Sex-related Genes Research Articles

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.

Proteome analysis provides insights into sex differences in Holothuria Scabra.

Sex-determining mechanism is still ambiguous for sea cucumber Holothuria scabra which only manifests gonochorism in gonad. In this study, proteomic analysis was employed to delineate sex-related proteins and genes in gonads of H. scabra, subsequently validated through Quantitative real-time polymerase chain reaction (qRT-PCR). A total of 5,313 proteins were identified via proteome sequencing. Among these, 817 proteins exhibited expression in both the ovary and testis, with 445 proteins displaying up-regulation and 372 proteins showing down-regulation (ovary vs testis). Furthermore, 136 and 69 proteins were identified as ovary-specific and testis-specific Differentially Abundant Proteins (DAPs), respectively. And 9 DAP coding genes which play crucial role in ovary and testis were verified by qRT-PCR. Notably, 24 ovary-bias proteins enriched in ribosome pathway strongly indicated the crucial role of ribosome in ovary. This study serves to furnish novel evidence pertaining to sex differences in H. scabra.

Production of neofemale by 17β-estradiol and YY super-male breeding in mandarin fish (Siniperca chuatsi)

It is well known that mono-sex culture has the advantages of a more rapid growth rate, more uniform size and lower energy consumption of gonadal development in fish. In production practice, male mandarin fish (Siniperca chuatsi) show great aquaculture value after the overwintering stage. As a species with an XY-type sex determination system, feminizing genetically male fish into physiological female fish and breeding YY supermales is a common method used to produce all-male populations. In this study, we systematically investigated feminization of mandarin fish by indirect feeding of 17β-estradiol (E2) at different doses (200, 400 and 600 mg/kg). Neo-females were successfully yielded in all E2-treated concentrations. The sex-reversed ratios of the 200, 400 and 600 mg/kg E2-treated groups were 40%, 87% and 100%, respectively. From 45 to 135 dat (days after treatment), the gonads in the 600 mg/kg E2-treated group consisted of oogonium and previtellogenic oocytes. At 165 dat, early vitellogenic oocytes were observed in the gonads of the 600 mg/kg E2-treated group. Regarding the expression of sex-related genes, 600 mg/kg E2-treated mandarin fish presented a different gene expression pattern from that of normal males. Moreover, the growth and development status were also analysed in XY normal males and YY supermales at 360 dph, and YY supermales showed a higher number of lumina and significantly lower growth performance than XY normal males. In conclusion, we have established a technique for efficiently inducing male to female sex reversion and YY supermale breeding of mandarin fish, which provides a great foundation for the production of all-male mandarin fish.

Genome-wide investigation of the DMRT gene family sheds new insight into the regulation of sex differentiation in spotted knifejaw (Oplegnathus punctatus) with fusion chromosomes (Y)

The role of the DMRT family in male sex determination and differentiation is significant, but its regulatory role in spotted knifejaw with Y fusion chromosomes remains unclear. Through genome-wide scanning, transcriptome analysis, qPCR, FISH, and RNA interference (RNAi), we investigated the DMRT family and the dmrt1-based sex regulation network. Seven DMRTs were identified (DMRT1/2 (2a,2b)/6, DMRT4/5, DMRT3), and dmrt gene dispersion among chromosomes is possibly driven by three whole-genome duplications. Transcriptome analysis enriched genes were associated with sex regulation and constructed a network associated with dmrt1. qPCR and FISH results showed the expression dimorphism of sex-related genes in dmrt-related regulatory networks. RNAi experiments indicated a distinct sex regulation mode in spotted knifejaw. Dmrt1 knockdown upregulated male-related genes (sox9a, sox9b, dmrt1, amh, amhr2) and hsd11b2 expression, which is critical for androgen synthesis. Amhr2 is located on the heterozygous chromosome (Y) and is specifically localized in primary spermatocytes, and is extremely upregulated after dmrt1 knockdown which suggested besides the important role of dmrt1 in male differentiation, the amhr2 along with amhr2/amh system, also play important regulatory roles in maintaining high expression of the hsd11b2 and male differentiation. This study aims to further investigate sex regulatory mechanisms in species with fusion chromosomes.

A MADS-Box Gene-Based InDel Marker Discriminating Sex in Actinidia arguta

Gene-based markers are valuable tools in breeding programs due to their direct linkage to traits of interest. In dioecious plants, such as kiwifruit (Actinidia spp.), sex-discriminating markers can shorten the breeding cycle by enabling the selection of preferred sexes at the juvenile stage. To develop a gene-based sex-discriminating marker, resequencing was conducted on female and male A. arguta accessions, and insertion and deletion (InDel) variations within sex-related genes were explored. A total of 203,116 InDels were detected between female and male A. arguta accessions, and 118,865 InDels were heterozygous between the two accessions. Sequence similarity between thirty-seven sex-related genes from seven dioecious species and the kiwifruit reference genome was investigated, revealing that ten genes exhibited similarities ranging from 59 to 79%. Among the 118,865 InDels, seven InDels were located on four sex-related genes encoding agamous-like MADS-box genes and hypothetical proteins. A 20 bp insertion in male A. arguta located in the agamous-like MADS-box gene was converted into an InDel marker, which clearly discriminates female and male A. arguta accessions and the interspecific hybrid cultivar. The InDel marker was designated CBk25id01 and produced approximately 350 bp amplicon only in the male A. arguta. The CBk25id01 linked to the agamous-like MADS-box gene involved in floral organ development may help understand sex differentiation and accelerate the breeding of kiwifruits.

Transcriptomics analysis of allergen-induced inflammatory gene expression in the Four-Core Genotype mouse model.

Sex differences in allergic inflammation have been reported, but the mechanisms underlying these differences remain unknown. Contributions of both sex hormones and sex-related genes to these mechanisms have been previously suggested in clinical and animal studies. Here, Four-Core Genotypes (FCG) mouse model was used to study the inflammatory response to house dust mite (HDM) challenge and identify differentially expressed genes (DEGs) and regulatory pathways in lung tissue. Briefly, adult mice (8-10 wk old) of the FCG (XXM, XXF, XYM, XYF) were challenged intranasally with 25 μg of HDM or vehicle (PBS-control group) 5 days/wk for 5 wk (n = 3/10 group). At 72 h after the last exposure, we analyzed the eosinophils and neutrophils in the bronchoalveolar lavage (BAL) of FCG mice. We extracted lung tissue and determined DEGs using Templated Oligo-Sequencing (TempO-Seq). DEG analysis was performed using the DESeq2 package and gene enrichment analysis was done using Ingenuity Pathway Analysis. A total of 2,863 DEGs were identified in the FCG. Results revealed increased eosinophilia and neutrophilia in the HDM-treated group with the most significantly expressed genes in XYF phenotype and a predominant effect of female hormones vs. chromosomes. Regardless of the sex hormones, mice with female chromosomes had more downregulated genes in the HDM group but this was reversed in the control group. Interestingly, genes associated with inflammatory responses were overrepresented in the XXM and XYF genotypes treated with HDM. Sex hormones and chromosomes contribute to inflammatory responses to HDM challenge, with female hormones exerting a predominant effect mediated by inflammatory DEGs.NEW & NOTEWORTHY Gene expression profiling helps to provide deep insight into the global view of disease-related mechanisms and responses to therapy. Using the Four-Core Genotype mouse model, our findings revealed the influence of sex hormones and sex chromosomes in the gene expression of lungs exposed to an aeroallergen (House Dust Mite) and identified sex-specific pathways to better understand sex disparities associated with allergic airway inflammation.

Comparative Transcriptome Analysis Reveals Sexually Dimorphic Gene Expression in the Gonads of Brachymystax tsinlingensis Li

Brachymystax tsinlingensis Li is an endangered cold-water salmonid fish native to China. This study aimed to identify sex-related genes and biological pathways via gonadal transcriptome sequencing of B. tsinlingensis Li. A total of 167,904 unigenes were identified with an average length of 836 bp and an N50 of 1452 bp, of which 84,977 (50.61%) unigenes were successfully annotated in six major databases. Comparative transcriptome analysis identified 22,864 differentially expressed genes (DEGs), of which 17,231 were up-regulated (male-biased genes, mDEGs) and 5633 were down-regulated (female-biased genes, fDEGs). Several DEGs associated with gonadal development were found through Gene Ontology enrichment analysis, such as ccnb1, zp3, bmp15, dmrt1, and psmc3ip. Signaling pathways related to gonadal development were found to be enriched through analysis using the Kyoto Encyclopedia of Genes and Genomes Pathway database, such as genes involves in base excision repair, the notch signaling pathway, neuroactive ligand-receptor interaction, the VEGF signaling pathway, and the estrogen signaling pathway. In addition, mRNA expression levels of 19 DEGs were determined to validate the reliability of the transcriptomic data by quantitative real-time polymerase chain reaction. These results revealed genes and signaling pathways potentially involved in gonadal development in B. tsinlingensis Li and provided basic molecular data for future research on reproductive regulation and breeding of B. tsinlingensis Li.

Insulin-like Androgenic Gland Hormone Induced Sex Reversal and Molecular Pathways in Macrobrachium nipponense: Insights into Reproduction, Growth, and Sex Differentiation.

This study investigated the potential to use double-stranded RNA insulin-like androgenic gland hormone (dsIAG) to induce sex reversal in Macrobrachium nipponense and identified the molecular mechanisms underlying crustacean reproduction and sex differentiation. The study aimed to determine whether dsIAG could induce sex reversal in PL30-male M. nipponense during a critical period. The sex-related genes were selected by performing the gonadal transcriptome analysis of normal male (dsM), normal female (dsFM), neo-female sex-reversed individuals (dsRM), and unreversed males (dsNRM). After six injections, the experiment finally resulted in a 20% production of dsRM. Histologically, dsRM ovaries developed slower than dsFM, but dsNRM spermathecae developed normally. A total of 1718, 1069, and 255 differentially expressed genes were identified through transcriptome sequencing of the gonads in three comparison groups, revealing crucial genes related to reproduction and sex differentiation, such as GnRHR, VGR, SG, and LWS. Principal Component Analysis (PCA) also distinguished dsM and dsRM very well. In addition, this study predicted that the eyestalks and the "phototransduction-fly" photoperiodic pathways of M. nipponense could play an important role in sex reversal. The enrichment of related pathways and growth traits in dsNRM were combined to establish that IAG played a significant role in reproduction, growth regulation, and metabolism. Finally, complete sex reversal may depend on specific stimuli at critical periods. Overall, this study provides valuable findings for the IAG regulation of sex differentiation, reproduction, and growth of M. nipponense in establishing a monoculture.

Identification of male-specific markers by 2b-RAD sequencing reveals an XX/XY sex-determination system in cultured lined seahorse, Hippocampus erectus

Seahorses (Hippocampus spp.), a group of male ovoviviparous fish, are of high commercial value in traditional Chinese medicine and aquarium trades. The lined seahorse (Hippocampus erectus), an easily domesticated species, was introduced to China and quickly became a popular farmed species to relieve the conflict between supply and demand. Although the sexual dimorphism in size and immunocompetence has been observed, the sex determination mechanism in the lined seahorse remains unclear. In the present study, we applied the 2b-RAD-seq approach for the first time to successfully identify 6 male-specific tags and 4 sex-specific SNP loci in the lined seahorse, which were further validated in 108 individuals by PCR amplification based on the flanking sequences of sex-specific markers. The male-specific tags and heterozygous SNP loci indicate that a male heterozygous XX/XY sex-determination system is employed in the lined seahorse. Based on the annotation and analysis of the sex-specific sequences, two sex-related candidate genes, lrriq1 and il34, were identified. The results provide novel insights into sex-determination mechanisms and promising methods for all-male breeding of Hippocampus erectus.

Effects of dietary supplementation of estradiol-17β during fry stage on growth, physiological and immune parameters and gonadal gene expression in adult snakeskin gourami.

In snakeskin gourami (Trichopodus pectoralis), females are generally larger than males, and estradiol-17β (E2)-sex reversal to produce female monosex has gained interest in this species. In this study, we aimed to investigate the effects of E2-induced sex reversal on growth, physiological and immune parameters, and gonadal gene expression in adult snakeskin gourami. Fry (7days posthatching) were divided into different experimental groups based on the dose of E2: control (no E2 (0mgkg-1) supplementation), E2-100 (100mgkg-1), E2-200 (200mgkg-1), and E2-300 (300mgkg-1), fed with the E2 doses for 90 d and cultured for 11months (adult stage). The findings revealed that E2 supplementation produced 88.89-100% of female population. After 11months of culture, the effects of sexual dimorphism on the growth performance of the E2-100 group were not significant compared to that on the growth performance of the control male and female groups; however, it improved significantly in the E2-200 and E2-300 groups (P<0.05). E2 elevated the CP and fat contents in body in E2-200 and E2-300 groups (P<0.05) compared to that in the control group. No sex differences in blood metabolites, haematological values, or immune parameters were identified. Nevertheless, E2-200 and E2-300 groups showed increased blood glucose, triglyceride, haemoglobin, and total immunoglobulin (P<0.05) compared to control male fish. In addition, all concentrations of E2 increased alternative complement 50 (P<0.05). Several genes, including bHLH, cyp19a1, daz, deadend, esrb, esrrg, gnrhr, gpa, gsg1l, hsd17β, mospd1, nanos2, p53, piwi2, rerg, rps6ka, tgfb, and vgr, showed differential expression between testis and ovary in control female and E2-treated groups. The expression patterns of the genes were similar in the ovary of the control female and E2-200-treated fish. In conclusion, the findings demonstrate that a feminisation duration of 7-97days and two doses of E2 at 200 or 300mgkg-1 successfully produced all-female stocks in snakeskin gourami. Furthermore, the findings showed that E2-treated females were maintained throughout adulthood and exhibited several superior characteristics to male fish. Together with the information generated on differentially expressed sex-related genes, these findings could enable the culturing of faster-growing sex to increase productivity and contribute to the development of intensive snakeskin gourami farming.

Identification of Male Sex-Related Genes Regulated by SDHB in Macrobrachium nipponense Based on Transcriptome Analysis after an RNAi Knockdown.

The oriental river prawn (Macrobrachium nipponense) is a commercially important species in Asia. A previous study showed that the succinate dehydrogenase complex iron sulfur subunit B (SDHB) gene participates in testes development in this species through its effect on the expression of the insulin-like androgenic gland hormone gene. This study knocked-down the Mn-SDHB genes in M. nipponense using RNAi. A transcriptome analysis of the androgenic gland and testes was then performed to discover the male sex-related genes regulated by SDHB and investigate the mechanism of male sexual development in this species. More than 16,623 unigenes were discovered in each sample generated. In the androgenic gland, most of the differentially expressed genes were enriched in the hypertrophic cardiomyopathy pathway, while in the testes, they were enriched in the citrate cycle pathway. In addition, after Mn-SDHB knockdown, five genes were found to be downregulated in the androgenic gland in a series of biological processes associated with phosphorylated carbohydrate synthesis and transformations in the glycolysis/gluconeogenesis pathway. Moreover, a total of nine male sex-related genes were identified including Pro-resilin, insulin-like androgenic gland hormone, Protein mono-ADP-ribosyltransferase PAPR11, DNAJC2, C-type Lectin-1, Tyrosine-protein kinase Yes, Vigilin, and Sperm motility kinase Y-like, demonstrating the regulatory effects of Mn-SDHB, and providing a reference for the further study of the mechanisms of male development in M. nipponense.

Gonadal transcriptome analysis of paradise fish Macropodus opercularis to reveal sex-related genes

Macropodus opercularis is an ornamental fish species endemic to China, with obvious sexual dimorphism in phenotype. To obtain the gene expression profile of the gonads of M. opercularis and explore its sex-related genes, six cDNA libraries were constructed from the sexually mature M. opercularis, and RNA-seq analysis was performed. The sequenced clean data were assembled by de novo splicing to generate 171,415 unigenes, and differentially expressed genes (DEGs) screening revealed that there were 41,638 DEGs in the gonads of M. opercularis. By comparing those DEGS in the ovary with the testis, we found 29,870 DEGs were upregulated and 11,768 DEGs were downregulated. Kyoto Encyclopedia of Genes and Genomes (KEGG) and gene ontology (GO) enrichment analysis showed that GO terms related to cell cycle and gamete formation were enriched, and pathway signals related to sex differences, such as FoxO signalling pathway and PI3K-Akt signalling pathway, were also detected. Reverse transcript fluorescence quantitative PCR (RT-qPCR) validation of 14 DEGs associated with sex differences showed that the RT-qPCR results were consistent with RNA-Seq analysis, and five genes, foxl2, sox3, foxo, zar1, cyp19a1, were significantly expressed in the ovaries. dmrt1, cyp11b, amh, sf1, sox9, gdf6, dmrt3, fstl1 and hsd11b2, a total of nine genes were significantly expressed in the testis. The results of this study provide a basis for the study of gonadal differentiation, developmental mechanisms and related functional genes in M. opercularis.

17β-Estradiol Induced Sex Reversal and Gonadal Transcriptome Analysis in the Oriental River Prawn (Macrobrachium nipponense): Mechanisms, Pathways, and Potential Harm.

Sex reversal induced by 17β-estradiol (E2) has shown the potential possibility for monoculture technology development. The present study aimed to determine whether dietary supplementation with different concentrations of E2 could induce sex reversal in M. nipponense, and select the sex-related genes by performing the gonadal transcriptome analysis of normal male (M), normal female (FM), sex-reversed male prawns (RM), and unreversed male prawns (NRM). Histology, transcriptome analysis, and qPCR were performed to compare differences in gonad development, key metabolic pathways, and genes. Compared with the control, after 40 days, feeding E2 with 200 mg/kg at PL25 (PL: post-larvae developmental stage) resulted in the highest sex ratio (female: male) of 2.22:1. Histological observations demonstrated the co-existence of testis and ovaries in the same prawn. Male prawns from the NRM group exhibited slower testis development without mature sperm. RNA sequencing revealed 3702 differentially expressed genes (DEGs) between M vs. FM, 3111 between M vs. RM, and 4978 between FM vs. NRM. Retinol metabolism and nucleotide excision repair pathways were identified as the key pathways for sex reversal and sperm maturation, respectively. Sperm gelatinase (SG) was not screened in M vs. NRM, corroborating the results of the slice D. In M vs. RM, reproduction-related genes such as cathepsin C (CatC), heat shock protein cognate (HSP), double-sex (Dsx), and gonadotropin-releasing hormone receptor (GnRH) were expressed differently from the other two groups, indicating that these are involved in the process of sex reversal. Exogenous E2 can induce sex reversal, providing valuable evidence for the establishment of monoculture in this species.

Identification and dimorphic expression of sex-related genes in Pacific abalone (Haliotis discus hannai)

Sex determination is a hot topic in evolutionary biology. The mechanism of sex determination is complex and diverse in shellfish, while no sex chromosomes have been found in mollusks. The Pacific abalone (Haliotis discus hannai) is one of the most economically important cultured shellfish species in China, and its sex determination mechanism is still unclear. Here, we identified the sex-determination region (SDR) of Pacific abalone and developed five stable sex-specific markers through genome-wide association studies (GWAS) based on re-sequencing data from 803 abalones. These markers can be used for sex discrimination in polyploid and sexually immature individuals in abalone breeding projects. In addition, haplotype analysis and quantitative expression of candidate sex-related genes showed that RhoGAPp190, Skp1, Cul2, and Otulin were potentially involved in the sex determination of Pacific abalone by the ubiquitin-mediated proteolysis pathway. Our study could help to better understand the molecular mechanisms that underpin sex determination and reproductive regulation in mollusks.

Expression pattern analysis of anti-Mullerian hormone in testis development of pearlscale angelfish (Centropyge vrolikii).

In vertebrates, anti-Mullerian hormone (Amh) secreted by Sertoli cells (SC) performs a pivotal function in male sex differentiation. Compared with that of higher vertebrates, the expression pattern of Amh is more diversified in fish. In this study, the full-length complementary DNA (cDNA) of Amh in Centropyge vrolikii (Cv-Amh) was cloned and analysed, which was 2,470 bp, including a 238 bp 5'UTR, a 1,602 bp ORF and a 633 bp 3'UTR; the similarity of Amh between Cv-Amh and other fish is relatively high. The quantitative real-time PCR (qRT-PCR) results of healthy tissues and gonads at sex reversal stages in C. vrolikii showed that the expression level of Amh in the testis was significantly higher than that in other tissues (P < 0.05). Amh was weakly expressed in the vitellogenic stage ovary and perinucleolus stage ovary, but its expression significantly increased in the gonads at the hermaphroditic stage, and finally reached the highest in the pure testis after sexual reversal. The results of in situ hybridization indicated that the positive signal of Amh was strongly concentrated in SCs of testis. After Amh knockdown in the gonads, the effect on sex-related genes was tested using qRT-PCR. Among these, the expression of Dmrt1, Cyp11a, Hsd11b2, Sox8 and Sox9 significantly decreased, whereas that of Cyp19a, Sox4, Foxl2 and Sox3 increased. These results suggested that Amh could be the pivotal gene in reproductive regulation in C. vrolikii, and the data will contribute to sex-related research of C. vrolikii in the future.

Identifying Genes Associated with Female Flower Development of Phellodendron amurense Rupr. Using a Transcriptomics Approach.

Phellodendron amurense Rupr., a species of Rutaceae, is a nationally protected and valuable medicinal plant. It is generally considered to be dioecious. With the discovery of monoecious P. amurense, the phenomenon that its sex development is regulated by epigenetics has been revealed, but the way epigenetics affects the sex differentiation of P. amurense is still unclear. In this study, we investigated the effect of DNA methylation on the sexual development of P. amurense. The young inflorescences of male plants were treated with the demethylation agent 5-azaC, and the induced female flowers were obtained. The induced female flowers' morphological functions and transcriptome levels were close to those of normally developed plants. Genes associated with the development of female flowers were studied by comparing the differences in transcriptome levels between the male and female flowers. Referring to sex-related genes reported in other plants, 188 candidate genes related to the development of female flowers were obtained, including sex-regulating genes, genes related to the formation and development of sexual organs, genes related to biochemical pathways, and hormone-related genes. RPP0W, PAL3, MCM2, MCM6, SUP, PIN1, AINTEGUMENTA, AINTEGUMENTA-LIKE6, AGL11, SEUSS, SHI-RELATED SEQUENCE 5, and ESR2 were preliminarily considered the key genes for female flower development. This study has demonstrated that epigenetics was involved in the sex regulation of P. amurense, with DNA methylation as one of its regulatory modes. Moreover, some candidate genes related to the sexual differentiation of P. amurense were obtained with analysis. These results are of great significance for further exploring the mechanism of sex differentiation of P. amurense and studying of sex differentiation of plants.

The Role of the Heat Shock Cognate Protein 70 Genes in Sex Determination and Differentiation of Chinese Tongue Sole (Cynoglossus semilaevis).

Fish sex determination can be affected by environmental temperature. This process relies on temperature-sensitive proteins such as heat shock proteins (HSPs). Our previous work found that heat shock cognate proteins (HSCs) may participate in high-temperature associated sex reversal of Chinese tongue sole (Cynoglossus semilaevis). However, the role of hsc genes in responding to high temperature and affecting sex determination/differentiation remains unclear. Here, by using C. semilaevis as model, we identified hsc70 and hsc70-like. hsc70 was abundant in the gonads with a testicular-higher expression at all gonadal development stages except for 6 months post fertilization (mpf). Intriguingly, hsc70-like showed higher expression in testes from 6 mpf on. Both long-term heat treatment during the temperature-sensitive sex-determining period and short-term heat stress at the end of this period caused different expression of hsc70/hsc70-like between sexes. The dual-luciferase assay results also suggested that these genes can respond to high temperature rapidly in vitro. Heat treatment of C. semilaevis testis cells overexpressed with hsc70/hsc70-like could affect the expression of sex-related genes sox9a and cyp19a1a. Our results indicated that hsc70 and hsc70-like were key regulators linking external high-temperature signals with sex differentiation in vivo and provide a new idea for understanding the mechanism by which high temperature affects sex determination/differentiation in teleosts.

Sex-Inclined Piwi-Interacting RNAs in Serum Exosomes for Sex Determination in the Greater Amberjack (Seriola dumerili).

The greater amberjack (Seriola dumerili) is a gonochoristic fish with no sexual dimorphism in appearance, making sex identification difficult. Piwi-interacting RNAs (piRNAs) function in transposon silencing and gametogenesis and are involved in various physiological processes, including sex development and differentiation. Exosomal piRNAs can be indicators for the determination of sex and physiological status. In this study, four piRNAs were differentially expressed in both serum exosomes and gonads between male and female greater amberjack. Three piRNAs (piR-dre-32793, piR-dre-5797, and piR-dre-73318) were significantly up-regulated and piR-dre-332 was significantly down-regulated in serum exosomes and gonads of male fish, compared to female fish, consistent with the serum exosomal results. According to the relative expression of four marker piRNAs derived from the serum exosomes of greater amberjack, the highest relative expression of piR-dre-32793, piR-dre-5797, and piR-dre-73318 in seven female fish and that of piR-dre-332 in seven male fish can be used as the standard for sex determination. The method of sex identification can ascertain the sex of greater amberjack by blood collection from the living body, without sacrificing fish. The four piRNAs did not show sex-inclined expression in the hypothalamus, pituitary, heart, liver, intestine, and muscle tissue. A piRNA-target interaction network involving 32 piRNA-mRNA pairs was generated. Sex-related target genes were enriched in sex-related pathways, including oocyte meiosis, transforming growth factor-beta signaling pathway, progesterone-mediated oocyte maturation, and gonadotropin releasing hormone signaling pathway. These results provide a basis for sex determination in greater amberjack and improve our understanding of the mechanisms underlying sex development and differentiation in the species.

Amh/Amhr2 Signaling Causes Masculinization by Inhibiting Estrogen Synthesis during Gonadal Sex Differentiation in Japanese Flounder (Paralichthys olivaceus).

The anti-Müllerian hormone (Amh) is a protein belonging to the TGF-β superfamily, the function of which has been considered important for male sex differentiation in vertebrates. The Japanese flounder (Paralichthys olivaceus) is a teleost fish that has an XX/XY sex determination system and temperature-dependent sex determination. In this species, amh expression is up-regulated in genetic males and in temperature-induced masculinization during the sex differentiation period. However, to the best of our knowledge, no reports on the Amh receptor (Amhr2) in flounder have been published, and the details of Amh signaling remain unclear. In this study, we produced amhr2-deficient mutants using the CRISPR/Cas9 system and analyzed the gonadal phenotypes and sex-related genes. The results revealed that the gonads of genetically male amhr2 mutants featured typical ovaries, and the sex differentiation-related genes showed a female expression pattern. Thus, the loss of Amhr2 function causes male-to-female sex reversal in Japanese flounder. Moreover, the treatment of genetically male amhr2 mutants with an aromatase inhibitor fadrozole, which inhibits estrogen synthesis, resulted in testicular formation. These results strongly suggest that Amh/Amhr2 signaling causes masculinization by inhibiting estrogen synthesis during gonadal sex differentiation in the flounder.

Transcriptome analysis of differentially expressed circular RNAs in the testis and ovary of golden pompano (Trachinotus blochii)

The artificial breeding of golden pompano (Trachinotus blochii) has expanded greatly in recent years, and after long-term breeding efforts, clear sexual dimorphisms have been observed in T. blochii growth traits, with females growing faster. As sponges of microRNA (miRNAs), circular RNAs (CircRNAs) can alleviate miRNA inhibition of target mRNA. However, few studies have examined sex-related CircRNAs and none of those have looked at T. blochii. To further understand the role of CircRNAs in sex differentiation and sexual size dimorphism in T. blochii, six CircRNA libraries were constructed from the testes and ovaries of T. blochii. A total of 1522 CircRNAs were found distributed over all 24 chromosomes of T. blochii. 135 differentially expressed CircRNAs (DECs) were identified by screening, These DECs were then subjected to GO enrichment, which found 47 enriched pathways. A number of CircRNAs were enriched in cellular processes and metabolic processes. According to the KEGG pathway analysis, a series of sex differentiation pathways were enriched, including the GnRH, calcium, and MAPK signaling pathways. Furthermore, we selected two CircRNAs from the DECs named circ-cacna1b and circ-octc. We found that the cacna1b gene is regulated by 7 miRNAs, 3 of which were regulated by circ-cacna1b, i.e., mmu-miR-138-5p, fru-miR-138, and pma-miR-138b. In addition, the miRNA named pma-miR-138b can regulate sex-related genes, such as sox9 and dmrt1, among others. The co-expression network of CircRNA-miRNA-mRNA showed circ-cacna1b may play a crucial role in T. blochii sex differentiation by regulating pma-miR-138b to affect the expression of sex differentiation genes. The circ-octc may be one of the largest contributors to sexual size dimorphism during growth through its effect on lipid metabolism. These findings could broaden our understanding of CircRNAs and provide new insight into their function in sex differentiation and growth.