNext\u0026reg; Multiplex Small RNA Library Prep Set for Illumina\u0026reg; kit strictly following the operation instructions. After the quality inspection, the qualified libraries were sequenced on the Illumina Hiseq 2000 (Illumina, San Diego, CA, USA).\u003c/p\u003e \u003cp\u003eThe raw sequences were processed through the Illumina pipeline program. After eliminating contaminated reads, such as adapter dimers, junk sequences, those with low complexity, members of common RNA families (rRNA, tRNA, snRNA, snoRNA), and repeats, the clean reads were further filtered by the software package ACGT101 - miR - v4.2 (LC Sciences, Houston, Texas, USA).\u003c/p\u003e \u003cp\u003eThe clean sequence reads were aligned with miRBase 21.0[\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e], permitting a mismatch of one or two nucleotide bases, to identify both known miRNAs and novel 3p - and 5p - derived miRNAs. EdgeR[\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e] was used to analyze the differential expression of the identified miRNA (dispersion\u0026thinsp;=\u0026thinsp;0.1, and the rest were the default parameters). The screening criteria are that the difference multiple is greater than 2 and \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003e2.5. miRNA target predictions and integrate analysis of miRNA and mRNA\u003c/h2\u003e \u003cp\u003eMiranda[\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e] (parameter: set score threshold to 140, set energy threshold to -10 kcal/mol, demand strict 5' seed pairing, set gap-open penalty to -4.0 and set gap-extend penalty to -9.0) (v3.3a) and TargetScan[\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e] (V 7.0) were used to predict target genes, and the prediction result shared by the two softwires as the final results. In addition, the differential expression mRNAs of the concurrent period were used to integrate analysis in omicshare tools (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e\u003ca href=\"https://github.com/OpenGene/fastp\" target=\"_blank\"\u003ewww.omicshare.com/tools\u003c/a\u003e\u003c/span\u003e\u003cspan address=\"http://www.omicshare.com/tools\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) with two demands; 1: There is a targeted relationship between miRNA and mRNA; 2: miRNA was negatively correlated with mRNA expression.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003e2.6. The verification of miRNA and mRNA with qRT-PCR\u003c/h2\u003e \u003cp\u003eThere were 7 genes and 9 miRNAs were selected to verify the sequencing data. For mRNA, the quantitative real-time PCR (qRT-PCR) primers were designed in primer 5.0, and \u003cem\u003eβ-actin\u003c/em\u003e was used as reference gene (The primers were shown in Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e). The cDNA of mRNA was synthesized with the PrimeScript\u0026trade; FAST RT reagent Kit and the express level of mRNAs were determined using TB Green\u0026reg; Fast qPCR Mix (TaKaRa, Beijing, China). All primers used five serially diluted cDNA as templates to construct standard curves, and determine the amplification efficiency of the primer. For miRNA, the Mir-XTM miRNA qRT-PCR TB GreenTM Kit and TB Green\u0026reg; Premix Ex Taq\u0026trade; II (Tli RNaseH Plus) (TaKaRa, Beijing, China) were used to complete real-time fluorescence quantitative analysis, and the reverse primer is the universal primer included in the kit, the forward primer is the full length of miRNA sequence (U base instead T base substitution sequence), and the reference U6 are included in the kit (The primers were shown in Table \u003cspan refid=\"MOESM2\" class=\"InternalRef\"\u003eS2\u003c/span\u003e). The relative expression level of mRNA and miRNA were calculated by the 2^-\u003csup\u003eΔΔCt\u003c/sup\u003e method.\u003c/p\u003e \u003c/div\u003e"},{"header":"3. Results","content":"\u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003e3.1. The gonadal differentiation process of \u003cem\u003eP. mera\u003c/em\u003e\u003c/h2\u003e \u003cp\u003eThe histological results showed that the gonadal differentiation of \u003cem\u003eP. mera\u003c/em\u003e was completed at 150 dph. At 15 dph, the single PGC was observed above the intestine, it has arrived the genital crest (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA). With the development of gonad, the primordial germ was surrounded by somatic cell and formed primitive undifferentiated gonads (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB, C). At 55 dph, there are two sections of undifferentiated gonad, one of them was long shape which will develop into ovaries, another one still was water drops shape which will develop into testis. At 60 dph, the long gonads begin to have a small cavity between the back of gonad and mesentery, suggesting the initiation of the gonad histological differentiation, and the cavity gradually develop into ovarian cavity and enlarge. At 110 dph, the oogoniums have presented the morphology of primary oocyte, it marked the beginning of ovarian cytological differentiation (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eD, E, F). The sperm primordium appeared at the base of gonad at 95 dph in the gonads of water drops shape, which suggested the initiation of the testis histological differentiation, and spermatogonia cells presented the morphology of primary spermatocytes at 150 dph, indicating the initiation of testicular cytological differentiation (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eG, H, I).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eA: Single primordial germ cell in genital crest at 15 dph; B: Primitive gonadal were formed at 20 dph; C: The primordial germ cell numbers increased at 25 dph; D: Ovary histological differentiation with ovarian cavity appeared at 60 dph; E, F: Ovary development with enlargement of ovarian cavity, the primary oocytes appeared at 110dph, and ovarian cytological differentiation began; G: Sperm primordium appears at the base of gonad, and histological differentiation of testis begins at 95 dph; H, I: Testis developed, and the spermatogonia appeared spermatocyte-like at 150 dph, indicating that the cytological differentiation of testis began;\u003c/p\u003e \u003cp\u003ePGC: Primordial germ cells; AB: air bladder; G: gut; PG: primary gonad; OC: Ovarian cavity; BV: blood vessel; PO: primary oocyte; SA: Sperm anlage; PS: Primary spermatocyte.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003e3.2. Overview of transcriptome sequencing data\u003c/h2\u003e \u003cp\u003eA total of 24 samples from four stages of gonadal differentiation of \u003cem\u003eP. mera\u003c/em\u003e were sequenced using Illumina NovaSeq sequencing platform, and 1149852254 raw reads were obtained. After quality control, 1137396262 clean reads were obtained, the average Q30 ratio of clean reads is 93.46%, which indicates that the clean reads can be used for the subsequent analysis. The data were compared to the reference genome of \u003cem\u003eP. mera\u003c/em\u003e, and the average total comparison rate was 93.26%, the average unique comparison rate was 88.74%. A total of 47541 genes were detected in the sequencing data (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eSummary statistics of sequencing data\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"6\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eSample\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eRaw reads\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eClean reads\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eQ30 (%)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eTotal mapping ratio (%)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\"\u003e \u003cp\u003eUniquely mapping ratio (%)\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003em60 dph-1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e48554896\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e47732030\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e92.42%\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e91.73%\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e88.01%\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003em60 dph-2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e47324756\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e45990558\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e91.72%\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e87.45%\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e83.56%\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003em60 dph-3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e47717928\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e46622366\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e91.93%\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e89.56%\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e84.65%\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003em95 dph-1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e57509484\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e56890792\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e91.47%\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e93.70%\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e88.54%\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003em95 dph-2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e43823372\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e43340520\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e89.35%\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e92.63%\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e88.75%\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003em95 dph-3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e57012282\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e56545560\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e94.05%\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e94.75%\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e90.35%\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003em110 dph-1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e40379064\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e40164198\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e95.39%\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e95.21%\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e91.30%\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003em110 dph-2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e39704502\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e39500594\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e95.38%\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e95.53%\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e91.62%\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003em110 dph-3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e43282212\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e42970878\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e94.02%\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e95.05%\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e91.17%\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003em150 dph-1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e57994336\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e57584718\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e94.18%\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e95.07%\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e91.13%\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003em150 dph-2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e69825146\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e69298336\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e94.10%\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e94.95%\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e91.06%\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003em150 dph-3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e37826662\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e37617134\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e94.97%\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e94.59%\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e88.86%\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ef60 dph-1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e45399048\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e44105590\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e91.48%\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e83.10%\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e79.01%\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ef60 dph-2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e45941564\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e45070634\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e91.62%\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e89.79%\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e84.87%\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ef60 dph-3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e47918168\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e47259338\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e90.38%\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e90.14%\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e86.21%\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ef95 dph-1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e38107292\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e37861510\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e95.02%\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e94.80%\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e90.88%\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ef95 dph-2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e42335224\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e42034914\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e94.33%\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e95.15%\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e91.31%\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ef95 dph-3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e37670940\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e37444940\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e94.95%\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e95.14%\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e86.81%\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ef110 dph-1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e46973400\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e46530270\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e92.93%\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e94.33%\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e89.98%\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ef110 dph-2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e50275914\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e49950856\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e94.79%\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e95.02%\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e90.67%\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ef110 dph-3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e45963804\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e45783608\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e95.67%\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e95.44%\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e91.13%\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ef150 dph-1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e42003708\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e41703404\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e94.27%\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e94.99%\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e91.05%\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ef150 dph-2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e59059068\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e58658882\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e94.68%\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e94.91%\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e91.37%\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ef150 dph-3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e57249484\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e56734632\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e93.82%\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e95.22%\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e87.40%\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003e3.3. Identification, annotation and expression analysis of DEGs between testis and ovary at four differentiation stages\u003c/h2\u003e \u003cp\u003eThe edgeR was used to identify the DEGs of four stages, a total of 8755 DEGs were identified, of which 2372 were up-regulated and 6383 were down-regulated. The specific number of DEGs of different stages of differentiation were displayed in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA and B. DEGs between testis and ovary gradually increased following the development of gonad, indicating that more and more gender-related genes were involved in the differentiation process. Additionally, DEGs of each stage were used to KEGG enrichment analyse to explore its potential function. Interestingly, the steroid hormone synthesis relate pathway was significantly enriched in four periods, it suggested steroid hormone synthesis pathway probably was the key pathway to lead the gonadal differentiation of \u003cem\u003eP. mera\u003c/em\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC, D, E, F). In order to further understand the function of this pathway, the genes related to steroid synthesis with differential expression were screened to applied in the expression profile analysis and protein-protein interaction(PPI) network analysis, the results showed that there were different dominant genes in different stages of differentiation, such as: \u003cem\u003ecyp19a1a\u003c/em\u003e, \u003cem\u003eldlr\u003c/em\u003e and \u003cem\u003ecyp27a1\u003c/em\u003e of 60 dph; \u003cem\u003ehsd3b\u003c/em\u003e, \u003cem\u003epla2g4d\u003c/em\u003e and \u003cem\u003ecyp11a1\u003c/em\u003e of 95 dph; \u003cem\u003efoxl2\u003c/em\u003e, \u003cem\u003estar\u003c/em\u003e and \u003cem\u003ecyp11b\u003c/em\u003e of 110 dph; \u003cem\u003eesr1\u003c/em\u003e, \u003cem\u003edmrt1\u003c/em\u003e and \u003cem\u003ecyp17a1\u003c/em\u003e of 150 dph. There were 8 genes including \u003cem\u003ecyp17a1\u003c/em\u003e, \u003cem\u003ehsd3b1\u003c/em\u003e, \u003cem\u003ecyp11b\u003c/em\u003e, \u003cem\u003ecyp3a27\u003c/em\u003e, \u003cem\u003ehsd3b2\u003c/em\u003e, \u003cem\u003ecyp2c8\u003c/em\u003e and \u003cem\u003ecyp19a1a\u003c/em\u003e were identified as hub genes in PPI network, which may play a key role in gonadal differentiation of \u003cem\u003eP. mera\u003c/em\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eG, H).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003e3.4. Identification, spatiotemporal and trend analysis of DEGs in testis and ovary differentiation\u003c/h2\u003e \u003cp\u003eIn order to further explore the molecular regulatory network of gonadal differentiation in \u003cem\u003eP. mera\u003c/em\u003e, neighbor periods in testis or ovary were compared and analyzed respectively.\u003c/p\u003e \u003cp\u003eIn testis, a total of 13466 DEGs were identified, in which 107 DEGs were overlapped. As the differentiation of gonads, the number of DEGs between adjacent days decreased gradually, which may result from the gradual completion of gonadal differentiation and the steady expression of differentiation-related genes, it is helpful to maintain the development of testis (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eC, D). Additionally, the expression thermogram showed that there were significantly different expression profiles at different stages, which further indicated that gonadal differentiation was regulated by a complex network (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA). Further trend analysis of DEGs shows that these DEGs are significantly enriched in 9 plates (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB). The DEGs including \u003cem\u003edmrtb1\u003c/em\u003e, \u003cem\u003edmrt2\u003c/em\u003e, \u003cem\u003esox9-b\u003c/em\u003e, \u003cem\u003ezar\u003c/em\u003e and \u003cem\u003ecyp26a1\u003c/em\u003e etc in profile17 was stably up-regulated after testis differentiation, and maintained at a certain level in the later stage, indicating that they may be closely related to testis differentiation, whereas the expression level of \u003cem\u003edmrt1\u003c/em\u003e, \u003cem\u003ehsd11b2\u003c/em\u003e, and \u003cem\u003edmrt3a\u003c/em\u003e etc in profile 10 were low in the early stage of differentiation and started to rise after 110 dph, which demonstrated that these genes may play a key role in the development and maintenance of testis.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eIn ovary, there were 14198 DEGs between ovaries in several stages, and 273 DEGs were overlap (Fig.\u0026nbsp;4C, D). Consistent with the testis, the number of differential genes between adjacent ovarian ages decreased gradually, and the expression thermogram of each period was significantly different. The trend analysis showed that these DEGs significantly enriched in 6 plates(Fig.\u0026nbsp;4A, B). The expression level of \u003cem\u003efoxl2\u003c/em\u003e, \u003cem\u003ekdm2b\u003c/em\u003e, \u003cem\u003ewnt4a\u003c/em\u003e, \u003cem\u003emap2k6\u003c/em\u003e and \u003cem\u003etdrd9\u003c/em\u003e in profile 18 increased gradually and reached the highest at 110 dph, it is proved that these genes mainly play a role in the late stage of ovarian differentiation. In addition, \u003cem\u003esox11\u003c/em\u003e, \u003cem\u003ewt1\u003c/em\u003e, \u003cem\u003efgf1, fgf13, fgf2, smad4\u003c/em\u003e and \u003cem\u003esmad6\u003c/em\u003e etc were enriched in profile 17, which began to up-regulated from 95 dph and keep highly expression, indicating that these genes were important for the later development and maintenance of the ovary.\u003c/p\u003e \u003cp\u003eFigure 4 Heatmap of all DEGs between each ovary differentiation stages. B: Trend diagram of different expressed genes. C, D: UpSet and Venn plots showing the distribution of DEGs at 4 differentiation periods. The bar chart above represents the number of genes contained in each type of group. The bar chart at the bottom left represents the number of DEGs included in each stage of gonadal differentiation.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003e3.5. Combination analysis of miRNA and mRNA\u003c/h2\u003e \u003cp\u003eFurthermore, we selected the 150 dph gonads for small RNA sequencing, which is helpful to further explore the regulatory network in the later stage of gonadal differentiation, because this is the key period to control gonadal maturation. The details of the original sequencing data and clean tags of each sample were displayed in Table S3, the clean tags of each sample were compared with the genome of \u003cem\u003eP.mera\u003c/em\u003e, and the comparison rate was between 73.71% and 78.11%, and the gap rate between samples was small, indicating that the samples were not polluted (Table S4).\u003c/p\u003e \u003cp\u003eThe aligned sequences were utilized for miRNA identification, resulting in the discovery of 1561 miRNAs, among which 924 were novel miRNAs (Figure \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e). These identified miRNAs were subsequently subjected to differential expression analysis, revealing a total of 224 miRNAs that exhibited significant differential expression between the testis and ovary. To further investigate the regulatory network during the late differentiation stage, differentially expressed mRNAs from the same stage were employed for target gene prediction. The analysis demonstrated a notable negative regulatory relationship between 189 miRNAs and 3,813 mRNAs (Figure \u003cspan refid=\"MOESM2\" class=\"InternalRef\"\u003eS2\u003c/span\u003e). Furthermore, given the pivotal role of steroid hormone synthesis-related pathways, core genes involved in steroid hormone synthesis and significantly different expression of gender-biased miRNA were selected to construct the mRNA-miRNA regulatory axis (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e5\u003c/span\u003e), thereby providing insights into the intricate regulatory mechanisms underlying late meiosis. The results showed that miRNA-200-y was regulated by several genes related to sex differentiation and gametogenesis, it was highly expressed in testis, while its corresponding target genes including \u003cem\u003esox9-b\u003c/em\u003e, \u003cem\u003ezp3\u003c/em\u003e, \u003cem\u003espdya\u003c/em\u003e, \u003cem\u003eago3\u003c/em\u003e and \u003cem\u003etyro3\u003c/em\u003e were down-regulated in males. Concordantly, miRA-144-y was highly expressed in ovary and its target genes of \u003cem\u003ecamk2a, ppp2r5b\u003c/em\u003e and \u003cem\u003ehsd11b2\u003c/em\u003e were down-regulated expression in ovary. Single miRNA could be regulated by multiple mRNA and single mRNA also could be regulated by multiple miRNAs, which emphasized the complexity of gonadal differentiation.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003erepresent miRNA, and blue indicate their target genes)\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003e3.6 Validation of differential expression mRNA and miRNA\u003c/h2\u003e \u003cp\u003eDifferentially expressed genes and miRNAs in gonads were randomly selected for verification. The results showed that the expression levels of mRNA and miRNAs were consistent with RNA-seq data, which ensured the reliability of the above sequencing results.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"4. Discussion","content":"\u003cp\u003eSex determination and sex differentiation were an intricate and highly regulated processes. Upon initiation of the sexual developmental pathway, a network of genes involved in sex differentiation emerges, which was two mutually antagonistic regulatory systems[\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e, \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e]. These opposing networks govern the fate of bipotential gonadal primordium, which possess the potential for either testicular or ovarian differentiation, ultimately driving their commitment to one of the two reproductive organ destinies. This process ensures the precise establishment of male or female gonadal identity, a critical step in sexual development[\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e]. Monitoring this complex developmental process could provide invaluable data support for subsequent breeding efforts.\u003c/p\u003e \u003cp\u003eTo elucidate the entire process of gonadal differentiation and the sex-specific molecular regulatory mechanisms of \u003cem\u003eP. mera\u003c/em\u003e, we collected gonads from 5 dph, from the appearance of the ovarian cavity at 60 dph to the presence of spermatocytes at 150 dph. The early sex differentiation of \u003cem\u003eP. mera\u003c/em\u003e was essentially complete by this stage. Based on the differentiation time, the gonad differentiation of \u003cem\u003eP. mera\u003c/em\u003e occurs later compared to other carp species within the same family, such as common carp and rare gudgeon (\u003cem\u003eGobiocypris rarus\u003c/em\u003e)[\u003cspan additionalcitationids=\"CR36\" citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e]. This delay may be attributed to the extended early development and life cycles of \u003cem\u003eP. mera\u003c/em\u003e, thus resulting in a relatively later differentiation time.\u003c/p\u003e \u003cp\u003eAdditionally, we utilized the testis and ovary at a critical stage of sex differentiation to sequence the transcriptome and systematically analyzed these dynamic transcriptional data. Among the findings, we observed that genes associated with steroid hormones synthesis played a significant role in the gonadal differentiation of \u003cem\u003eP. mera\u003c/em\u003e. These genes exhibited sexual dimorphism across four distinct periods. Steroids, being the most crucial precursors of synthetic sex hormones, are essential for the development of male or female characteristics. Previous studies have indicated that the balance of sex hormones can determine the direction of sex differentiation[\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e]. Fish, in particular, were highly sensitive to the perception of sex hormones, while this balance was regulated by aromatase[\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e]. In this study, the expression of \u003cem\u003ecyp19a1a\u003c/em\u003e during four differentiation periods was significantly higher in ovary than in testis at all stages of differentiation, and it reached its peak at 110 dph. Early studies have shown that aromatase was mainly used to hydrolyze conjugated steroids and promote the synthesis of synthetic estrogens[\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e]. Thus, vitellogenesis and steroid hormone synthesis were the reasons of the up-regulation of \u003cem\u003ecyp19a1a\u003c/em\u003e expression. In this study, the oocyte of \u003cem\u003eP. mera\u003c/em\u003e began meiosis at 110 dph, and with the maturation of oocytes, vitellogenesis was gradually completed, accompanied by the completion of gonadal differentiation. Therefore, the expression of \u003cem\u003ecyp19a1a\u003c/em\u003e in the gonads of \u003cem\u003eP. mera\u003c/em\u003e reached the highest at 110 dph and decreased at 150 dph. These results emphasize the importance of \u003cem\u003ecyp19a1a\u003c/em\u003e in ovarian differentiation of \u003cem\u003eP. mera\u003c/em\u003e. Accumulating evidence of previous investigations had unequivocally demonstrated the pivotal role of \u003cem\u003ecyp19a1a\u003c/em\u003e in the differentiation and maintenance of fish ovaries. Such as \u003cem\u003eDanio rerio\u003c/em\u003e[\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e], \u003cem\u003eOreochromis nilotica\u003c/em\u003e[\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e], and \u003cem\u003eScatophagus argus\u003c/em\u003e[\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e], genetic mutations in \u003cem\u003ecyp19a1a\u003c/em\u003e consistently result in ovarian-to-testicular sex reversal. This conserved phenomenon across diverse fish species highlights the evolutionary significance of \u003cem\u003ecyp19a1a\u003c/em\u003e in regulating gonadal development, underscoring its indispensable function in maintaining the ovarian fate of germ cells. Interestingly, \u003cem\u003efoxl2\u003c/em\u003e, one of the ovary makers, has showed great similarity to the expression trend of \u003cem\u003ecyp19a1a\u003c/em\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e6\u003c/span\u003e). Previous research has demonstrated that \u003cem\u003efoxl2\u003c/em\u003e played a crucial role too in the differentiation and maintenance of ovarian, with mutations in \u003cem\u003efoxl2\u003c/em\u003e leading to ovarian-to-testicular sex reversal[\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e, \u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e]. Notably, substantial evidence indicates that \u003cem\u003efoxl2\u003c/em\u003e binds to the promoter region of \u003cem\u003ecyp19a1a\u003c/em\u003e, thereby promoting its transcription[\u003cspan additionalcitationids=\"CR46\" citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e]. Consequently, we propose that during the differentiation of \u003cem\u003eP. mera\u003c/em\u003e, \u003cem\u003efoxl2\u003c/em\u003e may similarly enhance the transcription of \u003cem\u003ecyp19a1a\u003c/em\u003e, facilitating the production of estrogen to finish the commitment to ovarian differentiation. In addition to the classical sex differentiation genes, we found some genes that exhibited significantly up-regulated expression during the late stage of ovarian differentiation. Including \u003cem\u003ekdm2b\u003c/em\u003e, \u003cem\u003ewnt4a\u003c/em\u003e, \u003cem\u003emap2k6\u003c/em\u003e, and \u003cem\u003etdrd9\u003c/em\u003e, as well as s\u003cem\u003ex11\u003c/em\u003e, \u003cem\u003ewt1\u003c/em\u003e, \u003cem\u003esmad6\u003c/em\u003e and \u003cem\u003esmad4\u003c/em\u003e. Notably, both \u003cem\u003ewt1\u003c/em\u003e and \u003cem\u003ewnt4a\u003c/em\u003e are components of the WNT signaling pathway. Researches in mammals has demonstrated that the WNT signaling pathway plays a pivotal role in ovarian determination and differentiation through the stable activity of WNT ligand on β-catenin, which regulates downstream target genes essential for ovarian development[\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e, \u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e]. Furthermore, the WNT signaling pathway could suppress the expression of \u003cem\u003esox9\u003c/em\u003e, a key transcription factor involved in male differentiation[\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e]. These findings highlight the central role of the WNT signaling pathway in regulating ovarian fate and inhibiting male-specific pathways during sex differentiation.\u003c/p\u003e \u003cp\u003eIn comparison to females, males exhibited a variety of genes with specific high-expression patterns. Among these, \u003cem\u003edmrt1\u003c/em\u003e, a member of the \u003cem\u003edmrt\u003c/em\u003e gene family, was recognized as a conserved male differentiation gene that plays an essential role in gonadal differentiation and maintenance across fish and even many vertebrates[\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e, \u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e, \u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e]. In our study, \u003cem\u003edmrt1\u003c/em\u003e was found to be virtually non-expressed in females, which contrasts sharply with the expression patterns of \u003cem\u003efoxl2\u003c/em\u003e and \u003cem\u003ecyp19a1a\u003c/em\u003e in the gonads. This suggested that \u003cem\u003edmrt1\u003c/em\u003e may serve as a key gene for testis differentiation in \u003cem\u003eP. mera\u003c/em\u003e. Furthermore, drawing from studies in other species[\u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e], it is speculated that \u003cem\u003edmrt1\u003c/em\u003e might inhibit the expression of \u003cem\u003efoxl2\u003c/em\u003e and \u003cem\u003ecyp19a1a\u003c/em\u003e in the testes of \u003cem\u003eP. mera\u003c/em\u003e while promoting testis differentiation. Additionally, \u003cem\u003esox9-b\u003c/em\u003e demonstrated male-biased expression at all developmental stages examined in this study, aligning with the male-positive feedback regulatory pathway (\u003cem\u003edmrt1\u003c/em\u003e-\u003cem\u003esox9-b\u003c/em\u003e-\u003cem\u003efgf\u003c/em\u003e) reported in numerous studies[\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e, \u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e]. This indicates that \u003cem\u003esox9-b\u003c/em\u003e plays a crucial role in the testis differentiation of \u003cem\u003eP. mera\u003c/em\u003e. Although no significant difference was observed in the expression of \u003cem\u003efgf9\u003c/em\u003e between male and female individuals in this study, genes such as \u003cem\u003efgf1\u003c/em\u003e, \u003cem\u003efgf13\u003c/em\u003e, and \u003cem\u003efgfbp3\u003c/em\u003e were up-regulated in males during all four periods analyzed. It is possible that other members of the \u003cem\u003efgf\u003c/em\u003e family also contribute to this regulation through specific expression patterns. These findings collectively underscore the complex interplay of genetic factors in sex determination and differentiation pathways, highlighting the conservation and divergence of mechanisms across different species.\u003c/p\u003e \u003cp\u003eTo further elucidate the regulatory network underlying gonadal differentiation and maturation in \u003cem\u003eP. mera\u003c/em\u003e, we conducted miRNA sequencing of gonads at 150 dph and integrated these data with existing transcriptomic data. The results revealed a total of 224 differentially expressed miRNAs between testes and ovaries at this stage, among these, several miRNAs known to be involved in gonadal differentiation regulation were identified, such as lett-7, miR-21-x and miR-184[\u003cspan additionalcitationids=\"CR55\" citationid=\"CR54\" class=\"CitationRef\"\u003e54\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e56\u003c/span\u003e]. Notably, miR-21-x, is essential for porcine oocytes entering meiosis, showed upregulated expression from the onset of oocyte entry into meiosis until metaphase II of the second meiotic division[\u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e54\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eTarget gene prediction analysis indicated a significant negative correlation between the expression of 189 miRNAs and 3,813 genes, underscoring the critical role of miRNAs during late-stage gonadal differentiation in \u003cem\u003eP. mera\u003c/em\u003e. The intricate regulatory network formed by miRNA-mRNA interactions reveals that individual miRNAs can regulate multiple target genes simultaneously, while multiple miRNAs may converge on a single target gene. This suggests an exceedingly complex regulatory framework within the gonadal development process. Among the identified miRNAs, miR-200-y, miR-144-y and miR-141-x emerged as key regulators, potentially targeting several genes associated with gonadal differentiation. Specifically, miR-200-y and miR-141, members of the miR-200 family, exhibited ovary-specific upregulation. Importantly, their predicted targets include male pathway genes such as \u003cem\u003esox9\u003c/em\u003e, \u003cem\u003etyro3\u003c/em\u003e, \u003cem\u003edmrt2\u003c/em\u003e and \u003cem\u003edmrt2a\u003c/em\u003e. This implies that they may suppress male-related pathways in the ovary, thus promoting ovarian differentiation. Supporting evidence comes from studies in zebrafish, where knockout of the miR-200 family impaired oocyte maturation[\u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e57\u003c/span\u003e], and research in sheep indicating that miR-200b regulated follicular granulosa cell development[\u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e58\u003c/span\u003e]. Collectively, these findings suggest that the miR-200 family may represent a pivotal factor in oocyte maturation in \u003cem\u003eP. mera\u003c/em\u003e. In addition to known miRNAs, our study identified several novel gender biased miRNAs, including novel-m0207, novel-m0123-5p, novel-m0123-5p and novol-m0122-5p. Their predicted target genes encompass key regulators of gonadal differentiation, such as \u003cem\u003esox9-b\u003c/em\u003e, \u003cem\u003ecyp19a1a\u003c/em\u003e, and \u003cem\u003ewnt4a\u003c/em\u003e. While these newly discovered miRNAs are speculated to play potential roles in testicular or ovarian differentiation, further functional validation experiments are warranted to confirm their specific roles. In summary, our study provides valuable insights into the regulatory mechanisms of gonadal differentiation and maturation in \u003cem\u003eP. mera\u003c/em\u003e, highlighting the importance of both conserved and novel miRNAs in this process. Future investigations should focus on unraveling the precise regulatory mechanisms of these miRNAs and their downstream target genes to deepen our understanding of sex determination and gonadal development in fish.\u003c/p\u003e"},{"header":"5. Conclusions","content":"\u003cp\u003eIn this study, we conducted a comprehensive analysis of the gonadal differentiation process in \u003cem\u003eP. mera\u003c/em\u003e using histology, transcriptomics, and miRNA sequencing. Our findings provide a detailed description of the genes and miRNAs expressed during sexual dimorphism in \u003cem\u003eP. mera\u003c/em\u003e differentiation. We identified that the genes involved in sex steroid synthesis play a crucial role in the sex differentiation of \u003cem\u003eP. mera\u003c/em\u003e. Notably, the key rate-limiting gene for estrogen synthesis, \u003cem\u003ecyp19a1a\u003c/em\u003e, exhibited significant differential expression between testes and ovaries across four differentiation stages. This suggests that cyp19a1a is critical for ovarian differentiation in \u003cem\u003eP. mera\u003c/em\u003e. Additionally, other potential female candidate genes, including \u003cem\u003efoxl2\u003c/em\u003e, \u003cem\u003eesr1\u003c/em\u003e, \u003cem\u003ewnt4a\u003c/em\u003e, \u003cem\u003ewt1\u003c/em\u003e, \u003cem\u003esmad6\u003c/em\u003e, and \u003cem\u003esmad4\u003c/em\u003e, may also play essential roles in ovarian differentiation. In testis, potential male candidate genes such as \u003cem\u003edmrt1\u003c/em\u003e, \u003cem\u003esox9-b\u003c/em\u003e, \u003cem\u003edmrt2a\u003c/em\u003e, \u003cem\u003efgf1\u003c/em\u003e, \u003cem\u003efgf13\u003c/em\u003e, and \u003cem\u003ecyp17a1\u003c/em\u003e, are likely to be important for testis differentiation. Furthermore, we discovered numerous miRNAs implicated in gonadal maturation and differentiation in the later stages in \u003cem\u003eP. mera\u003c/em\u003e. These include members of the well-known miR-200 family, as well as newly identified miRNAs such as novel-m0207, novel-m0123-5p, and novel-m0122-5p. The dynamic changes in these genetic factors may provide valuable insights into the regulatory network underlying gonadal differentiation in \u003cem\u003eP. mera\u003c/em\u003e. Understanding these mechanisms will aid in improving all-female culture technologies for \u003cem\u003eP. mera\u003c/em\u003e in aquaculture, thereby enhancing breeding efficiency and productivity. This study contributes significantly to deciphering the complex regulatory network governing gonadal differentiation in \u003cem\u003eP. mera\u003c/em\u003e, offering promising prospects for advancing aquaculture practices.\u003c/p\u003e"},{"header":"Declarations","content":"\u003ch2\u003eFunding\u003c/h2\u003e \u003cp\u003eThis research was supported by the Investigation of Fishery Resources in Guangxi (GXZC2022-G3-001062-ZHZB) and Shuangcheng Cooperative Agreement Research Grant of Yibin, China (XNDX2022020004).\u003c/p\u003e\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eZhenlin Ke: Conceptualization, Methodology, Formal analysis, Visualization, Investigation, Writing - Original Draft, Writing - Review \u0026amp; EditinWeijun Wu: Methodology, Visualization, Investigation, Writing - Review \u0026amp; EditingZhe Li: Formal analysis, InvestigationYusen Li: Formal analysis, Writing - Review \u0026amp; EditingYaoquan Han: Formal analysis, Writing - Review \u0026amp; EditingJun Shi: Formal analysis, Writing - Review \u0026amp; EditingLilong Chen: Methodology, InvestigationDapeng Wang: Methodology, Writing\u0026mdash;Review \u0026amp; EditingYong Lin: Conceptualization, methodologyMin Li: Conceptualization, , Writing\u0026mdash;Review \u0026amp; EditingHua Ye: Conceptualization, Methodology, Writing - Review \u0026amp; Editing\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eKitano J, Ansai S, Takehana Y, Yamamoto Y (2024) Diversity and Convergence of Sex-Determination Mechanisms in Teleost Fish. Annu Rev Anim Biosci 12:233\u0026ndash;259\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eDevlin RH, Nagahama Y (2002) Sex determination and sex differentiation in fish: an overview of genetic, physiological, and environmental influences. Aquaculture 208:191\u0026ndash;364\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLobo IKC, Nascimento \u0026Aacute;R, do, Yamagishi MEB, Guiguen Y, Silva GF da, Severac D et al (2020) Transcriptome of tambaqui \u003cem\u003eColossoma macropomum\u003c/em\u003e during gonad differentiation: Different molecular signals leading to sex identity. Genomics. ; 112:2478\u0026ndash;88\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eNakamoto M, Shibata Y, Ohno K, Usami T, Kamei Y, Taniguchi Y et al (2018) Ovarian aromatase loss-of-function mutant medaka undergo ovary degeneration and partial female-to-male sex reversal after puberty. Mol Cell Endocrinol 460:104\u0026ndash;122\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eJiang D-N, Yang H-H, Li M-H, Shi H-J, Zhang X-B, Wang D-S (2016) gsdf is a downstream gene of dmrt1 that functions in the male sex determination pathway of the Nile tilapia. Mol Reprod Dev 83:497\u0026ndash;508\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHayashida T, Soma S, Nakamura Y, Higuchi K, Kazeto Y, Gen K (2023) Transcriptome characterization of gonadal sex differentiation in Pacific bluefin tuna, \u003cem\u003eThunnus orientalis\u003c/em\u003e (Temminck et Schlegel). Sci Rep 13:13867\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSun D, Yu H, Li Q (2022) Examination of the roles of \u003cem\u003eFoxl2\u003c/em\u003e and \u003cem\u003eDmrt1\u003c/em\u003e in sex differentiation and gonadal development of oysters by using RNA interference. Aquaculture 548:737732\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBartel DP, MicroRNAs (2004) Genomics, Biogenesis, Mechanism, and Function. Cell 116:281\u0026ndash;297\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eTao W, Sun L, Shi H, Cheng Y, Jiang D, Fu B et al (2016) Integrated analysis of miRNA and mRNA expression profiles in tilapia gonads at an early stage of sex differentiation. BMC Genomics 17:328\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eJuanchich A, Le Cam A, Montfort J, Guiguen Y, Bobe J (2013) Identification of differentially expressed miRNAs and their potential targets during fish ovarian development. Biol Reprod 88(128):1\u0026ndash;11\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSex-Biased (2014) miRNAs in gonad and their potential roles for testis development in Yellow Catfish. PLoS ONE\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZhao H, Zhang L, Li Q, Zhao Z, Duan Y, Huang Z et al (2022) Integrated analysis of the miRNA and mRNA expression profiles in \u003cem\u003eLeiocassis longirostris\u003c/em\u003e at gonadal maturation. Funct Integr Genomics 22:655\u0026ndash;667\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGao Y, Wu Q, Wang G, Zhang S, Ma W, Shi X et al (2024) Histomorphic analysis and expression of mRNA and miRNA in embryonic gonadal differentiation in Chinese soft-shelled turtle (\u003cem\u003ePelodiscus sinensis\u003c/em\u003e). Gene 893:147913\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eJiang M, Jia S, Chen J, Chen K, Ma W, Wu X et al (2020) Timing of gonadal development and dimorphic expression of sex-related genes in gonads during early sex differentiation in the Yellow River carp. Aquaculture 518:734825\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWang Y (1979) On the classification, distribution, origin and evolution of the fishes referred to the subfamily cyprininae of China, with description of a new species. Acta Hydrobiol Sin 3:419\u0026ndash;438\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZhu Y (2006) Cyprinidae subfamily Abainae (except Haliotis), Cynanchinae and Cyprininae. Guangxi people\u0026rsquo;s publishing house, NanNing\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZhai G, Shu T, Chen K, Lou Q, Jia J, Huang J et al (2022) Successful Production of an All-Female Common Carp (\u003cem\u003eCyprinus carpio\u003c/em\u003e L.) Population Using \u003cem\u003ecyp17a1\u003c/em\u003e-Deficient Neomale Carp. Engineering 8:181\u0026ndash;189\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWang S, Le P, Chen Y (1988) China Red Data Book of Endangered Animals-Fish. Science, Beijing, China\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWu W, Tan H, Han Y, Yao M, Deng D, Chen J (2024) Identification and virulence gene analysis of pathogenic Aeromonas veronii isolated from \u003cem\u003eProcypris merus\u003c/em\u003e. 55(5):1520\u0026ndash;1529\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHan Y, He A, Lan J, Wu W, Li Y (2018) Individual fecundity and phenotypic traits of \u003cem\u003eProcypris merus\u003c/em\u003e. Jiangsu Agricultural Sci 46:134\u0026ndash;137\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLi Y, Lei J, Han Y, Shi J, Wu W, Wang D et al (2019) Effect of temperature and light intensity on oxygen consumption rate and critical asphyxiation point of Chinese ink carp (\u003cem\u003eProcypris merus\u003c/em\u003e). J South Agric 50:418\u0026ndash;423\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eXu H, Lu Z, Lin B, Liu Q, Tan D, Wei X et al (2021) Domestication and artificial reproduction of Chinese ink carp (\u003cem\u003eProcypris mera\u003c/em\u003e). Hubei Agricultural Sci 60:118\u0026ndash;120\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWu Q, Sun H, Li Y (2011) Effects of Different K Sperm Motility in Chinese Ink Carp, Ca and Glucose Concentrations on. Fish Sci 30:202\u0026ndash;205\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHan Y, He A, Lan J, Wu W, Li Y, Wang D et al (2018) Embryonic Development of Chinese Ink Carp. Fish Sci 37:368\u0026ndash;373\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eChen L, Wu W, Ye H, Li Z, Ke Z, Zhou K et al (2024) Changes in growth, the activities of major digestive enzymes and intestinal tissues during the early development of \u003cem\u003eProcypris merus\u003c/em\u003e. Freshw Fisheries 54:85\u0026ndash;92\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLi J, Que Y, Xu L, Zhu B, Lian H, Wang L et al (2023) Effects of initial diet and weaning strategies on the growth and survival of \u003cem\u003ePercocypris merus\u003c/em\u003e. Freshwtaer Fisheries 53:55\u0026ndash;60\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLi H, Yang B, Dong W, Que Y, Zhu B, Qiao Y (2023) Feeding and Starvation Tolerance of Yolk-sac Larvae of \u003cem\u003ePercocypris mera\u003c/em\u003e. J Hydroecology 44:89\u0026ndash;95\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eChen S, Zhou Y, Chen Y, Jia G (2018) fastp: an ultra-fast all-in-one FASTQ preprocessor. Bioinformatics 34(17):i884\u0026ndash;i890\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKim D, Paggi JM, Park C, Bennett C, Salzberg SL (2019) Graph-based genome alignment and genotyping with HISAT2 and HISAT-genotype. Nat Biotechnol 37(8):907\u0026ndash;915\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003ePertea M, Pertea GM, Antonescu CM, Chang T-C, Mendell JT, Salzberg SL (2015) StringTie enables improved reconstruction of a transcriptome from RNA-seq reads. Nat Biotechnol 33:290\u0026ndash;295\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eRobinson MD, McCarthy DJ, Smyth GK (2010) edgeR: a Bioconductor package for differential expression analysis of digital gene expression data. Bioinformatics 26:139\u0026ndash;140\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGriffiths-Jones S, Saini HK, Van DS, Enright AJ (2008) Mirbase: tools for microRNA genomics. Nucleic Acids Res 36:154\u0026ndash;158\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eEdris Badreddin (2011) A comparison of the Oligomap and TargetScan algorithms for miRNA target analysis. bmi231.stanford.edu\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eUngewitter EK, Yao HH-C (2012) How to Make a Gonad: Cellular Mechanisms Governing Formation of the Testes and Ovaries. Sex Dev 7:7\u0026ndash;20\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWang M, Chen L, Zhou Z, Xiao J, Chen B, Huang P et al (2023) Comparative transcriptome analysis of early sexual differentiation in the male and female gonads of common carp (\u003cem\u003eCyprinus carpio\u003c/em\u003e). Aquaculture 563:738984\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eJia Y, Nan P, Zhang W, Wang F, Zhang R, Liang T et al (2018) Transcriptome analysis of three critical periods of ovarian development in Yellow River carp (\u003cem\u003eCyprinus carpio\u003c/em\u003e). Theriogenology 105:15\u0026ndash;26\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eDai L, Liu B, Wang Z (2013) Histological observation of gonadal differentiation of \u003cem\u003eGobiocypris rarus\u003c/em\u003e. J Southwest China Normal Univ Nat Sci Ed. ;38\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBogart MH (1987) Sex determination: A hypothesis based on steroid ratios. J Theor Biol 128:349\u0026ndash;357\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSimpson ER, Clyne C, Rubin G, Boon WC, Robertson K, Britt K et al (2002) Aromatase\u0026mdash;A Brief Overv Annual Rev Physiol 64:93\u0026ndash;127\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGuiguen Y, Fostier A, Piferrer F, Chang C-F (2010) Ovarian aromatase and estrogens: A pivotal role for gonadal sex differentiation and sex change in fish. Gen Comp Endocrinol 165:352\u0026ndash;366\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eChen W, Liu L, Ge W (2023) Expression analysis of growth differentiation factor 9 (\u003cem\u003eGdf9\u003c/em\u003e/\u003cem\u003egdf9\u003c/em\u003e), anti-m\u0026uuml;llerian hormone (\u003cem\u003eAmh\u003c/em\u003e/\u003cem\u003eamh\u003c/em\u003e) and aromatase (\u003cem\u003eCyp19a1a\u003c/em\u003e/\u003cem\u003ecyp19a1a\u003c/em\u003e) during gonadal differentiation of the zebrafish, \u003cem\u003eDanio rerio\u003c/em\u003e. Freshwtaer Fisheries 53:55\u0026ndash;60\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZhang X, Li M, Ma H, Liu X, Shi H, Li M et al (2017) Mutation of \u003cem\u003efoxl2\u003c/em\u003e or \u003cem\u003ecyp19a1a\u003c/em\u003e Results in Female to Male Sex Reversal in XX Nile Tilapia. Endocrinology 158:2634\u0026ndash;2647\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMustapha UF, Peng Y-X, Huang Y-Q, Assan D, Zhi F, Shi G et al (2022) Comparative transcriptome analysis of the differentiating gonads in \u003cem\u003eScatophagus argus\u003c/em\u003e. Front Mar Sci. ;9\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eRen Z, Ye D, Su N, Wang C, He L, Wang H et al (2024) \u003cem\u003efoxl2l\u003c/em\u003e is a germ cell-intrinsic gatekeeper of oogenesis in zebrafish. Zoological Res 45:1116\u0026ndash;1130\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZhang C, He Q, Cheng H, Li L, Ruan X, Duan X et al (2022) Transcription factors \u003cem\u003efoxl2\u003c/em\u003e and \u003cem\u003efoxl3\u003c/em\u003e regulate \u003cem\u003ecyp19a1a\u003c/em\u003e and \u003cem\u003ecyp11b\u003c/em\u003e in orange-spotted grouper (\u003cem\u003eEpinephelus coioides\u003c/em\u003e). Aquaculture Reports. ; 25:101243\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLee KH, Yamaguchi A, Rashid H, Kadomura K, Yasumoto S, Matsuyama M (2009) Germ cell degeneration in high-temperature treated pufferfish, \u003cem\u003eTakifugu rubripes\u003c/em\u003e. Sex Dev 3:225\u0026ndash;232\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003ePannetier M, Fabre S, Batista F, Kocer A, Renault L, Jolivet G et al (2006) \u003cem\u003eFOXL2\u003c/em\u003e activates P450 aromatase gene transcription: towards a better characterization of the early steps of mammalian ovarian development. 36(3):399\u0026ndash;413\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKim K-A, Wagle M, Tran K, Zhan X, Dixon MA, Liu S et al (2008) R-Spondin Family Members Regulate the Wnt Pathway by a Common Mechanism. Mol Biol Cell 19:2588\u0026ndash;2596\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eTevosian SG (2012) Genetic Control of Ovarian Development. Sex Dev 7:33\u0026ndash;45\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMustapha UF, Peng Y-X, Huang Y-Q, Assan D, Zhi F, Shi G et al (2022) Comparative transcriptome analysis of the differentiating gonads in \u003cem\u003eScatophagus argus\u003c/em\u003e. Front Mar Sci. ;9\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eDai S, Qi S, Wei X, Liu X, Li Y, Zhou X et al (2021) Germline sexual fate is determined by the antagonistic action of \u003cem\u003edmrt1\u003c/em\u003e and \u003cem\u003efoxl3\u003c/em\u003e/\u003cem\u003efoxl2\u003c/em\u003e in tilapia. Development 48:8\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eAugstenov\u0026aacute; B, Ma W-J (2025) Decoding \u003cem\u003eDmrt1\u003c/em\u003e: insights into vertebrate sex determination and gonadal sex differentiation\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLavery R, Chassot A-A, Pauper E, Gregoire EP, Klopfenstein M, de Rooij DG et al (2012) Testicular differentiation occurs in absence of R-spondin1 and \u003cem\u003eSox9\u003c/em\u003e in mouse sex reversals. Plos Genetic 8:1003170\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSong C, Yao J, Cao C, Liang X, Huang J, Han Z et al (2016) PPARγ is regulated by miR-27b-3p negatively and plays an important role in porcine oocyte maturation. Biochem Biophys Res Commun 479:224\u0026ndash;230\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWu Y, Wang Y, Xue Y, Wu N, Wu J, Zhang J (2023) \u003cem\u003ecbx2\u003c/em\u003e is a functional target of the let-7 family in the gonad of Japanese flounder (\u003cem\u003eParalichthys olivaceus\u003c/em\u003e). Comparative Biochemistry and Physiology Part B: Biochemistry and Molecular Biology. ; 263:110799\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWu J, Bao J, Wang L, Hu Y, Xu C (2011) MicroRNA-184 downregulates nuclear receptor corepressor 2 in mouse spermatogenesis. BMC Dev Biol 11:64\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eXiong S, Tian J, Ge S, Li Z, Long Z, Guo W et al (2020) The microRNA-200 cluster on chromosome 23 is required for oocyte maturation and ovulation in zebrafish. Biol Reprod 103:769\u0026ndash;778\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSong P, Xi J, Zhang Z, Fu Q, Yue Q, Zhang P et al Effects of Ovine miR-200b on Cell Cycle and Apoptosis of Follicular Granulosa Cells. Acta veterinaria et zootechnica sinica. 52:3471\u0026ndash;3479\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"marine-biotechnology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"mbte","sideBox":"Learn more about [Marine Biotechnology](http://link.springer.com/journal/10126)","snPcode":"10126","submissionUrl":"https://submission.nature.com/new-submission/10126/3","title":"Marine Biotechnology","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"Procypris mera, Gonadal differentiation, Transcriptome, miRNA","lastPublishedDoi":"10.21203/rs.3.rs-8288461/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-8288461/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eThe Chinese ink carp (\u003cem\u003eProcypris mera\u003c/em\u003e), a primitive species within the Cyprinidae family, exhibits sexual dimorphism in growth, with females growing significantly faster than males. Due to the depletion of wild resources and limitations in breeding technology, its commercial viability has become concerning. Research into all-female breeding and germplasm resource recovery is crucial for addressing these challenges, with sex control intervention playing a pivotal role. In this study, we conducted histological observations of \u003cem\u003eP. mera\u003c/em\u003e gonads using Hematoxylin-Eosin (HE) staining and identified four key stages of gonadal differentiation (60 dph, 95 dph, 110 dph, and 150 dph,) for transcriptome sequencing. Additionally, miRNA sequencing was performed at the late differentiation stage (150 dph). Our results indicate that gonadal differentiation in \u003cem\u003eP. mera\u003c/em\u003e is largely completed by 150 dph. During ovarian differentiation, the estrogen synthesis rate-limiting gene \u003cem\u003ecyp19a1a\u003c/em\u003e is promoted by \u003cem\u003efoxl2\u003c/em\u003e, facilitating estrogen production to drive ovarian differentiation while antagonizing male pathway genes. In contrast, during testicular differentiation, genes such as \u003cem\u003edmrt1\u003c/em\u003e, \u003cem\u003esox9-b\u003c/em\u003e, and \u003cem\u003efgf1\u003c/em\u003e are upregulated across all four stages, inhibiting the expression of female pathway genes dominated by \u003cem\u003efoxl2\u003c/em\u003e and \u003cem\u003ecyp19a1a\u003c/em\u003e to ensure commitment to testis differentiation. Furthermore, miRNAs also play a critical role in gonadal differentiation and maturation during the later stages. The miR-200 family regulates multiple genes associated with gonadal differentiation, potentially serving as core regulators. Additionally, some newly identified miRNAs (novell-m0207, novell-m0123-5P, and novell-m0122-5P) may also contribute to the regulation of gonadal differentiation processes. Overall, this study may provide valuable insights into the gonadal differentiation process and its underlying molecular regulatory network in \u003cem\u003eP. mera\u003c/em\u003e. These findings offer a solid foundation for improving production performance and advancing aquaculture practices for this species.\u003c/p\u003e","manuscriptTitle":"Integrated analysis of mRNA-seq and miRNA-seq reveal the dynamics of the sexual differentiation of Procypris mera","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-12-17 11:20:31","doi":"10.21203/rs.3.rs-8288461/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2026-01-03T00:42:52+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-01-02T23:25:51+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-12-27T05:50:39+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"308525268461064660893854501341787732114","date":"2025-12-13T19:27:09+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"7805123193820194770133501800960290983","date":"2025-12-13T05:03:30+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"55675544159813120147138657887327536503","date":"2025-12-13T03:15:47+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-12-12T19:17:51+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-12-12T00:44:38+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-12-12T00:44:02+00:00","index":"","fulltext":""},{"type":"submitted","content":"Marine Biotechnology","date":"2025-12-05T13:39:31+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"marine-biotechnology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"mbte","sideBox":"Learn more about [Marine Biotechnology](http://link.springer.com/journal/10126)","snPcode":"10126","submissionUrl":"https://submission.nature.com/new-submission/10126/3","title":"Marine Biotechnology","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"cd5ee367-9ab9-4c32-acfb-321ea44cb033","owner":[],"postedDate":"December 17th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2026-03-16T16:16:40+00:00","versionOfRecord":{"articleIdentity":"rs-8288461","link":"https://doi.org/10.1007/s10126-026-10581-x","journal":{"identity":"marine-biotechnology","isVorOnly":false,"title":"Marine Biotechnology"},"publishedOn":"2026-03-13 15:58:02","publishedOnDateReadable":"March 13th, 2026"},"versionCreatedAt":"2025-12-17 11:20:31","video":"","vorDoi":"10.1007/s10126-026-10581-x","vorDoiUrl":"https://doi.org/10.1007/s10126-026-10581-x","workflowStages":[]},"version":"v1","identity":"rs-8288461","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-8288461","identity":"rs-8288461","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}