Metzincin metalloproteases in PGC migration and gonadal sex conversion.

Development of a functional gonad includes migration of primordial germ cells (PGCs), differentiations of somatic and germ cells, formation of primary follicles or spermatogenic cysts with somatic gonadal cells, development and maturation of gametes, and subsequent releasing of mature germ cells. These processes require extensive cellular and tissue remodeling, as well as broad alterations of the surrounding extracellular matrix (ECM). Metalloproteases, including MMPs (matrix metalloproteases), ADAMs (a disintegrin and metalloproteinases), and ADAMTS (a disintegrin and metalloproteinase with thrombospondin motifs), are suggested to have critical roles in the remodeling of the ECM during gonad development. However, few research articles and reviews are available on the functions and mechanisms of metalloproteases in remodeling gonadal ECM, gonadal development, or gonadal differentiation. Moreover, most studies focused on the roles of transcription and growth factors in early gonad development and primary sex determination, leaving a significant knowledge gap on how differentially expressed metalloproteases exert effects on the ECM, cell migration, development, and survival of germ cells during the development and differentiation of ovaries or testes. We will review gonad development with focus on the evidence of metalloprotease involvements, and with an emphasis on zebrafish as a model for studying gonadal sex differentiation and metalloprotease functions.

Role of Sox3 in Estradiol-Induced Sex Reversal in Pelodiscus sinensis.

The Chinese soft-shelled turtle Pelodiscus sinensis, an economically important species in China, exhibits significant sexual dimorphism. Males are more valuable than females owing to their wider calipash and faster growth. Estradiol (E2)-induced sex reversal is used to achieve all-male breeding of turtles; however, the mechanism of this sex reversal remains unclear. In this study, we characterized the Sox3 gene, whose expression level was high in the gonads and brain and exhibited significant sexual dimorphism in the ovary. During embryonic development, Sox3 was highly expressed at the initiation of ovarian differentiation. E2 and Sox3-RNAi treatment before sexual differentiation led to 1352, 908, 990, 1011, and 975 differentially expressed genes in five developmental stages, respectively, compared with only E2 treatment. The differentially expressed genes were clustered into 20 classes. The continuously downregulated and upregulated genes during gonadal differentiation were categorized into Class 0 (n = 271) and Class 19 (n = 606), respectively. KEGG enrichment analysis showed that Sox3 significantly affected sexual differentiation via the Wnt, TGF-ß, and TNF signaling pathways and mRNA surveillance pathway. The expression of genes involved in these signaling pathways, such as Dkk4, Nog, Msi1, and Krt14, changed significantly during gonadal differentiation. In conclusion, the deletion of Sox3 may lead to significant upregulation of the mRNA surveillance pathway and TNF and Ras signaling pathways and downregulation of the Wnt and TGF-ß signaling pathways, inhibiting E2-induced sex reversal. These findings suggest that Sox3 may play a certain promoting effect during E2-induced sex reversal in P. sinensis.

Circular RNA expression profiles and CircSnd1-miR-135b/c-foxl2 axis analysis in gonadal differentiation of protogynous hermaphroditic ricefield eel Monopterus albus.

BACKGROUND: The expression and biological functions of circular RNAs (circRNAs) in reproductive organs have been extensively reported. However, it is still unclear whether circRNAs are involved in sex change. To this end, RNA sequencing (RNA-seq) was performed in gonads at 5 sexual stages (ovary, early intersexual stage gonad, middle intersexual stage gonad, late intersexual stage gonad, and testis) of ricefield eel, and the expression profiles and potential functions of circRNAs were studied. RESULTS: Seven hundred twenty-one circRNAs were identified, and the expression levels of 10 circRNAs were verified by quantitative real-time PCR (qRT-PCR) and found to be in accordance with the RNA-seq data, suggesting that the RNA-seq data were reliable. Then, the sequence length, category, sequence composition and the relationship between the parent genes of the circRNAs were explored. A total of 147 circRNAs were differentially expressed in the sex change process, and GO and KEGG analyses revealed that some differentially expressed (such as novel_circ_0000659, novel_circ_0004005 and novel_circ_0005865) circRNAs were closely involved in sex change. Furthermore, expression pattern analysis demonstrated that both circSnd1 and foxl2 were downregulated in the process of sex change, which was contrary to mal-miR-135b. Finally, dual-luciferase reporter assay and RNA immunoprecipitation showed that circSnd1 and foxl2 can combine with mal-miR-135b and mal-miR-135c. These data revealed that circSnd1 regulates foxl2 expression in the sex change of ricefield eel by acting as a sponge of mal-miR-135b/c. CONCLUSION: Our results are the first to demonstrate that circRNAs have potential effects on sex change in ricefield eel; and circSnd1 could regulate foxl2 expression in the sex change of ricefield eel by acting as a sponge of mal-miR-135b/c. These data will be useful for enhancing our understanding of sequential hermaphroditism and sex change in ricefield eel or other teleosts.

Loss of Nobox prevents ovarian differentiation from juvenile ovaries in zebrafish.

As a species without master sex-determining genes, zebrafish displays high plasticity in sex differentiation, making it an excellent model for studying the regulatory mechanisms underlying gonadal differentiation and gametogenesis. Despite being a gonochorist, zebrafish is a juvenile hermaphrodite that undergoes a special phase of juvenile ovary before further differentiation into functional testis and ovary. The mechanisms underlying juvenile ovary formation and subsequent gonadal differentiation remain largely unknown. In this study, we explored the role of Nobox/nobox (new born ovary homeobox protein), another oocyte-specific transcription factor in females, in early zebrafish gonadogenesis using CRISPR/Cas9 technology. As in mammals, nobox is specifically expressed in zebrafish gonads with a dimorphic pattern at juvenile stage. In contrast to the mutant of figla (factor in the germline alpha, another oocyte-specific transcription factor), the nobox mutants showed formation of typical perinucleolar (PN) follicles at primary growth (PG) stage in juvenile gonads, suggesting occurrence of follicle assembly from cystic oocytes (chromatin nucleolar stage, CN). These follicles, however, failed to develop further to form functional ovaries, resulting in all-male phenotype. Despite its expression in adult testis, the loss of nobox did not seem to affect testis development, spermatogenesis and male spawning. In summary, our results indicate an important role for Nobox in zebrafish ovarian differentiation and early folliculogenesis.

Effects of endocrine disrupting chemicals on gonad development: Mechanistic insights from fish and mammals.

Over the past century, evidence has emerged that endocrine disrupting chemicals (EDCs) have an impact on reproductive health. An increased frequency of reproductive disorders has been observed worldwide in both wildlife and humans that is correlated with accidental exposures to EDCs and their increased production. Epidemiological and experimental studies have highlighted the consequences of early exposures and the existence of key windows of sensitivity during development. Such early in life exposures can have an immediate impact on gonadal and reproductive tract development, as well as on long-term reproductive health in both males and females. Traditionally, EDCs were thought to exert their effects by modifying the endocrine pathways controlling reproduction. Advances in knowledge of the mechanisms regulating sex determination, differentiation and gonadal development in fish and rodents have led to a better understanding of the molecular mechanisms underlying the effects of early exposure to EDCs on reproduction. In this manuscript, we review the key developmental stages sensitive to EDCs and the state of knowledge on the mechanisms by which model EDCs affect these processes, based on the roadmap of gonad development specific to fish and mammals.

Timing of early gonadal differentiation and effects of estradiol-17ß treatments on the sex differentiation in Largemouth bass (Micropterus salmoides).

In this study, an efficient estradiol-17ß (E2)-induced feminization method was established based on the timing of early gonadal differentiation in Largemouth bass (Micropterus salmoides). Histological section results showed that from 20 days post-hatch (dph) to 30 dph, the germ cells gradually differentiated into oogonium and spermatic deferent, respectively. Moreover, female-biased genes Foxl2 and Cyp19a1a were up-regulated to the first peak at 20 dph, while the male-biased genes Dmrt1 were up-regulated to the first peak at 30 dph. These results indicated that the timing of early gonadal differentiation in Largemouth bass was between 20 and 30 dph. Therefore, 15 dph Largemouth bass with a body length of 15.10 ± 0.09 mm were chosen, and four E2-treated diets were set as 0 (E0, control), 50 mg/kg E2 (E50), 100 mg/kg E2 (E100), and 200 mg/kg E2 (E200). After feeding with E2-treated diets for 60 days, female ratios were 55%, 100%, 100%, and 100% in E0, E50, E100, and E200 groups, respectively. No intersex fish were observed in all the groups. However, 30% of females in the E200 group possessed thinner ovaries, with smaller ovary cavity structures and a decreased number of primary oocyte cells than those in other groups. Besides, the Largemouth bass in the E0 group grew more than those in E50, E100, and E200 groups during the E2 treatments period (P < 0.05). In conclusion, our study suggested that 50-100 mg/kg E2-treated diets could effectively induce the feminization of 15 dph Largemouth bass within 60 days duration time, which provided valuable information for the breeding of the all-male Largemouth bass population.

Molecular characterization and functional analysis of cyp11a and cyp11b in black rockfish (Sebastes schlegelii).

The cyp11 includes cyp11a and cyp11b in most mammals and teleosts, encoded cholesterol side chain lyase and 11ß-hydroxylase, respectively. It is essential in steroid hormone synthesis. However, studies on the regulation of cyp11 are limited, especially in teleosts. In this study, the molecular characterization and function of cyp11a and cyp11b of black rockfish was investigated. Both of them showed high homology with other teleost counterparts by phylogenetic analysis. The expression of cyp11a and cyp11b exhibited a clear sexually dimorphic pattern, with a higher expression level in testis than that of in ovaries. During the different developmental stages (40 dpf, 80 dpf, 190 dpf, 360 dpf, 720 dpf), the expression of cyp11a was earlier than cyp11b. In situ hybridization results showed that cyp11a and cyp11b were mainly expressed in oogonia and oocytes of the ovary. They were located in spermatogonia and interstitial compartment in the 1.5-year-old gonads, and spermatocytesgonia and the peritubular myoid cell of the testis in the 2.5-year-old gonads. To explore the distinct roles of cyp11a and cyp11b in gonads, oestrogen and androgens were used to stimulate the primary testicular and ovarian cells. The expressions of cyp11a and cyp11b were tested under different dose of 17α-methyltestosterone (17α-MT) and 17ß-estradiol (E2). The results showed cyp11a was significantly increased at 10-6  mol ml-1 of 17α-MT and 10-8  mol ml-1 of E2 in ovary and 10-10  mol ml-1 of 17α-MT and E2 in testis, while cyp11b was significantly decreased after 17α-MT and E2 treatment. These results indicated that cyp11a and cyp11b were likely to have different functions, and also implied they might play an important roles in the differentiation of gonads and the synthesis of steroids in black rockfish.

Analyses of mRNA-seq and miRNA-seq of the brain reveal the sex differences of gene expression and regulation before and during gonadal differentiation in 17ß-estradiol or 17α-methyltestosterone-induced olive flounder (Paralichthys olivaceus).

Olive flounder (Paralichthys olivaceus) is a commercially important flatfish species cultured in East Asia. Female flounders generally grow more rapidly than males, therefore control of the sex ratio seems to be a proposed way to increase production. However, the sex determination gene and sex determination mechanism have yet been elucidated. The brain is an important organ that is involved in gonadal development. To explore the sex differences of gene expression in the brain before and during the flounder gonadal differentiation, we used messenger RNA (mRNA)-seq technology to investigate transcriptomes of male and female brains. Between female and male brains, 103 genes were differentially expressed before ovarian differentiation, 16 genes were differentially expressed before testicular differentiation, and 64 genes were differentially expressed during gonadal differentiation. According to annotation and Kyoto Encyclopedia of Genes and Genomes information, the differentially expressed genes (DEGs) were involved in circadian rhythm, circadian rhythm-fly, circadian entrainment, dopaminergic synapse, calcium signaling, glutamatergic synapse, taste transduction, herpes simplex infection, long-term depression, retrograde endocannabinoid signaling, and the synaptic vesicle cycle pathways. MicroRNA (miRNA)-seq was performed during the gonadal differentiation and the target genes of miRNAs were predicted. Integrated analysis of mRNA-seq and miRNA-seq showed that 29 of the 64 DEGs were regulated by the differentially expressed miRNAs during the gonadal differentiation. Our study provides a basis for further studies of brain sex differentiation and the molecular mechanism of sex determination in olive flounder.

Tissues injury and pathological changes in Hyla intermedia juveniles after chronic larval exposure to tebuconazole.

Tebuconazole (TBZ), an azole pesticide, is one of the most frequently detected fungicides in surface water. Despite its harmful effects, mainly related to endocrine disturbance, the consequences of TBZ exposure in amphibians remain poorly understood. Here, we investigated the adverse and delayed effects of TBZ chronic exposure on a native anuran species, often inhabiting cultivated areas, the Italian tree frog (Hyla intermedia). To disclose the multiple mechanisms of action through which TBZ exerts its toxicity we exposed tadpoles over the whole larval period to two sublethal TBZ concentrations (5 and 50 µg/L), and we evaluated histological alterations in three target organs highly susceptible to xenobiotics: liver, kidney, and gonads. We also assessed morphometric and gravimetric parameters: snout-vent length (SVL), body mass (BM), liver somatic index (LSI), and gonad-mesonephros complex index (GMCI) and determined sex ratio, gonadal development, and differentiation. Our results show that TBZ induces irreversible effects on multiple target organs in H. intermedia, exerting its harmful effects through several pathological pathways, including a massive inflammatory response. Moreover, TBZ markedly affects sexual differentiation also by inducing the appearance of sexually undetermined individuals and a general delay of germ cell maturation. Given the paucity of data on the effects of TBZ in amphibians, our results will contribute to a better understanding of the environmental risk posed by this fungicide to the most endangered group of vertebrates.

Active feminization of the preoptic area occurs independently of the gonads in Amphiprion ocellaris.

Sex differences in the anatomy and physiology of the vertebrate preoptic area (POA) arise during development, and influence sex-specific reproductive functions later in life. Relative to masculinization, mechanisms for feminization of the POA are not well understood. The purpose of this study was to induce sex change from male to female in the anemonefish Amphiprion ocellaris, and track the timing of changes in POA cytoarchitecture, composition of the gonads and circulating sex steroid levels. Reproductive males were paired together and then sampled after 3 weeks, 6 months, 1 year and 3 years. Results show that as males change sex into females, number of medium cells in the anterior POA (parvocellular region) approximately double to female levels over the course of several months to 1 year. Feminization of gonads, and plasma sex steroids occur independently, on a variable timescale, up to years after POA sex change has completed. Findings suggest the process of POA feminization is orchestrated by factors originating from within the brain as opposed to being cued from the gonads, consistent with the dominant hypothesis in mammals. Anemonefish provide an opportunity to explore active mechanisms responsible for female brain development in an individual with male gonads and circulating sex steroid levels.

Activation of the brain-pituitary-gonadotropic axis in the black porgy Acanthopagrus schlegelii during gonadal differentiation and testis development and effect of estradiol treatment.

Previous studies revealed an estradiol (E2)-dependent peak in brain activity, including neurosteroidogenesis and neurogenesis in the black porgy during the gonadal differentiation period. The brain-pituitary-gonadotropic axis is a key regulator of reproduction and may also be involved in gonadal differentiation, but its activity and potential role in black porgy during the gonadal differentiation period is still unknown. The present study analyzed the expression of regulatory factors involved in the gonadotropic axis at the time of gonadal differentiation (90, 120, 150 days after hatching [dah]) and subsequent testicular development (180, 210, 300 dah). In agreement with previous studies, expression of brain aromatase cyp19a1b peaked at 120 dah, and this was followed by a gradual increase during testicular development. The expression of gonadotropin subunits increased slightly but not significantly during gonadal differentiation and then increased significantly at 300 dah. In contrast, the expression of brain gnrh1 and pituitary gnrh receptor 1 (gnrhr1) exhibited a pattern with two peaks, the first at 120 dah, during the period of gonadal differentiation, and the second peak during testicular development. Gonad fshr and lhcgr increased during gonadal differentiation period with highest transcript level in prespawning season during testicular development. This suggests that the early activation of brain gnrh1, pituitary gnrhr1 and gths, and gonad gthrs might be involved in the control of gonadal differentiation. E2 treatment increased brain cyp19a1b expression at each sampling time, in agreement with previous studies in black porgy and other teleosts. E2 also significantly stimulated the expression of pituitary gonadotropin subunits at all sampling times, indicating potential E2-mediated steroid feedback. In contrast, no significant effect of E2 was observed on gnrh1. Moreover, treatment of AI or E2 had no statistically significant effect on brain gnrh1 transcription levels during gonadal differentiation. This indicated that the early peak of gnrh1 expression during the gonadal differentiation period is E2-independent and therefore not directly related to the E2-dependent peak in brain neurosteroidogenesis and neurogenesis also occurring during this period in black porgy. Both E2-independent and E2-dependent mechanisms are thus involved in the peak expression of various genes in the brain of black porgy at the time of gonadal differentiation.

Alternative splicing of GnRH2 and GnRH2-associated peptide plays roles in gonadal differentiation of the rice field eel, Monopterus albus.

The rice field eel, Monopterus albus, is a protogynous hermaphrodite fish, in which the gonads are initially female ovaries which then transform into male testes. The exact mechanisms governing sex reversal in the rice field eel are unknown. In this study, a novel alternative splicing variant of GnRH2 (GnRH2-SV), retaining the second intron, was discovered in the gonad of the rice field eel. Compared to GnRH2, GnRH2-SV may give rise to a novel truncated GnRH2-associated peptide (New GAP2). The normal transcript of GnRH2 was primarily expressed in the brain, and could also be detected in the liver, spleen, ovary, and testis. However, GnRH2-SV was only expressed in the ovary and testis. During sex reversal, GnRH2 expression levels increased significantly at late stages; however, expression levels of GnRH2-SV were lower in ovary than in ovotestis and testis. We also examined the effect of three peptides (GnRHa, GAP2, and New GAP2) on gonadal sex differentiation during the third stage of ovarian development of the rice field eel. Compared to the control group, the expression of amh increased significantly following incubation with each of the three peptides. However, only New GAP2 stimulated the expression of sox9a1 mRNA in vitro. After intraperitoneal injection of GAP2, the expression of amh, foxl2, and cyp19a1a increased significantly after 12 h; the concentration of serum 11-KT was also significantly increased at the 12 h time point. Treatment with New GAP2 significantly increased the expression of amh, dmrt1a, and sox9a1, and also increased the concentration of serum 11-KT. After treated with GnRHa, the expression of amh, dmrt1a, sox9a1, cyp19a1a, and foxl2 increased significantly, as did the level of serum E2. These results indicated that both GAP2 and New GAP2 play a crucial role in inducing expression changes of sex-differentiation related genes, and may be involved in the gonadal development and sex reversal in the rice field eel.

Influence of DEHP on thyroid, sex steroid-related genes and gonadal differentiation in Rana chensinensis tadpoles.

In the present study, responses of the Chinese brown frog (Rana chensinensis) to exposure to di-2-ethylhexyl phthalate (DEHP), a common plasticizer, during the larval period were characterized. The effects of DEHP on metamorphosis rate, thyroid hormone, thyroid histology and the expression of genes involved in the steroid hormone synthesis in gonad were investigated. Metamorphosis rate and 50 percent of the tadpoles to reach Gosner stage 42 (T0.5 ) were significantly slower in all DEHP groups. The thyroid glands of the tadpoles exposed to DEHP clearly exhibited colloid depletion. In addition, decreased concentrations of T4 and T3 were observed in the tadpoles exposed to DEHP. Moreover, the highest DEHP exposure (10 µmol/L DEHP) showed increased ratio of females significantly. Also, up-regulation significantly of transcripts of cytochrome P450 aromatase (CYP19) gene was detected in male tadpoles exposed to DEHP. The present results indicate that this increase in estrogens could lead to female-biased sex ratio in DEHP exposure group. Taken together, the present study indicates that DEHP disrupt thyroid hormone and sex steroid signaling in R. chensinensis tadpoles. Our present observations support evidence of a crosstalk between TH and sex steroids in gonad differentiation.

Expression analysis of growth differentiation factor 9 (Gdf9/gdf9), anti-müllerian hormone (Amh/amh) and aromatase (Cyp19a1a/cyp19a1a) during gonadal differentiation of the zebrafish, Danio rerio.

In the zebrafish, no sex-determining gene has been identified, while some sex-related genes, such as cyp19a1a and amh, show sexually dimorphic expression. Interestingly, most of these genes are expressed in the somatic cells. With increasing evidence suggesting roles of germ cells in gonadal differentiation, there is an increasing interest in the factors released by the germ cells for the bidirectional communication between the two compartments. We have reported that Gdf9/gdf9 is an oocyte-specific factor in the zebrafish, similar to that of mammals. Whether and how Gdf9 is involved in gonadal differentiation is unknown. In this study, we compared the expression levels of gdf9, cyp19a1a, and amh among several other sex-related genes in the gonads before, during, and after sex differentiation. The expression of gdf9 started in the gonads before sex differentiation, and its level surged in the differentiated ovary. Its expression pattern was similar to that of cyp19a1a, but reciprocal to amh expression. Using recombinant zebrafish Gdf9 (rzfGdf9), we further showed that Gdf9 significantly suppressed the expression of amh while increased that of activin beta subunits (inhbaa and inhbb) in vitro. Although gdf9 and cyp19a1a showed co-expression during gonadal differentiation, we only observed a slight but not significant response of cyp19a1a to rzfGdf9. Knocking down the expression of gdf9 and cyp19a1a with vivo-morpholinos caused a male-skewed sex ratio. Our data suggested that Gdf9 is likely involved in promoting oocyte/ovary differentiation in the zebrafish and it may act by suppressing amh expression, at least partly, in the somatic cells.

Significant association of cyp19a promoter methylation with environmental factors and gonadal differentiation in olive flounder Paralichthys olivaceus.

The sex ratio of olive flounder Paralichthys olivaceus is sensitive to temperature or exogenous hormone exposures in the period of gonadal differentiation. Among sex-related genes, cyp19a, encoding cytochrome P450 aromatase, exhibits significant sex-dimorphic expression pattern and plays an important role in fish gonadal differentiation and development. The present study investigated the expression levels and promoter methylation dynamics of cyp19a and its regulators (nr5a2 and nr0b1), and sex-steroid hormone levels during flounder gonadal differentiation under the treatments of high temperature and estradiol-17ß (E2). The results showed that levels of flounder cyp19a expression and estradiol-17ß were repressed by high temperature treatment during this period. The up-regulation of nr5a2 by E2 treatment may be related to the all-female formation, and up-regulation of nr0b1 by high temperature treatment may be associated with masculinization. Co-transfection assay indicated that nr5a2 and nr0b1 were antagonist regulators of cyp19a. Furthermore, cyp19a promoter exhibited significant demethylation phenomenon at early stage of ovarian differentiation. While, high temperature could repress the demethylation process, resulting in hypermethylation maintenance in cyp19a promoter. The hypermethylation promoter was able to suppress cyp19a expression by blocking the nr5a2-mediated transactivation activity in vitro. The DNA methylation of epigenetic modification in cyp19a promoter might be the vital way linking environmental factors and gonadal differentiation in flounder.

Are sex steroids essential for gonadal differentiation of the ornate frog, Microhyla ornata?

Knowledge about sensitivities and responses of amphibian larvae to sex steroids and the chemicals alike is the first step towards understanding and assessing the effect of diverse chemicals that interfere with gonadal development and other endocrine functions. Herein, we used Microhyla ornata to determine the role of sex steroids on its gonad differentiation and sex ratio. Our results show that the exposure to increasing concentrations of estradiol-17ß throughout larval development did not affect gonad differentiation resulting in 1:1 sex ratio at metamorphosis. But, females emerging from estradiol-17ß treatment had larger ovaries with larger sized follicles. Further, testes of some males contained lumens, the number of which was dose dependent. Similarly, exposure to testosterone propionate had negligible effects on gonad differentiation. However, the mean diameter of the largest follicles was smaller in treated ovaries. Treatment of tadpoles with tamoxifen had no effect on gonad differentiation and ovary development while testicular development was accelerated at the highest concentration. Similarly, treatment of tadpoles with cyproterone acetate had little effect on gonad differentiation as well as development, hence the sex ratios at the end of metamorphosis. Further, in tadpoles exposed to increasing concentrations of formestane, gonad differentiation was normal resulting in 1:1 sex ratio. Thus, in M. ornata, both estradiol and testosterone are essential for the development of ovaries and testes respectively but, they are not critical to gonadal differentiation. Hence, the effects of sex steroids and other endocrine disrupting chemicals could be species-specific; different species may have differential sensitivities to such chemicals.

Molecular cloning and sexually dimorphic expression of wnt4 in olive flounder (Paralichthys olivaceus).

WNT4 (wingless-type MMTV integration site family, member 4) is regarded as a key regulator of gonad differentiation in mammalians. However, the potential role of wnt4 in teleosts during gonad differentiation and development is still unclear. The full-length cDNA sequence of wnt4 in olive flounder (Paralichthys olivaceus) was obtained using RACE (rapid amplification of cDNA ends) technique. The wnt4 ORF contains 1059 nucleotides, encoding a protein with a signal peptide domain and a wnt family domain. Expression in tissues of adult flounders was analyzed by real-time RT-PCR. The results showed that wnt4 was widely expressed in multiple tissues of flounders, and the expression level was significantly higher in ovary than in testis. Then wnt4 expression pattern was investigated during gonadal differentiation period and at gonadal development stages (I-V). The results showed the expression levels were significantly higher in testis than in ovary during gonadal differentiation. Notably, wnt4 expression had a very significant increase before testis differentiation. At gonad different developmental stages, there was no expression signal at stage I or stage II, and the expression of wnt4 was much stronger in ovary than in testis at stage III and stage IV, followed by a faint expression in stage V in both sexes. Our results imply that cloned wnt4 could be wnt4a. It is a sex-related gene and its expression pattern in gonadal differentiation period of flounder is different from that in mammalians or other teleosts. Flounder wnt4 might play more important role in testis than in ovary during gonadal differentiation.

Long-term exposures to di-n-butyl phthalate inhibit body growth and impair gonad development in juvenile Murray rainbowfish (Melanotaenia fluviatilis).

The aim of the present study was to evaluate whether long-term exposures to environmentally relevant concentrations of di-n-butyl phthalate (DnBP) disrupt the reproduction-based endpoints in juvenile Murray rainbowfish (Melanotaenia fluviatilis). Fish were exposed to 5, 15 or 50 µg l(-1) DnBP for 30, 60 and 90 days each, and the effects on survival, body growth, whole-body concentrations of sex steroid hormones and gonadal development were investigated. The lowest observed effective concentration to affect the condition factor after 90 days was 5 µg l(-1). Complete feminization of the gonad was noted in fish exposed to 5 µg l(-1) for 90 days and to 15 and 50 µg l(-1) of DnBP for 30 or 60 days. After 90 days of exposure to DnBP, the ovaries were regressed and immature as opposed to the control fish which were in early-vitellogenic stage. Testes, present only in fish exposed to 5 µg l(-1) of DnBP for 30 or 60 days, were immature in comparison to the control fish that contained testes in the mid-spermatogenic phase. The E2/11-KT ratio was significantly higher only after exposures to 5 µg l(-1) DnBP for 90 days and 50 µg l(-1) DnBP for 30 days. Our data suggest that exposures to 5 µg l(-1) DnBP for 30 days did not have profound effects on body growth and gonadal differentiation of fish. However, 30 days of exposure to 15 µg l(-1) could interfere with the gonad development and to 50 µg l(-1) could compromise the hormonal profile of juvenile fish.

Genome-wide identification and expression profiles of sex-related gene families in the Pacific abalone Haliotis discus hannai.

In recent years, members of the Dmrt family, TGF-ß superfamily and Sox family have been recognized as crucial genes for sex determination/differentiation across diverse animal species. Nevertheless, knowledge regarding the abundance and potential functions of these genes in abalone remains limited. In this study, a total of 5, 10, and 7 members of the Dmrt family, the TGF-ß superfamily and the Sox family, respectively, were identified in the Pacific abalone Haliotis discus hannai. Sequence characteristics, phylogenetic relationships and spatiotemporal expression profiles of these genes were investigated. Notably, HdDmrt-04 (Dmrt1/1L-like) emerged as a potential mollusc-specific gene with a preponderance for expression in the testis. Interestingly, none of the TGF-ß superfamily members exhibited specific or elevated expression in the gonads, highlighting the need for further investigation into their role in abalone sex differentiation. The Sox proteins in H. discus hannai were categorized into 7 subfamilies: B1, B2, C, D, E, F, and H. Among them, HdSox-07 (SoxH-like) was observed to play a crucial role in testis development, while HdSox-03 (SoxB1-like) and HdSox-04 (SoxC-like) probably cooperate in abalone ovary development. Taken together, the results of the present study suggested that HdDmrt-04 and HdSox-07 can be used as male-specific markers for gonad differentiation in H. discus hannai and imply conservation of their functions across invertebrates and vertebrates. Our findings provide new insights into the evolution and genetic structure of the Dmrt family, the TGF-ß superfamily and the Sox family in abalone and pave the way for a deeper understanding of sex differentiation in gastropods.

[Estrogen receptor Esr1 induces early ovarian differentiation in Trachemys scripta].

This study aims to explore the roles of three estrogen receptors (Esr1, Esr2, and Gper1) in early differentiation of embryonic gonads of Trachemys scripta. The expression characteristics of the receptor genes were studied first. The Esr1, Esr2, and Gper1 agonists PPT, WAY 200070, and G-1 were respectively injected into the embryos at the male-producing temperature (MPT) before initiation of gonadal differentiation. The sex reversal of the treated embryonic gonads was analyzed in terms of morphological structure of gonads, distribution pattern of germ cells, and expression of key genes and proteins involved in sex differentiation. The expression level of esr1 during the critical stage of sex differentiation was higher than those of esr2 and gper1 (very low expression) and was particularly high in the gonads at the female-producing temperature (FPT). After treatment with PPT, the MPT gonads presented obviously feminized morphology and structure, with the germ cells exhibiting a female distribution pattern. Furthermore, the mRNA expression levels of the key genes (dmrt1, amh, and sox9) for male differentiation were down-regulated significantly, while those of the key genes (foxl2 and cyp19a1) for female differentiation were up-regulated observably. The fluorescent signals of Amh and Sox9 expression almost disappeared, while Foxl2 and Arom were activated to express abundantly, which fully demonstrated the sex reversal of the gonads from male to female (sex reversal rate: 70.27%). However, the MPT gonads treated with WAY 200070 and G-1 still differentiated into testes, and the expression patterns of the key genes and proteins were similar to those in male gonads. The above results demonstrate that activation of Esr1 alone can fully initiate the early female differentiation process of gonads, suggesting that estrogen may induce early ovarian differentiation via Esr1 in Trachemys scripta. The findings provide a basis for further revealing the mechanisms of estrogen regulation in sex determination and differentiation of turtles.