Mechanisms Of Sex Differentiation Research Articles

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.

Genome-wide identification of STATs and analysis of their role in sex determination in Pacific oysters (Crassostrea gigas)

STAT (signal transducer and activator of the transcription) proteins, are a group of highly conserved transcription factors and fundamental components of the JAK-STAT signaling pathway. They play crucial roles in a variety of biological processes, such as immunity, proliferation, differentiation, and growth. However, little information is known regarding their role in gonad development and sex determination in mollusks. In this study, we identified 3 STAT genes in Pacific Oyster Crassostrea gigas. Phylogenetic analysis showed that STATs from mollusks were highly conserved, and most of them had four identical motif regions, except for the STAT1 and STAT3 predicted sequences from Crassostrea hongkongensis. Tissue expression analysis indicated CgSTAT1 had a high expression level in most tissues, while CgSTAT3 had a low expression level in most tissues. Expression analysis of early developmental stages showed CgSTAT1 had a higher expression level from egg to D shaped larva and a lower expression level in subsequent stages. In contrast CgSTAT1, CgSTAT2 had a reverse expression pattern. Expression analysis of different developmental stages of diploid gonads indicated that CgSTAT1 had a higher expression level at the S1 and S3 stages relative to the S2 stage in females, while in males the S3 stage had a higher expression than than the S2 stage. The expression level of CgSTAT1 between diploids and triploids in females differed significantly, but there were no significant differences in males. Expression of CgSTAT2 differed significantly between diploid and triploid males. These data suggest an important role for STATs in sex differentiation in diploid and triploid oysters. Our study is the first to explore the role of STATs in sex differentiation and gonadal development in oysters, and will help us better understand the molecular mechanisms of sex differentiation in shellfish.

Multi-Omics Analysis Reveals Intricate Gene Networks Involved in Female Development in Melon

Sexual differentiation is an important developmental phenomenon in cucurbits that directly affects fruit yield. The natural existence of multiple flower types in melon offers an inclusive structure for studying the molecular basis of sexual differentiation. The current study aimed to identify and characterize the molecular network involved in sex determination and female development in melon. Male and female pools separated by the F2 segregated generation were used for sequencing. The comparative multi-omics data revealed 551 DAPs and 594 DEGs involved in multiple pathways of melon growth and development, and based on functional annotation and enrichment analysis, we summarized four biological process modules, including ethylene biosynthesis, flower organ development, plant hormone signaling, and ubiquitinated protein metabolism, that are related to female development. Furthermore, the detailed analysis of the female developmental regulatory pathway model of ethylene biosynthesis, signal transduction, and target gene regulation identified some important candidates that might have a crucial role in female development. Two CMTs ((cytosine-5)-methyltransferase), one AdoHS (adenosylhomocysteinase), four ACSs (1-aminocyclopropane-1-carboxylic acid synthase), three ACOs (ACC oxidase), two ARFs (auxin response factor), four ARPs (auxin-responsive protein), and six ERFs (Ethylene responsive factor) were identified based on various female developmental regulatory models. Our data offer new and valuable insights into female development and hold the potential to offer a deeper comprehension of sex differentiation mechanisms in melon.

Regulatory mechanism of LncRNAs in gonadal differentiation of hermaphroditic fish, Monopterusalbus

BackgroundMonopterusalbus is a hermaphroditic fish with sex reversal from ovaries to testes via the ovotestes in the process of gonadal development, but the molecular mechanism of the sex reversal was unknown.MethodsWe produced transcriptomes containing mRNAs and lncRNAs in the crucial stages of the gonad, including the ovary, ovotestis and testis. The expression of the crucial lncRNAs and their target genes was detected using qRT‒PCR and in situ hybridization. The methylation level and activity of the lncRNA promoter were analysed by applying bisulfite sequencing PCR and dual-luciferase reporter assays, respectively.ResultsThis effort revealed that gonadal development was a dynamic expression change. Regulatory networks of lncRNAs and their target genes were constructed through integrated analysis of lncRNA and mRNA data. The expression and DNA methylation of the lncRNAs MSTRG.38036 and MSTRG.12998 and their target genes Psmβ8 and Ptk2β were detected in developing gonads and sex reversal gonads. The results showed that lncRNAs and their target genes exhibited consistent expression profiles and that the DNA methylation levels were negatively regulated lncRNA expression. Furthermore, we found that Ptk2β probably regulates cyp19a1 expression via the Ptk2β/EGFR/STAT3 pathway to reprogram sex differentiation.ConclusionsThis study provides novel insight from lncRNA to explore the potential molecular mechanism by which DNA methylation regulates lncRNA expression to facilitate target gene transcription to reprogram sex differentiation in M.albus, which will also enrich the sex differentiation mechanism of teleosts.

Transcriptome profiling of banana shrimp (Fenneropenaeus merguiensis) ovaries and testes: Insights into FoxL2.

The banana shrimp is found in the Pacific and Indian Oceans. Female shrimp are preferred for consumption because they are larger than males. Understanding the mechanism of sex differentiation is important for developing techniques to increase the number of female shrimp for economic benefits. This study investigates the reproductive development of F. merguiensis using transcriptome analysis. Sxl2, dsx, AGH, FEM-1, and Nrg-X2 were classified as essential genes for testes development during the juvenile stage. Several genes were required for both juvenile and adult male development. Additionally, the expression of several genes was shown to be required for juvenile and adult ovarian development, including SOP1, SOP2, Ptgr1, EST, Vgr, Vmol1, and TR-beta A. Interestingly, high levels of FoxL2 expression were observed in the testes, in contrast to previous studies in humans and other mammals. The binding of FoxL2 to the Vtg promoter was demonstrated in silico with the highest relative binding score (RS = 0.89) using the JASPAR program. Knock-down of the FoxL2 gene with dsRNA significantly suppressed FoxL2 at 2, 4, and 6 d. As a result, Vtg expression increased when compared with the control at 2, 4, and 6 d, indicating that FoxL2 plays an important role in Vtg expression in the ovary. Our findings highlight the role of FoxL2 in banana shrimp reproduction and provide valuable information on the genes associated with the F. merguiensis reproductive system.

Sex biased expression of Fem-1 in larval stages suggests its function in early sex differentiation of Litopeneaus vannamei

The Pacific white shrimp Litopenaeus vannamei (L. vannamei) is an economically important species worldwide. As females exhibit a faster growth rate than males, it is crucial to understand the mechanism of sex determination and differentiation for the purpose of mono-sex breeding in shrimp. The feminization-1 gene (Fem-1) serves as a sex-determining gene in Caenorhabditis elegans, and the Fem-1 family is implicated in sex development in several crustaceans. In this study, three Fem-1 family members, namely LvFem-1a, LvFem-1b, and LvFem-1c, were identified and characterized in L. vannamei. Based on genetic sex marker, individuals were separated by sex during the early developmental stages. The expression profiles of these genes were analyzed in female or male larvae. Significant differences between females and males were observed during the early stages of development. In Zoea III or earlier stages, the expressions levels in males were significantly higher than those in females. Intriguingly, there was a shift towards higher expression in females during the mysis and post-larval stages. These findings indicate that the Zoea III stage may be crucial for sex differentiation in shrimp. In addition, the analysis of gene expression across various ovarian stages and tissues was conducted. Results showed that all three genes were highly expressed in the ovary, with particularly elevated levels in ovarian development stage II. These findings suggest that the Fem-1 genes play a critical role in early ovary development. This study is the first research focusing on the sex-specific expression of sex differentiation genes in shrimp. The obtained results provide new insights into to the role of the Fem-1 gene family in sex differentiation and gonadal development.

Identification of Crustacean Female Sex Hormone Receptor Involved in Sexual Differentiation of a Hermaphroditic Shrimp.

The neurohormone crustacean female sex hormone (CFSH) contains a highly conserved interleukin-17 (IL-17) domain in the mature peptide. Although CFSH has been demonstrated to stimulate female sexual differentiation in crustaceans, its receptors (CFSHR) have been poorly reported. The present study identified an IL-17 receptor (named Lvit-IL-17R), a candidate of CFSHR, from the protandric simultaneous hermaphroditic (PSH) shrimp Lysmata vittata through GST pulldown assays and RNAi experiments. Lvit-IL-17R is a transmembrane protein with an SEFIR (similar expression as the fibroblast growth factor and IL-17R) domain, as determined through sequence analysis. A GST pulldown experiment confirmed the interactions between the type I CFSHs (CFSH1a and CFSH1b) and Lvit-IL-17R. Meanwhile, the RNAi results revealed that Lvit-IL-17R displays similar functions to type I CFSHs in regulating sexual differentiation and gonad development. In brief, Lvit-IL-17R is a potential receptor for type I CFSHs aimed at regulating the sexual differentiation of the PSH species. This study helps shed new light on the mechanism of sexual differentiation among crustaceans.

Transcriptome characterization of gonadal sex differentiation in Pacific bluefin tuna, Thunnus orientalis (Temminck et Schlegel)

Tunas (genus Thunnus) are one of the most ecologically and commercially important fish worldwide. To establish a biological basis for reproduction in this globally essential species, we have recently studied crucial reproductive aspects of the Pacific bluefin tuna (T. orientalis; PBT), as a model of tuna species, based on our closed-cycle aquaculture technology. In this study, we clarified the global expression profile of the genes regulating gonadal sex differentiation in PBT, as this developmental process is vital to sexual reproduction. Based on the results of our comparative (RNA-sequencing) and temporal (qRT-PCR) transcriptome analyses using the updated genome dataset, we propose the molecular mechanisms of gonadal sex differentiation in PBT. In female gonads, foxl2 and cyp19a1a (coding aromatase) are expressed at the onset of sex differentiation. Active aromatase-mediated estrogen biosynthesis, which includes positive regulation of cyp19a1a expression by Foxl2, induces ovarian differentiation. By contrast, dmrt1 and gsdf are upregulated in differentiating male gonads lacking active estrogen synthesis. Dmrt1 and Gsdf would mainly promote testicular differentiation. Furthermore, androgen biosynthesis is upregulated in differentiating male gonad. Endogenous androgens may also be vital to testicular differentiation. This study provides the first comprehensive data clarifying the molecular basis for gonadal sex differentiation in tunas.

Genome-wide association study reveals genomic loci of sex differentiation and gonadal development in Plectropomus leopardus.

Introduction: Plectropomus leopardus, a commercially significant marine fish, is primarily found in the Western Pacific regions and along the coast of Southeast Asia. A thorough analysis of the molecular mechanisms involved in sex differentiation is crucial for gaining a comprehensive understanding of gonadal development and improving sex control breeding. However, the relevant fundamental studies of P. leopardus are relatively lacking. Methods: In this study, a genome-wide association study (GWAS) was conducted to investigate the genetic basis mechanism of sex differentiation and gonadal developmental traits in P. leopardus utilizing about 6,850,000 high-quality single-nucleotide polymorphisms (SNPs) derived from 168 individuals (including 126 females and 42 males) by the genome-wide efficient mixed-model association (GEMMA) algorithm. Results: The results of these single-trait GWASs showed that 46 SNP loci (-log10 p > 7) significantly associated with sex differentiation, and gonadal development traits were distributed in multiple different chromosomes, which suggested the analyzed traits were all complex traits under multi-locus control. A total of 1,838 potential candidate genes were obtained by considering a less-stringent threshold (-log10 p > 6) and ±100kb regions surrounding the significant genomic loci. Moreover, 31 candidate genes were identified through a comprehensive analysis of significant GWAS peaks, gene ontology (GO) annotations, and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analyses, including taf7, ddx6, apoeb, sgk1, a2m, usf1, hsd3b7, dll4, xbp1, tet3, esr1, and gli3. These trait-associated genes have been shown to be involved in germline development, male sex differentiation, gonad morphogenesis, hormone receptor binding, oocyte development, male gonad development, steroidogenesis, estrogen-synthetic pathway, etc. Discussion: In the present study, multiple genomic loci of P. leopardus associated with sex differentiation and gonadal development traits were identified for the first time by using GWAS, providing a valuable resource for further research on the molecular genetic mechanism and sex control in P. leopardus. Our results also can contribute to understanding the genetic basis of the sex differentiation mechanism and gonadal development process in grouper fish.

Effect of Exogenous Hormone on R-Spondin 2 (Rspo2) and R-Spondin 3 (Rspo3) Gene Expression and Embryo Development in Chinese Soft-Shelled Turtle (Pelodiscus sinensis).

The Chinese soft-shelled turtle, Pelodiscus sinensis, is an important aquaculture species in China that exhibits distinct sexual dimorphism; male individuals are economically more valuable than females. In vertebrates, several R-spondin family proteins have been associated with sex differentiation mechanisms; however, their involvement in P. sinensis sex differentiation is unclear. Exogenous hormones such as estradiol (E2) also influence the sex differentiation of P. sinensis and induce sexual reversal. In the present study, we investigated the effects of E2 on the embryonic development of P. sinensis and the expression of R-spondin 2 (Rspo2) and R-spondin 3 (Rspo3). We amplified P. sinensis Rspo2 and Rspo3 and analyzed their expression patterns in different tissues. Comparative analyses with protein sequences from other species elucidated that P. sinensis RSPO2 and RSPO3 sequences were conserved. Moreover, phylogenetic analysis revealed that P. sinensis RSPO2 and RSPO3 were closely related to these two proteins from other turtle species. Furthermore, Rspo2 and Rspo3 were highly expressed in the brain and gonads of adult turtles, with significantly higher expression in the ovaries than in the testes (p < 0.05). We also evaluated the expression of Rspo2 and Rspo3 after the administration of three concentrations of E2 (1.0, 5.0, and 10.0 mg/mL) to turtle eggs during embryonic development. The results revealed that E2 upregulated Rspo2 and Rspo3, and the expression trends varied during different embryonic developmental stages (stages 13-20). These findings lay the groundwork for future investigations into the molecular mechanisms involved in the sex differentiation of Chinese soft-shelled turtles.

Characterization of Early Gonadal Differentiation and Induction of Sex Reversal in the Rock Bream Oplegnathus fasciatus.

Rock bream (Oplegnathus fasciatus) is a typical fish that has a unique multiple sex chromosome system (♀X1X1X2X2/♂X1X2Y). We examined the early gonadal development in rock bream via continuous histological observations of the gonads at 40-120days post hatching (dph). The fish was identified as a typical gonochorist, and female gonads were found to differentiate earlier than male gonads. The ovarian cavity of the female was initially observed at 80 dph, whereas the efferent duct of the male was not observed until 100 dph. Immunofluorescence with the vasa-antibody revealed that germ cells were predominantly distributed around the ovarian cavity in females and on the edge of the gonad in males during the early stages of sex differentiation. Sex reversal was induced via the oral administration of letrozole (LTZ), 17α-methyltestosterone (MT), and 17β-estradiol (E2), respectively, during the labile period of gonadal development. LTZ and MT induced 100% masculinization of genotype-females, whereas E2 induced only 50-60% feminization of genotype-males. Such findings suggest that the fish retained high sexual plasticity despite the existence of the neo-Y chromosome. MT and E2 had negative effect on fish growth, whereas LTZ did not exert such side effect. LTZ and MT could accelerate gonadal development in sex-reversed genotype-males, whereas E2 inhibited gonadal development in genotype-females of rock bream. These findings provide a basis for further research on the mechanisms of sex determination and differentiation in fishes with X1X2Y sex chromosome system and provide a sex reversal protocol for rock bream.