Mechanisms Of Sex Differentiation Research Articles

Understanding Genetic Regulation of Sex Differentiation in Hermaphroditic Fish.

As a fundamental taxonomic group within vertebrates, fish represent an invaluable resource for investigating the mechanisms underlying sex determination and differentiation owing to their extensive geographical distribution and rich biodiversity. Within this biological cohort, the processes of sex determination and differentiation are intricately governed by both genetic factors and the complex interplay of environmental cues. While variations in external environmental factors, particularly temperature, can exert a modulatory influence on sex differentiation in fish to a limited degree, genetic factors remain the primary determinants of sexual traits. Hermaphroditic fish display three distinct types of sexual transitions: protandry (male to female), protogyny (female-to-male), bidirectional sex change (both directions serially). These fish, characterized by their unique reproductive strategies and sexual plasticity, serve as exemplary natural models for elucidating the mechanisms of sex differentiation and sexual transitions in fish. The present review delves into the histological dynamics during gonadal development across three types of sequential hermaphroditic fish, meticulously delineating the pivotal characteristics at each stage, from the inception of primordial gonads to sexual specialization. Furthermore, it examines the regulatory genes and associated signaling pathways that orchestrate sex determination and differentiation. By systematically synthesizing these research advancements, this paper endeavors to offer a comprehensive and profound insight into the intricate mechanisms governing sex differentiation in sequential hermaphroditic fish.

Insights into Solea senegalensis Reproduction Through Gonadal Tissue Methylation Analysis and Transcriptomic Integration.

Fish exhibit diverse mechanisms of sex differentiation and determination, shaped by both external and internal influences, often regulated by distinct DNA methylation patterns responding to environmental changes. In S. senegalensis aquaculture, reproductive issues in captivity pose significant challenges, particularly the lack of fertilization capabilities in captive-bred males, hindering genetic improvement measures. This study analyzed the methylation patterns and transcriptomic profiles in gonadal tissue DNA from groups differing in rearing conditions and sexual maturity stages. RRBS (Reduced Representation Bisulfite Sequencing) was employed to detect notable methylation variations across groups, while RNA was extracted and sequenced for differential expression analysis. Our findings suggest that DNA methylation significantly regulates gene expression, acting as a mechanism that can both repress and enhance gene expression depending on the genomic context. The complexity of this epigenetic mechanism is evident from the varying levels of methylation and correlation rates observed in different CpGs neighboring specific genes linked to reproduction. Differential methylation comparisons revealed the highest number of differently methylated CpGs between maturation stages, followed by rearing conditions, and lastly between sexes. These findings underscore the crucial role of methylation in regulating gene expression and its potential role in sex differentiation, highlighting the complex interplay between epigenetic modifications and gene expression.

SpMS1, a male sterility factor, interacts with SpAP1 to regulate unisexual flower development in dioecious spinach.

The emergence of unisexual flower is an important event during plant evolution. The molecular mechanism underlying the formation of unisexual flowers remains unclear in dioecious spinach. In this study, we identified the spinach MALE STERILITY1 gene, SpMS1, which serves as a masculine factor to regulate male fertility and sex reversion. Silencing SpMS1 led to stamen sterility in male flowers and the development of masculine traits in female flowers. Overexpression of SpMS1 in wild-type Arabidopsis resulted in sterile stamens and irregular pollen exine. Notably, ectopic expression of SpMS1 in Arabidopsis ms1 mutants restored pollen viability and flower fertility. Furthermore, our findings demonstrate that SpMS1 interacts with MADS-box transcription factor SpAP1 to regulate unisexual flower development. Thus, SpMS1 exhibits a conserved function in pollen fertility akin to bisexual flowers, while also acting as a key regulator of unisexual flower development in spinach. This study sheds light on the mechanism of sex differentiation in dioecious plants and also provides valuable insights for manipulating male sterility in plant breeding programs.

Comparative Transcriptome Analysis of Sexual Differentiation in Male and Female Gonads of Nao-Zhou Stock Large Yellow Croaker (Larimichthys crocea).

The Nao-zhou stock large yellow croaker (Larimichthys crocea) is a unique economic seawater fish species in China and exhibits significant dimorphism in both male and female phenotypes. Cultivating all-female seedlings can significantly improve breeding efficiency. To accelerate the cultivation process of all female seedlings of this species, it is necessary to deeply understand the regulatory mechanisms of sexual differentiation and gonadal development. This study used Illumina high-throughput sequencing to sequence the transcriptome of the testes and ovaries of Nao-zhou stock large yellow croaker to identify genes and molecular functions related to sex determination. A total of 10,536 differentially expressed genes were identified between males and females, including 5682 upregulated and 4854 downregulated genes. Functional annotation screened out 70 important candidate genes related to sex, including 34 genes highly expressed in the testis (including dmrt1, foxm1, and amh) and 36 genes highly expressed in the ovary (including gdf9, hsd3b1, and sox19b). Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis found that differentially expressed genes were significantly enriched in nine signaling pathways related to sex determination and gonadal development, including steroid hormone biosynthesis, MAPK signaling pathway, and the TGF-beta signaling pathway. By screening sex-related differentially expressed genes and mapping protein-protein interaction networks, hub genes such as dmrt1, amh, and cyp19a1a were found to be highly connected. The expression levels of 15 sex-related genes, including amh, dmrt1, dmrt2a, foxl1, and zp3b, were determined by qRT-PCR and RNA sequencing. This study screened for differentially expressed genes related to sex determination and differentiation of Nao-zhou stock large yellow croaker and revealed the signaling pathways involved in gonad development of male and female individuals. The results provide important data for future research on sex determination and differentiation mechanisms, thereby providing a scientific basis for the cultivation of all-female seedlings.

Identification of Genes and Long Non-Coding RNAs Putatively Related to Portunus trituberculatus Sex Determination and Differentiation Using Oxford Nanopore Technology Full-Length Transcriptome Sequencing.

The swimming crab (Portunus trituberculatus) is an economically important species in China, and its growth traits show obvious sexual dimorphism. Thus, it is important to study the mechanism of sex determination and differentiation in this species. Herein, we identified 2138 differentially expressed genes and 132 differentially expressed long non-coding RNAs (lncRNAs) using Oxford Nanopore Technology full-length transcriptome sequencing. We predicted 561 target genes of the differentially expressed lncRNAs according to their location and base pair complimentary principles. Furthermore, pathways related to sex determination, differentiation, and reproduction were enriched for lncRNAs according to gene ontology and Kyoto Encyclopedia of Genes and Genomes analyses. This indicated that lncRNAs might play regulatory roles in these pathways. Our results could form the basis for future studies of sex determination and differentiation in P. trituberculatus.

CRISPR-Mediated SRY Gene Mutation Increases the Expression of Female Lineage-Specific Gene in Pre-Implantation Buffalo Embryo.

In mammals, sex determination is governed by the SRY gene on the Y chromosome, redirecting gonadal development from forming ovaries to testes. Mutations or alterations in the SRY gene can significantly affect phenotypic changes and lineage-specific markers. This study aims to elucidate the role of the SRY gene in buffalo embryos using CRISPR-Cas9 technology. We designed a crRNA targeting the HMG domain of the SRY gene using the CRISPOR algorithm. Nucleofection of sgRNA-Cas9 RNPs into buffalo fibroblasts confirmed efficient cleavage at the targeted site. Using this validated guide, we investigated the role of the SRY gene in sexual determination by electroporating CRISPR-Cas9-RNPs into single-stage zygotes of buffalo. Genetic changes in the SRY gene were confirmed through sequencing, revealing mosaic blastocysts with multiple alleles and non-mosaic mutants. Mutations in SRY gene increased the expression of female lineage-specific gene Wnt4 whereas decreased the expression of male specific gene SOX9 in blastocysts, suggesting reprogramming towards female sex determination pathways. Our findings provide insights into buffalo sex differentiation mechanisms and potential applications in reproductive strategies for breeding programmes.

Reference-based genome assembly and comparative genomics of Calamus Brandisii Becc. for unveiling sex-specific genes for early gender detection.

Calamus brandisii Becc. is an endangered rattan species indigenous to the Western Ghats of India and used in the furniture and handicraft industries. However, its dioecious nature and longer flowering time pose challenges for conservation efforts. Developing markers for early gender detection in seedlings is crucial for maintaining viable populations for in-situ and ex-situ conservation. Currently, no sex chromosomes or gender-specific genes have been reported in the species. We report the first comprehensive comparative genomics study between the male and female genomes of C. brandisii to identify polymorphisms and potential genes for gender determination. Reference-based assembly was conducted and the male and female genomes were predicted to contain 43,810 and 50,493 protein-coding genes respectively. The haploid genome size was ∼691Mb and ∼884Mb for male and female genomes respectively. Comparative analysis revealed significant genetic variation between the two genomes including 619,776 SNPs, 73,659 InDels, 212,123 Structural variants (SVs) and 305 copy number variations (CNVs). A total of 5 male-specific and 11 female-specific genes linked to the sex determining region was predicted. The genomic variants identified between the two genomes could be used in development of markers for early gender identification in C. brandisii for restoration programs. The gender-specific genes identified in this study also provide new insights into the mechanisms of sex determination and differentiation in rattans.

Identification and mapping of sex-linked markers in giant abalone (Haliotis gigantea Gmelin)

Giant abalone (Haliotis gigantea Gmelin) is an economically important species within Haliotis genus, yet the genetic basis of sex in this species is still poorly understood. Therefore, we screened and mapped sex-linked SNPs and InDels, and subsequently developed a PCR-based method for genetic sex identification in giant abalone. Based on whole-genome sequencing reads, we obtained a total 6,159,941 high-quality SNPs and 2,623,263 high-quality InDels. Among them, 1,737 SNPs and 179 InDels were detected as sexually dimorphic, assuming a male heterogametic sex determination system. The majority of these variants, 99.1 % for SNPs and 99.4 % for InDels, were clustered on Chr.4 (0.02 Mb to 23.22 Mb). Furthermore, the estimation of genetic divergence between the sexes using FST showed that the genomic windows with the top 1 % FST values were concentrated on Chr.4 (0.5 Mb to 19.0 Mb) and Chr.5 (20.0 Mb to 35.0 Mb). Based on the sexually dimorphic variants on Chr.4, three PCR-based markers were successfully developed to determine the genetic sex of giant abalone. PCR validation using a tested panel of 67 females and 67 males from a cultivated population demonstrated compliance rates for these three markers ranging from 97.7 % to 99.3 %. These results suggest that the sex determination system is male heterogametic in giant abalone. Thus, this study provides valuable insights and tools for further research on the mechanisms of sex determination and differentiation, as well as for monitoring sex ratios in wild and cultivated populations in giant abalone.

Characterization and Functional Analysis of the 17-Beta Hydroxysteroid Dehydrogenase 2 (hsd17b2) Gene during Sex Reversal in the Ricefield Eel (Monopterus albus).

In mammals, 17-beta hydroxysteroid dehydrogenase 2 (Hsd17b2) enzyme specifically catalyzes the oxidation of the C17 hydroxyl group and efficiently regulates the activities of estrogens and androgens to prevent diseases induced by hormone disorders. However, the functions of the hsd17b2 gene involved in animal sex differentiation are still largely unclear. The ricefield eel (Monopterus albus), a protogynous hermaphroditic fish with a small genome size (2n = 24), is usually used as an ideal model to study the mechanism of sex differentiation in vertebrates. Therefore, in this study, hsd17b2 gene cDNA was cloned and its mRNA expression profiles were determined in the ricefield eel. The cloned cDNA fragment of hsd17b2 was 1230 bp, including an open reading frame of 1107 bp, encoding 368 amino acid residues with conserved catalytic subunits. Moreover, real-time quantitative reverse transcription polymerase chain reaction (RT-qPCR) analysis showed that hsd17b2 mRNA expressed strongly in the ovaries at early developmental stages, weakly in liver and intestine, and barely in testis and other tissues. In particular, hsd17b2 mRNA expression was found to peak in ovaries of young fish and ovotestis at the early stage, and eventually declined in gonads from the late ovotestis to testis. Likewise, chemical in situ hybridization results indicated that the hsd17b2 mRNA signals were primarily detected in the cytoplasm of oogonia and oocytes at stage I-II, subsequently concentrated in the granulosa cells around the oocytes at stage Ⅲ-Ⅳ, but undetectable in mature oocytes and male germ cells. Intriguingly, in ricefield eel ovaries, hsd17b2 mRNA expression could be significantly reduced by 17β-estradiol (E2) or tamoxifen (17β-estradiol inhibitor, E2I) induction at a low concentration (10 ng/mL) and increased by E2I induction at a high concentration (100 ng/mL). On the other hand, both the melatonin (MT) and flutamide (androgen inhibitor, AI) induction could significantly decrease hsd17b2 mRNA expression in the ovary of ricefield eel. This study provides a clue for demonstrating the mechanism of sexual differentiation in fish. The findings of our study imply that the hsd17b2 gene could be a key regulator in sexual differentiation and modulate sex reversal in the ricefield eel and other hermaphroditic fishes.

MiR-34 negatively regulates the expression of Dmrt and related genes in the testis of mud crab Scylla paramamosain

The mud crab (Scylla paramamosain) is a commercially significant marine decapod crustacean. Due to its obvious sexual dimorphism, the mechanism of sex differentiation and gonadal development has attracted significant research interest. The Dmrt (double-sex and mab-3 related transcription factor) genes are vital in animal gonadal development and sex differentiation. In the present study, miR-34 was predicted to target the 3′ end of Dmrt-1, idmrt-2, Dmrt-3, Dsx and Dmrt-like genes by prediction software, and the interactions between miR-34 and these Dmrt genes were validated by in vivo and in vitro experiments. Dual luciferase assay results indicated that miR-34 mimics/inhibitors co-transfected with plasmid vectors with 3′ end of Dmrt-1, idmrt-2, Dmrt-3, Dsx and Dmrt-like, respectively, led to a significant decrease/increase of fluorescence activity in HEK293T cells. In vivo experiments showed that injection of agomir-34 significantly inhibited Dmrt-1, idmrt-2, Dsx and Dmrt-like expression, while injection of antagomir-34 caused the opposite result. However, Dmrt-3 expression was not affected by injection of miR-34 reagents. Meanwhile, the expression of spermatogenesis and testicular development-related molecular marker genes (IAG, foxl2 and vasa) in mud crabs was significantly changed after injecting the miR-34 reagent in vivo. Furthermore, the result of immunoblotting proved that the expression level of Dmrt-like protein can be regulated by miR-34. These results imply that miR-34 is indirectly involved in sex differentiation and testicular development of S. paramamosain by regulating Dmrt-1, idmrt-2, Dsx and Dmrt-like genes.

Chromosome-level genome assembly of Hippophae rhamnoides variety

Fructus hippophae (Hippophae rhamnoides spp. mongolica×Hippophae rhamnoides sinensis), a hybrid variety of sea buckthorn that Hippophae rhamnoides spp. mongolica serves as the female parent and Hippophae rhamnoidessinensis serves as the male parent, is a traditional plant with great potentials of economic and medical values. Herein, we gained a chromosome-level genome of Fructus hippophae about 918.59 Mb, with the scaffolds N50 reaching 83.65 Mb. Then, we anchored 440 contigs with 97.17% of the total genome sequences onto 12 pseudochromosomes. Next, de-novo, homology and transcriptome assembly strategies were adopted for gene structure prediction. This predicted 36475 protein-coding genes, of which 36226 genes could be functionally annotated. Simultaneously, various strategies were used for quality assessment, both the complete BUSCO value (98.80%) and the mapping rate indicated the high assembly quality. Repetitive elements, which occupied 63.68% of the genome, and 1483600 bp of non-coding RNA were annotated. Here, we provide genomic information on female plants of a popular variety, which can provide data for pan-genomic construction of sea buckthorn and for the resolution of the mechanism of sex differentiation.

Molecular mechanisms of sex determination and differentiation in decapod crustaceans for potential aquaculture applications: An overview

AbstractCrustacean aquaculture, especially monosex culture, requires a comprehensive knowledge of the mechanisms regulating sex determination and differentiation. Decapod crustaceans display either female heterogamety (ZW/ZZ) or male heterogamety (XY/XX) as their mode of genotypic sex determination. However, some decapod species exhibit sexual plasticity via removing the androgenic gland, knockdown insulin receptor transcripts, administrating exogenous sex steroids, and altering environmental factors. Although the master sex‐determining gene in decapods remains elusive, various sexual genes, hormones, and neurotransmitter are emerging as important factors in sex differentiation. This review looks into recent advancements in studies related to the genetic sex determination system and factors that trigger sex reversal. Moreover, the elucidation of the role of genetic factors in sex differentiation in both females and males, as well as their underlying interrelated regulatory mechanisms, provides justification for three levels of application: the induction of monosex populations, detection of precocious sex and genotype, and prevention of uncontrolled reproduction. Finally, we proposed future directions of bioinformatics analysis and genome editing coupled with sex control to increase the yield for economic decapods. In conclusion, this review provides a foundation for future research on the reproductive biology and sexual regulation of decapods, with the aim of aiding practitioners in advancing the crustacean aquaculture industry.

Gonadal Transcriptome Analysis Reveals that SOX17 and CYP26A1 are Involved in Sex Differentiation in the Chinese Soft-Shelled Turtle (Pelodiscus sinensis).

The Chinese soft-shelled turtle (Pelodiscus sinensis) is an important aquaculture animal in China and exhibits growth dimorphism. Single-male cultures are often selected for higher economic efficiency. However, the mechanism of sex differentiation in P. sinensis is not well-known. In this study, a comparative transcriptome analysis of male (ZZ)- and 17β-oestradiol (E2)-induced pseudo-female (ZZ + E2)-stage embryonic gonads of P. sinensis was performed. A total of 420 differentially expressed genes (DEGs), which included 271 upregulated genes and 149 downregulated genes, were identified. These DEGs were mainly involved in several sex-related pathways, such as "ovarian steroidogenesis", "steroid hormone biosynthesis", "PPAR signalling pathway", and "metabolism of xenobiotics by cytochrome P450". In addition, 50 known and novel candidate genes involved in sex differentiation, such as the male-biased genes AMH, DMRT1, TBX1, and CYP26A1 and the female-biased genes CYP1A1, RASD1, and SOX17, were investigated and identified. For further verification, the full-length cDNAs of SOX17 and CYP26A1 were obtained. SOX17 contains a 1218-bp ORF and encodes 405 amino acids containing an HMG functional domain unique to the Sox superfamily. CYP26A1 contains a 1485-bp ORF and encodes 494 amino acids. Different expression levels of SOX17 and CYP26A1 could be detected in all the tested tissues of males and females. Notably, the expression of CYP26A1 was markedly greater in the gonads of male embryos (P < 0.05) than in those of female embryos, whereas the expression of SOX17 showed the opposite trend (P < 0.05). Taken together, the RNA-seq and qRT‒PCR results suggested potential roles for SOX17 and CYP26A1 in promoting female and male gonadal development, respectively, in P. sinensis. Our results provide new evidence for the mechanism of sex differentiation in P. sinensis.

Identification, Expression and Evolutional Analysis of Two cyp19-like Genes in Amphioxus.

The mechanism of sex determination and differentiation in animals remains a central focus of reproductive and developmental biology research, and the regulation of sex differentiation in amphioxus remains poorly understood. Cytochrome P450 Family 19 Subfamily A member 1 (CYP19A1) is a crucial sex differentiation gene that catalyzes the conversion of androgens into estrogens. In this study, we identified two aromatase-like genes in amphioxus: cyp19-like1 and cyp19-like2. The cyp19-like1 is more primitive and may represent the ancestral form of cyp19 in zebrafish and other vertebrates, while the cyp19-like2 is likely the result of gene duplication within amphioxus. To gain further insights into the expression level of these two aromatase-like, we examined their expression in different tissues and during different stages of gonad development. While the expression level of the two genes differs in tissues, both are highly expressed in the gonad primordium and are primarily localized to microsomal membrane systems. However, as development proceeds, their expression level decreases significantly. This study enhances our understanding of sex differentiation mechanisms in amphioxus and provides valuable insights into the formation and evolution of sex determination mechanisms in vertebrates.

New insights into the all-testis differentiation in zebrafish with compromised endogenous androgen and estrogen synthesis.

The regulatory mechanism of gonadal sex differentiation, which is complex and regulated by multiple factors, remains poorly understood in teleosts. Recently, we have shown that compromised androgen and estrogen synthesis with increased progestin leads to all-male differentiation with proper testis development and spermatogenesis in cytochrome P450 17a1 (cyp17a1)-/- zebrafish. In the present study, the phenotypes of female-biased sex ratio were positively correlated with higher Fanconi anemia complementation group L (fancl) expression in the gonads of doublesex and mab-3 related transcription factor 1 (dmrt1)-/- and cyp17a1-/-;dmrt1-/- fish. The additional depletion of fancl in cyp17a1-/-;dmrt1-/- zebrafish reversed the gonadal sex differentiation from all-ovary to all-testis (in cyp17a1-/-;dmrt1-/-;fancl-/- fish). Luciferase assay revealed a synergistic inhibitory effect of Dmrt1 and androgen signaling on fancl transcription. Furthermore, an interaction between Fancl and the apoptotic factor Tumour protein p53 (Tp53) was found in vitro. The interaction between Fancl and Tp53 was observed via the WD repeat domain (WDR) and C-terminal domain (CTD) of Fancl and the DNA binding domain (DBD) of Tp53, leading to the K48-linked polyubiquitination degradation of Tp53 activated by the ubiquitin ligase, Fancl. Our results show that testis fate in cyp17a1-/- fish is determined by Dmrt1, which is thought to stabilize Tp53 by inhibiting fancl transcription during the critical stage of sexual fate determination in zebrafish.

Estrogen Signaling Inhibits the Expression of anti-Müllerian hormone (amh) and gonadal-soma-derived factor (gsdf) during the Critical Time of Sexual Fate Determination in Zebrafish.

The mechanism of fish gonadal sex differentiation is complex and regulated by multiple factors. It has been widely known that proper steroidogenesis in Leydig cells and sex-related genes in Sertoli cells play important roles in gonadal sex differentiation. In teleosts, the precise interaction of these signals during the sexual fate determination remains elusive, especially their effect on the bi-potential gonad during the critical stage of sexual fate determination. Recently, all-testis phenotypes have been observed in the cyp17a1-deficient zebrafish and common carp, as well as in cyp19a1a-deficient zebrafish. By mating cyp17a1-deficient fish with transgenic zebrafish Tg(piwil1:EGFP-nanos3UTR), germ cells in the gonads were labelled with enhanced green fluorescent protein (EGFP). We classified the cyp17a1-deficient zebrafish and their control siblings into primordial germ cell (PGC)-rich and -less groups according to the fluorescence area of the EGFP labelling. Intriguingly, the EGFP-labelled bi-potential gonads in cyp17a1+/+ fish from the PGC-rich group were significantly larger than those of the cyp17a1-/- fish at 23 days post-fertilization (dpf). Based on the transcriptome analysis, we observed that the cyp17a1-deficient fish of the PGC-rich group displayed a significantly upregulated expression of amh and gsdf compared to that of control fish. Likewise, the upregulated expressions of amh and gsdf were observed in cyp19a1a-deficient fish as examined at 23 dpf. This upregulation of amh and gsdf could be repressed by treatment with an exogenous supplement of estradiol. Moreover, tamoxifen, an effective antagonist of both estrogen receptor α and β (ERα and Erβ), upregulates the expression of amh and gsdf in wild-type (WT) fish. Using the cyp17a1- and cyp19a1a-deficient zebrafish, we provide evidence to show that the upregulated expression of amh and gsdf due to the compromised estrogen signaling probably determines their sexual fate towards testis differentiation. Collectively, our data suggest that estrogen signaling inhibits the expression of amh and gsdf during the critical time of sexual fate determination, which may broaden the scope of sex steroid hormones in regulating gonadal sex differentiation in fish.

Temperature-Induced Sex Differentiation in River Prawn (Macrobrachium nipponense): Mechanisms and Effects.

Macrobrachium nipponense is gonochoristic and sexually dimorphic. The male prawn grows faster and usually has a larger size than the female. Therefore, a higher male proportion in stock usually results in higher yield. To investigate the impact of temperature on sexual differentiation in M. nipponense, two temperature treatments (26 °C and 31 °C) were conducted. The results showed that compared to the 31 °C treatment (3.20 ± 0.12), the 26 °C treatment displayed a lower female/male ratio (2.20 ± 0.11), which implied that a lower temperature could induce masculinization in M. nipponense. The temperature-sensitive sex differentiation phase was 25-35 days post hatching (DPH) at 26 °C while 15-20 DPH at 31 °C. Transcriptome and qPCR analysis revealed that a lower temperature up-regulated the expression of genes related to androgen secretion, and down-regulated the expressions of genes related to oogonia differentiation. Thirty-one temperature-regulated sex-differentiation genes were identified and the molecular mechanism of temperature-regulated sex differentiation was suggested. The finding of this study indicates that temperature regulation can be proposed as an innovative strategy for improving the culture yield of M. nipponense.

Identification of sex-specific DNA markers in the army fish (Spinibarbus hollandi) by whole genome re-sequencing method

With the development of high throughput sequencing technology, genome information for more and more fish species can be available. Based on these genomes, sex-specific marker of various fish species was developed and further applied in sex control breeding. Here, whole genome re-sequencing (WGS re-seq) method was used to screen sex-specific sequences in the army fish (Spinibarbus hollandi). A total of 34.34, 30.64, 11.58, 11.21 and 12.63 Gb were generated for five females, and 12.61, 11.12, 11.67, 13.01 and 11.63 Gb were generated for five males. Moreover, we generated 39.47 Gb data for pool sequencing of female samples and 32.17 Gb for male samples. After filtering and validating, 20 candidate sex-specific sequences were obtained. Further, by PCR confirmation, six male-specific markers (X11, X17, X22, C3, C7, C10) were successfully developed with 100% accuracy in eight females and eight males. Subsequently, in another two different farmed populations of army fish, the authenticity of sex identification remained to be 100% using these markers. Besides, after homology and coverage analysis, we found the candidate sequence corresponding to C3, C7 and C10 had a similar X chromosome homologous sequence, and the resulting C3, C7 and C10 markers also amplified two bands in males but one band in females. In all, these sex-specific markers developed in this study would assist in sex control breeding and promote further research on the sex determination and differentiation mechanism of S. hollandi.

Molecular and cellular regulation on sexual fate and gonadal development in hermaphrodite yellowfin seabream Acanthopagrus latus

Yellowfin seabream (Acanthopagrus latus) is a hermaphroditic fish with male prematurity, which is also an economically important mariculture species in coastal areas of China. Although the production yield has been increasing in recent years, artificial breeding of this species remains imperfect due to the lack understanding of its sex differentiation mechanism. In this study, we conducted transcriptomic sequencing to screen for sex-related genes in different stage of the testis, ovotestis, ovary, hypothalamus, and pituitary tissues of yellowfin seabream. During gonad development (from intersex to mature stage), Gnrh3, Kiss2, and Dbh were expressed in the hypothalamus and Lhb and Fshb were expressed in the pituitary. Sex-related genes such as Hsd11b1, Foxl2l, Amh, and Gsdf were among the top 10 downregulated genes in the intersex testis compared with the mature testis. The expression of Hsd11b, Wnt, and Sox families genes differed significantly in the sex change process among functional mature gonads. In addition, treatment with estradiol resulted in feminizing of the ovotestis. Inhibition of androgens or estrogens promoted ovarian or testicular steps of ovotestis development. In summary, we systematically evaluated gene expression profiles during yellowfin seabream gonad development from intersex to functional male or female. Our results confirmed that regulation by the hypothalamus-pituitary-gonad (HPG) axis and the promotion of sex steroid hormones acted on this process, and they provide a reference for further studies of the involvement of the HPG axis in sex change and of sex induction methods in yellowfin seabream.

Analysis of Chromatin Accessibility and DNA Methylation to Reveal the Functions of Epigenetic Modifications in Cyprinus carpio Gonads.

Epigenetic modifications are critical in precisely regulating gene expression. The common carp (Cyprinus carpio) is an economically important fish species, and females exhibit faster growth rates than males. However, the studies related to epigenetic modifications in the common carp gonads are limited. In this study, we conducted the Assay for Transposase Accessible Chromatin sequencing (ATAC-seq) and Bisulfite sequencing (BS-seq) to explore the roles of epigenetic modifications in the common carp gonads. We identified 84,207 more accessible regions and 77,922 less accessible regions in ovaries compared to testes, and some sex-biased genes showed differential chromatin accessibility in their promoter regions, such as sox9a and zp3. Motif enrichment analysis showed that transcription factors (TFs) associated with embryonic development and cell proliferation were heavily enriched in ovaries, and the TFs Foxl2 and SF1 were only identified in ovaries. We also analyzed the possible regulations between chromatin accessibility and gene expression. By BS-seq, we identified 2087 promoter differentially methylated genes (promoter-DMGs) and 5264 gene body differentially methylated genes (genebody-DMGs) in CG contexts. These genebody-DMGs were significantly enriched in the Wnt signaling pathway, TGF-beta signaling pathway, and GnRH signaling pathway, indicating that methylation in gene body regions could play an essential role in sex maintenance, just like methylation in promoter regions. Combined with transcriptomes, we revealed that the expression of dmrtb1-like, spag6, and fels was negatively correlated with their methylation levels in promoter regions. Our study on the epigenetic modifications of gonads contributes to elucidating the molecular mechanism of sex differentiation and sex maintenance in the common carp.