Screening of temperature-responsive signalling molecules during sex differentiation in Asian yellow pond turtle (Mauremys mutica).

BACKGROUND: The Asian yellow pond turtle (Mauremys mutica) is an important commercial freshwater aquaculture species in China. This species is a highly sexually dimorphic species, with males growing at a faster rate than females and exhibits temperature-dependent sex determination (TSD), in which the incubation temperature during embryonic development determines the sexual fate. However, the mechanisms of the sex determination or sex differentiation in the Asian yellow pond turtle are remain a mystery. RESULTS: Temperature-specific gonadal transcriptomics of the Asian yellow pond turtle were performed during the thermosensitive period (stage 15) using RNA-seq technology to identify candidate genes that initiate gonadal differentiation. We uncovered candidates that were the first to respond to temperature. These candidates were sexually dimorphic in expression, reflecting differences in gonadal (Cirbp, Runx1) and germline differentiation (Vasa, Nanos1, Piwil2), gametogenesis (Hmgb3, Zar1, Ovoinhibitor-like, Kif4), steroid hormone biosynthesis (Hsd17b5, Hsd17b6), heat shock (Dnajb6, Hsp90b1, Hsp90aa1) and transient receptor potential channel genes (Trpm1, Trpm4, Trpm6, Trpv1). CONCLUSIONS: Our work will provide important genetic information to elucidate the mechanisms of sex control in the Asian yellow pond turtles, and will contribute important genetic resources for further studies of temperature-dependent sex determination in turtles.

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.

Regulation of sexual differentiation initiation in Schizosaccharomyces pombe.

The fission yeast Schizosaccharomyces pombe is an excellent model organism to explore cellular events owing to rich tools in genetics, molecular biology, cellular biology, and biochemistry. Schizosaccharomyces pombe proliferates continuously when nutrients are abundant but arrests in G1 phase upon depletion of nutrients such as nitrogen and glucose. When cells of opposite mating types are present, cells conjugate, fuse, undergo meiosis, and finally form 4 spores. This sexual differentiation process in S. pombe has been studied extensively. To execute sexual differentiation, the glucose-sensing cAMP-PKA (cyclic adenosine monophosphate-protein kinase A) pathway, nitrogen-sensing TOR (target of rapamycin) pathway, and SAPK (stress-activating protein kinase) pathway are crucial, and the MAPK (mitogen-activating protein kinase) cascade is essential for pheromone sensing. These signals regulate ste11 at the transcriptional and translational levels, and Ste11 is modified in multiple ways. This review summarizes the initiation of sexual differentiation in S. pombe based on results I have helped to obtain, including the work of many excellent researchers.

Overview of chicken embryo genes related to sex differentiation.

Sex determination in chickens at an early embryonic stage has been a longstanding challenge in poultry production due to the unique ZZ:ZW sex chromosome system and various influencing factors. This review has summarized the genes related to the sex differentiation of chicken early embryos (mainly Dmrt1, Sox9, Amh, Cyp19a1, Foxl2, Tle4z1, Jun, Hintw, Ube2i, Spin1z, Hmgcs1, Foxd1, Tox3, Ddx4, cHemgn and Serpinb11 in this article), and has found that these contributions enhance our understanding of the genetic basis of sex determination in chickens, while identifying potential gene targets for future research. This knowledge may inform and guide the development of sex screening technologies for hatching eggs and support advancements in gene-editing approaches for chicken embryos. Moreover, these insights offer hope for enhancing animal welfare and promoting conservation efforts in poultry production.

Dmrt1 is the only male pathway gene tested indispensable for sex determination and functional testis development in tilapia.

Sex is determined by multiple factors derived from somatic and germ cells in vertebrates. We have identified amhy, dmrt1, gsdf as male and foxl2, foxl3, cyp19a1a as female sex determination pathway genes in Nile tilapia. However, the relationship among these genes is largely unclear. Here, we found that the gonads of dmrt1;cyp19a1a double mutants developed as ovaries or underdeveloped testes with no germ cells irrespective of their genetic sex. In addition, the gonads of dmrt1;cyp19a1a;cyp19a1b triple mutants still developed as ovaries. The gonads of foxl3;cyp19a1a double mutants developed as testes, while the gonads of dmrt1;cyp19a1a;foxl3 triple mutants eventually developed as ovaries. In contrast, the gonads of amhy;cyp19a1a, gsdf;cyp19a1a, amhy;foxl2, gsdf;foxl2 double and amhy;cyp19a1a;cyp19a1b, gsdf;cyp19a1a;cyp19a1b triple mutants developed as testes with spermatogenesis via up-regulation of dmrt1 in both somatic and germ cells. The gonads of amhy;foxl3 and gsdf;foxl3 double mutants developed as ovaries but with germ cells in spermatogenesis due to up-regulation of dmrt1. Taking the respective ovary and underdeveloped testis of dmrt1;foxl3 and dmrt1;foxl2 double mutants reported previously into consideration, we demonstrated that once dmrt1 mutated, the gonad could not be rescued to functional testis by mutating any female pathway gene. The sex reversal caused by mutation of male pathway genes other than dmrt1, including its upstream amhy and downstream gsdf, could be rescued by mutating female pathway gene. Overall, our data suggested that dmrt1 is the only male pathway gene tested indispensable for sex determination and functional testis development in tilapia.

Determination of the timing of early gonadal differentiation in silver pomfret, Pampus argenteus.

Silver pomfret is a species of global significance due to its high nutritional in fisheries sector. To accurately ascertain the timing of sex differentiation mechanism and mRNA level in this species, this study examined gonad morphology and patterns of gene expression related to sex differentiation in males and females from 51 to 180 days post hatch (dph), the temperature of water was maintained at 26 ± 1 â„ƒ. Distinct morphological differentiation of the silver pomfret ovaries, marked by the emergence of primary oocytes, became apparent from 68 dph. By 108 dph, the testes began to differentiate, as evidenced by the appearance of the efferent duct. Early oocytes exhibited a diameter ranged from 0.077 mm to 0.682 mm, with an average diameter of 0.343 ± 0.051 mm. The proportions of various types of germ cells within the testes were subjected to analysis. The localization of Vasa during the early stages of sexual differentiation was a subject to analysis as well. Vasa was predominantly localized within the cytoplasm of gonocyte, peri-nucleolus stage oocytes, primary oocytes and type A spermatogonocytes, indicating that Vasa is involved in the early gonadal differentiation of silver pomfret. The study investigated the expression patterns of dmrt1, gsdf, amh, foxl2, cyp19a1a, cyp11a, sox3 and vasa, all of which are involved in the sex differentiation of teleosts. Among these genes, amh, gsdf, sox3, foxl2, vasa were indentified as crucial contributors to the early gonadal development of silver pomfret. Significant sex-related differences were observed in the expression patterns of amh, dmrt1, gsdf, cyp11a, sox3, cyp19a1a, vasa. This study provides novel insights into the timing of physiological changes associated with the sexual differentiation of silver pomfret. Collectively, the present data indicates that the differentiation of ovaries and testes take place approximately at 68 dph in females and 108 dph in males.

Chromosome-Level Assembly of Artemia franciscana Sheds Light on Sex Chromosome Differentiation.

Since the commercialization of brine shrimp (genus Artemia) in the 1950s, this lineage, and in particular the model species Artemia franciscana, has been the subject of extensive research. However, our understanding of the genetic mechanisms underlying various aspects of their reproductive biology, including sex determination, is still lacking. This is partly due to the scarcity of genomic resources for Artemia species and crustaceans in general. Here, we present a chromosome-level genome assembly of A. franciscana (Kellogg 1906), from the Great Salt Lake, United States. The genome is 1 GB, and the majority of the genome (81%) is scaffolded into 21 linkage groups using a previously published high-density linkage map. We performed coverage and FST analyses using male and female genomic and transcriptomic reads to quantify the extent of differentiation between the Z and W chromosomes. Additionally, we quantified the expression levels in male and female heads and gonads and found further evidence for dosage compensation in this species.

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.

hsd17b1 is a key gene for ovarian differentiation of the Siberian sturgeon.

This is the first work using gonads from undifferentiated, genetically-sexed Siberian sturgeon describing expression changes in genes related to steroid synthesis and female and male sex differentiation. One factor identified as relevant for ovarian differentiation was the gene coding for the enzyme Hsd17b1, which converts estrone into estradiol-17ß. hsd17b1 was highly activated in female gonads at 2.5 months of age, around the onset of sex differentiation, preceding activation of two other genes involved in estrogen production (cyp19a1 and foxl2). hsd17b1 was also strongly repressed in males. Two known foxl2 paralogs are found in Siberian sturgeon-foxl2 and foxl2l-but only foxl2 appeared to be associated with ovarian differentiation. With regard to the male pathway, neither 11-oxygenated androgens nor classic male genes (amh, dmrt1, sox9, and dhh) were found to be involved in male sex differentiation, leaving open the question of which genes participate in early male gonad development in this ancient fish. Taken together, these results indicate an estrogen-dependence of female sex differentiation and 11-oxygenated androgen-independence of male sex differentiation.

Epigenetic mechanisms underlying sex differences in the brain and behavior.

Sex differences are found across brain regions, behaviors, and brain diseases. Sexual differentiation of the brain is initiated prenatally but it continues throughout life, as a result of the interaction of three major factors: gonadal hormones, sex chromosomes, and the environment. These factors are thought to act, in part, via epigenetic mechanisms which control chromatin and transcriptional states in brain cells. In this review, we discuss evidence that epigenetic mechanisms underlie sex-specific neurobehavioral changes during critical organizational periods, across the estrous cycle, and in response to diverse environments throughout life. We further identify future directions for the field that will provide novel mechanistic insights into brain sex differences, inform brain disease treatments and women's brain health in particular, and apply to people across genders.

Temperature- and genotype-dependent stress response and activation of the hypothalamus-pituitary-interrenal axis during temperature-induced sex reversal in pejerrey Odontesthes bonariensis, a species with genotypic and environmental sex determination.

In the pejerrey Odontesthes bonariensis (Atheriniformes, Atherinopsidae), exposure to high and low temperatures during the critical period of sex determination (CPSD) induce testicular and ovarian differentiation, respectively, regardless of the presence or not of the sex determining gene amhy, which is crucial for testis formation only at intermediate, sexually neutral temperatures. In this study we explored the existence of genotype-specific signaling of Crh (Corticotropin Releasing Hormone) family genes and their associated carrier protein, receptors, and other stress-related genes in response to temperature during the CPSD and the potential involvement of the central nervous system via the hypothalamus-pituitary-interrenal (HPI) axis in the sex determination of this species. The Crh family genes crhb, uts1, ucn3, the receptor crhr1 and the stress-related genes gr1, gr2, nr3c2 were transiently upregulated in the heads of pejerrey larvae during the CPSD by high temperature alone or in combination with other factors. Only crhr2 transcript abundance was not influenced by temperature but independently by time and genotype. In most cases, mRNA abundance was higher in the XX heads compared to that of XY individuals. The mRNAs of some of these genes were localized in the hypothalamus of pejerrey larvae during the CPSD. XX larvae also showed higher whole-body cortisol titers than the XY, downregulation of cyp19a1a and upregulation of the testis-related genes amhy/amha in trunks (gonads) and were 100% masculinized at the high temperature. In contrast, at the low temperature, crhbp and avt were upregulated in the heads, particularly the former in XY larvae. cyp19a1a and amhy/amha were up- and downregulated, respectively, in the gonads, and fish were 100% feminized. Signaling via the HPI axis was observed simultaneously with the first molecular signs of ongoing sex determination/differentiation in the gonads. Overall, the results strongly suggest a temperature-dependent, genotype-specific regulatory action of the brain involving the Crh family of stress-related genes on the process of environmental sex determination of pejerrey.

Sex-specific transcriptome of the chicken chorioallantoic membrane.

Dimorphism between male and female embryos has been demonstrated in many animal species, including chicken species. Likewise, extraembryonic membranes such as the chorioallantoic membrane (CAM) are likely to exhibit a sex-specific profile. Analysis of the previously published RNA-seq data of the chicken CAM sampled at two incubation times, revealed 783 differentially expressed genes between the CAM of male and female embryos. The expression of some of these genes is sex-dependant only at one or other stage of development, while 415 genes are sex-dependant at both developmental stages. These genes include well-known sex-determining and sex-differentiation genes (DMRT1, HEGM, etc.), and are mainly located on sex chromosomes. This study provides evidence that gene expression of extra-embryonic membranes is differentially regulated between male and female embryos. As such, a better characterisation of associated mechanisms should facilitate the identification of new sex-specific biomarkers.

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.

Histomorphic analysis and expression of mRNA and miRNA in embryonic gonadal differentiation in Chinese soft-shelled turtle (Pelodiscus sinensis).

The Chinese soft-shelled turtle (Pelodiscus sinensis) is extensively cultured in Asia for its nutritional and medical value. Gonadal differentiation is fantastic in turtles, whereas morphologic, mRNA, and miRNA expressions were insufficient in the turtle. In this study, ovaries and testes histomorphology analysis of 14-23 stage embryos were performed, and mRNA and miRNA expression profiles were analyzed. Histomorphology analysis revealed that gonads were undifferentiated at embryonic stage 14. Ovarian morphological differentiation became evident from stage 15, which was characterized by the development of the cortical region and degeneration of the medullary region. Concurrently, testicular morphological differentiation was apparent from stage 15, marked by the development of the medullary region and degeneration of the cortical region. qRT-PCR results showed that Cyp19a1 and Foxl2 exhibited female-specific expression at stage 15 and the expression increased throughout most of the embryonic development. Dmrt1, Amh, and Sox9 displayed male-specific expression at stage 15 and tended to increase substantially at later developmental stages. The expression of miR-8356 and miR-3299 in ZZ gonads were significantly higher than that in ZW gonads at stage 15, 17 and 19, and they had the highest expression at stage 15. While the expression of miR-8085 and miR-7982 had the highest expression at stage 19. Furthermore, chromatin remodeler genes showed differential expression in female and male P. sinensis gonads. These results of master sex-differentiation genes and morphological characteristics would provide a reference for the research of sex differentiation and sex reversal in turtles. Additionally, the expression of chromatin remodeler genes indicated they might be involved in gonadal differentiation of P. sinensis.

CRISPR/Cas9-mediated gene mutation of EcIAG leads to sex reversal in the male ridgetail white prawn Exopalaemon carinicauda.

In the culture of crustaceans, most species show sexual dimorphism. Monosex culture is an effective approach to achieve high yield and economic value, especially for decapods of high value. Previous studies have developed some sex control strategies such as manual segregation, manipulation of male androgenic gland and knockdown of the male sexual differentiation switch gene encoding insulin-like androgenic gland hormone (IAG) in decapods. However, these methods could not generate hereditable changes. Genetic manipulation to achieve sex reversal individuals is absent up to now. In the present study, the gene encoding IAG (EcIAG) was identified in the ridgetail white prawn Exopalaemon carinicauda. Sequence analysis showed that EcIAG encoded conserved amino acid structure like IAGs in other decapod species. CRISPR/Cas9-mediated genome editing technology was used to knock out EcIAG. Two sgRNAs targeting the second exon of EcIAG were designed and microinjected into the prawn zygotes or the embryos at the first cleavage with commercial Cas9 protein. EcIAG in three genetic males was knocked out in both chromosome sets, which successfully generated sex reversal and phenotypic female characters. The results suggest that CRISPR/Cas9-mediated genome editing technology is an effective way to develop sex manipulation technology and contribute to monosex aquaculture in crustaceans.

Alternative splicing of histone demethylase Kdm6bb mediates temperature-induced sex reversal in the Nile tilapia.

Sex determination in many fish species is remarkably plastic and temperature sensitive. Nile tilapia display a genetic sex-determination system (XX/XY). However, high-temperature treatment during critical thermosensitive periods can induce XX females into XXm pseudo-males, and this phenomenon is termed temperature-induced sex reversal (TISR). To investigate the molecular mechanism of TISR in Nile tilapia, we performed Iso-seq analysis and found a dramatic effect of high temperature on gene alternative splicing (AS). Kdm6bb histone demethylase showed a novel AS at intron 5 that generates Kdm6bb_tv1 transcripts without intron 5 and Kdm6bb_tv2 with intron 5. Kdm6bb_tv1 encodes a full-length protein while Kdm6bb_tv2 encodes a truncated protein. Expression analysis revealed that intron 5 splicing of Kdm6bb is male and gonad biased at larval stage, and only gonad biased at adult stage. High-temperature treatment induced intron 5 splicing in the gonads of XX and XY fish, resulting in increased Kdm6bb_tv1 expression. To directly test the role of Kdm6bb_tv1 in Nile tilapia TISR, we knocked out expression of Kdm6bb_tv1. However, Kdm6bb_tv1-/- homozygous mutants showed embryonic lethality. Overexpression of Kdm6bb_tv1, but not Kdm6bb_tv2, induced sex reversal of XX females into pseudo-males. Overexpression of Kdm6bb_tv1, as with high-temperature treatment, modified the promotor region of Gsdf and Dmrt1 by demethylating the trimethylated lysine 27 of histone 3 (H3K27me3), thereby increasing expression. Collectively, these studies demonstrate that AS of Kdm6bb intron 5 increases the expression of Kdm6bb_tv1, which acts as a direct link between high temperature and activation of Gsdf and Dmrt1 expression, leading to male sex determination.

Molecular biological, physiological, cytological, and epigenetic mechanisms of environmental sex differentiation in teleosts: A systematic review.

Human activities have been exerting widespread stress and environmental risks in aquatic ecosystems. Environmental stress, including temperature rise, acidification, hypoxia, light pollution, and crowding, had a considerable negative impact on the life histology of aquatic animals, especially on sex differentiation (SDi) and the resulting sex ratios. Understanding how the sex of fish responds to stressful environments is of great importance for understanding the origin and maintenance of sex, the dynamics of the natural population in the changing world, and the precise application of sex control in aquaculture. This review conducted an exhaustive search of the available literature on the influence of environmental stress (ES) on SDi. Evidence has shown that all types of ES can affect SDi and universally result in an increase in males or masculinization, which has been reported in 100 fish species and 121 cases. Then, this comprehensive review aimed to summarize the molecular biology, physiology, cytology, and epigenetic mechanisms through which ES contributes to male development or masculinization. The relationship between ES and fish SDi from multiple aspects was analyzed, and it was found that environmental sex differentiation (ESDi) is the result of the combined effects of genetic and epigenetic factors, self-physiological regulation, and response to environmental signals, which involves a sophisticated network of various hormones and numerous genes at multiple levels and multiple gradations in bipotential gonads. In both normal male differentiation and ES-induced masculinization, the stress pathway and epigenetic regulation play important roles; however, how they co-regulate SDi is unclear. Evidence suggests that the universal emergence or increase in males in aquatic animals is an adaptation to moderate ES. ES-induced sex reversal should be fully investigated in more fish species and extensively in the wild. The potential aquaculture applications and difficulties associated with ESDi have also been addressed. Finally, the knowledge gaps in the ESDi are presented, which will guide the priorities of future research.

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.

Waterborne Tebuconazole Exposure Induces Male-Biased Sex Differentiation in Zebrafish (Danio rerio) Larvae via Aromatase Inhibition.

Tebuconazole is a widely used fungicide for various crops that targets sterol 14-α-demethylase (CYP51) in fungi. However, attention has shifted to aromatase (CYP19) due to limited research indicating its reproductive impact on aquatic organisms. Herein, zebrafish were exposed to 0.5 mg/L tebuconazole at different developmental stages. The proportion of males increased significantly after long-term exposure during the sex differentiation phase (0-60, 5-60, and 19-60 days postfertilization (dpf)). Testosterone levels increased and 17ß-estradiol and cyp19a1a expression levels decreased during the 5-60 dpf exposure, while the sex ratio was equally distributed on coexposure with 50 ng/L 17ß-estradiol. Chemically activated luciferase gene expression bioassays determined that the male-biased sex differentiation was not caused by tebuconazole directly binding to sex hormone receptors. Protein expression and phosphorylation levels were specifically altered in the vascular endothelial growth factor signaling pathway despite excluding the possibility of tebuconazole directly interacting with kinases. Aromatase was selected for potential target analysis. Molecular docking and aromatase activity assays demonstrated the interactions between tebuconazole and aromatase, highlighting that tebuconazole poses a threat to fish populations by inducing a gender imbalance.

Morphological and transcriptional analysis of sexual differentiation and gonadal development in a burrowing fish, the four-eyed sleeper (Bostrychus sinensis).

Four-eyed sleeper (Bostrychus sinensis) is a commercially important sea water fish, and the male individuals exhibit significant advantages in somatic growth and stress resistance, so developing sex control strategy to create all-male progeny will produce higher economic value. However, little is known about the genetic background associated with sex differentiation in this species. In this study, we investigated gonadal development and uncovered critical window stages of sexual differentiation (about 2 mph), transition from proliferation to differentiation in female germ stem cells (GSCs) (2-3 mph) and male GSCs (3-4 mph). De novo transcriptome analysis revealed candidate genes and signaling pathways associated with sexual differentiation and gonadal development in four-eyed sleeper. The results showed that sox9 and zglp1 were the earliest sex-biased transcription factors during sex differentiation. Down-regulation of chemokine, cytokines-cytokine receptors and up-regulation of cellular senescence pathway might be involved in GSC differentiation. Weighted gene correlation network analysis showed that metabolic pathway and occludin were the hub signaling and gene in ovarian development, meanwhile the MAPK signaling pathways, cellular senescence pathway and ash1l (histone H3-lysine4 N-trimethyltransferase) were the hub pathways and gene in testicular development. The present work elucidated the developmental processes of sexual differentiation and gonadal development and revealed their associated revealed genes and signaling pathways in four-eyed sleeper, providing theoretical basis for developing sex-control techniques.