Transcriptome analysis of four types of gonadal tissues in largemouth bass (Micropterus salmoides) to reveal its sex-related genes.

The sex determination system of largemouth bass (Micropterus salmoides, LMB) is XX/XY; however, the underlying molecular mechanisms involved in early sex differentiation, gonadal development, and exogenous hormone-induced sex reversal remain unknown. In this study, LMB at 15 days post-hatching (dph) were fed diets containing 20 mg/kg of 17α-methyltestosterone (17α-MT) or 30 mg/kg of 17ß-estradiol (17ß-E2) for 60 days, respectively. Serum steroid levels, histological observations of the gonads, and identification of sex-specific markers were employed to screen the gonads of 60-day-old normal female fish (XX-F), normal male fish (XY-M), 17ß-E2 induced pseudo-female fish (XY-F), and 17α-MT-induced pseudo-male fish (XX-M) for transcriptome sequencing in order to uncover genes and pathway involved in the process of sexual reversal. The results from histology and serum sex steroid hormone analysis showed that both 17α-MT and 17ß-E2 were capable of inducing sex reversal of LMB at 15 dph. Transcriptome results revealed a total of 2,753 genes exhibiting differential expression, and the expression pattern of these genes in the gonads of XX-M or XY-F resembled that of normal females or males. The male sex-biased genes that are upregulated in XX-M and downregulated in XY-F are referred to as key genes for male reversal, while the female sex-biased genes that are upregulated in XY-F and downregulated in XX-M are referred to as key genes for female reversal. Finally, 12 differentially expressed genes (DEGs) related to male sex reversal were screened, including star2, cyp17a, cyp11b1, dmrt1, amh, sox9a, katnal1, spata4, spata6l, spata7, spata18 and foxl3. 2 DEGs (foxl2a and cyp19a1b) were found to be associated with female sex reversal. The changes in these genes collectively influence the direction of sex differentiation of LMB. Among them, star2, dmrt1 and cyp19a1b with significantly altered expression levels may play potentially crucial role in the process of gender reversal. The expression patterns of 21 randomly selected genes were verified using qRT-PCR which confirmed the reliability and accuracy of the RNA-seq results. These findings not only enhance our understanding of the molecular basis underlying sex reversal but also provide crucial data support for future breeding research on unisexual LMB.

Overexpression of Kdm6b induces testicular differentiation in a temperature-dependent sex determination system.

In reptiles, such as the red-eared slider turtle ( Trachemys scripta elegans), gonadal sex determination is highly dependent on the environmental temperature during embryonic stages. This complex process, which leads to differentiation into either testes or ovaries, is governed by the finely tuned expression of upstream genes, notably the testis-promoting gene Dmrt1 and the ovary-promoting gene Foxl2. Recent studies have identified epigenetic regulation as a crucial factor in testis development, with the H3K27me3 demethylase KDM6B being essential for Dmrt1 expression in T. s. elegans. However, whether KDM6B alone can induce testicular differentiation remains unclear. In this study, we found that overexpression of Kdm6b in T. s. elegans embryos induced the male development pathway, accompanied by a rapid increase in the gonadal expression of Dmrt1 at 31°C, a temperature typically resulting in female development. Notably, this sex reversal could be entirely rescued by Dmrt1 knockdown. These findings demonstrate that Kdm6b is sufficient for commitment to the male pathway, underscoring its role as a critical epigenetic regulator in the sex determination of the red-eared slider turtle.

Adamts9 is required for the development of primary ovarian follicles and maintenance of female sex in zebrafish.

Previous studies have suggested that adamts9 (a disintegrin and metalloprotease with thrombospondin type-1 motifs, member 9), an extracellular matrix (ECM) metalloprotease, participates in primordial germ cell (PGC) migration and is necessary for female fertility. In this study, we found that adamts9 knockout (KO) led to reduced body size, and female-to-male sex conversion in late juvenile or adult zebrafish; however, primary sex determination was not affected in early juveniles of adamts9 KO. Overfeeding and lowering the rearing density rescued growth defects in female adamts9 KO fish but did not rescue defects in ovarian development in adamts9 KO. Delayed PGC proliferation, significantly reduced number and size of Stage IB follicles (equivalent to primary follicles) in early juveniles of adamts9 KO, and arrested development at Stage IB follicles in mid- or late-juveniles of adamts9 KO are likely causes of female infertility and sex conversion. Via RNAseq, we found significant enrichment of differentially expressed genes involved in ECM organization during sexual maturation in ovaries of wildtype fish; and significant dysregulation of these genes in adamts9 KO ovaries. RNAseq analysis also showed enrichment of inflammatory transcriptomic signatures in adult ovaries of these adamts9 KO. Taken together, our results indicate that adamts9 is critical for development of primary ovarian follicles and maintenance of female sex, and loss of adamts9 leads to defects in ovarian follicle development, female infertility, and sex conversion in late juveniles and mature adults. These results show that the ECM and extracellular metalloproteases play major roles in maintaining ovarian follicle development in zebrafish.

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

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

[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.

CNV Analysis through Exome Sequencing Reveals a Large Duplication Involved in Sex Reversal, Neurodevelopmental Delay, Epilepsy and Optic Atrophy.

BACKGROUND: Duplications on the short arm of chromosome X, including the gene NR0B1, have been associated with gonadal dysgenesis and with male to female sex reversal. Additional clinical manifestations can be observed in the affected patients, depending on the duplicated genomic region. Here we report one of the largest duplications on chromosome X, in a Lebanese patient, and we provide the first comprehensive review of duplications in this genomic region. CASE PRESENTATION: A 2-year-old female patient born to non-consanguineous Lebanese parents, with a family history of one miscarriage, is included in this study. The patient presents with sex reversal, dysmorphic features, optic atrophy, epilepsy, psychomotor and neurodevelopmental delay. Single nucleotide variants and copy number variants analysis were carried out on the patient through exome sequencing (ES). This showed an increased coverage of a genomic region of around 23.6 Mb on chromosome Xp22.31-p21.2 (g.7137718-30739112) in the patient, suggestive of a large duplication encompassing more than 60 genes, including the NR0B1 gene involved in sex reversal. A karyotype analysis confirmed sex reversal in the proband presenting with the duplication, and revealed a balanced translocation between the short arms of chromosomes X and 14:46, X, t(X;14) (p11;p11) in her/his mother. CONCLUSIONS: This case highlights the added value of CNV analysis from ES data in the genetic diagnosis of patients. It also underscores the challenges encountered in announcing unsolicited incidental findings to the family.

5 mC modification of steroid hormone biosynthesis-related genes orchestrates feminization of channel catfish induced by high-temperature.

To elucidate the mechanism behind channel catfish feminization induced by high temperature, gonad samples were collected from XY pseudo-females and wild-type females and subjected to high-throughput sequencing for Whole-Genome-Bisulfite-Seq (WGBS) and transcriptome sequencing (RNA-Seq). The analysis revealed 50 differentially methylated genes between wild-type females and XY pseudo-females, identified through the analysis of KEGG pathways and GO enrichment in the promoter of the genome and differentially methylated regions (DMRs). Among these genes, multiple differential methylation sites observed within the srd5a2 gene. Repeatability tests confirmed 7 differential methylation sites in the srd5a2 gene in XY pseudo-females compared to normal males, with 1 specific differential methylation site (16608174) distinguishing XY pseudo-females from normal females. Interestingly, the expression of these genes in the transcriptome showed no difference between wild-type females and XY pseudo-females. Our study concluded that methylation of the srd5a2 gene sequence leads to decreased expression, which inhibits testosterone synthesis while promoting the synthesis of 17ß-estradiol from testosterone. This underscores the significance of the srd5a2 gene in the sexual differentiation of channel catfish, as indicated by the ipu00140 KEGG pathway analysis.

Molecular characterization and expression analysis of thyroid hormone receptors in protogynous rice field eel, Monopterus albus.

Thyroid hormones (THs) play important roles in growth, development, morphogenesis, reproduction, and so on. They are mainly meditated by binding to thyroid hormone receptors (TRs) in vertebrates. As important members of the nuclear receptor superfamily, TRs and their ligands are involved in many biological processes. To investigate the potential roles of TRs in the gonadal differentiation and sex change, we cloned and characterized the TRs genes in protogynous rice field eel (Monopterus albus). In this study, three types of TRs were obtained, which were TRαA, TRαB and TRß, encoding preproproteins of 336-, 409- and 415-amino acids, respectively. Multiple alignments of the three putative TRs protein sequences showed they had a higher similarity. Tissue expression analysis showed that TRαA mainly expressed in the gonad, while TRαB and TRß in the brain. During female-to-male sex reversal, the expression levels of all the three TRs showed a similar trend of increase followed by a decrease in the gonad. Intraperitoneal injection of triiodothyronine (T3) stimulated the expression of TRαA and TRαB, while it had no significant change on the expression of TRß in the ovary. Gonadotropin-releasing hormone analogue (GnRHa) injection also significantly upregulated the expression levels of TRαA and TRαB after 6 h, while it had no significant effect on TRß. These results demonstrated that TRs were involved in the gonadal differentiation and sex reversal, and TRα may play more important roles than TRß in reproduction by the regulation of GnRHa in rice field eel.

Molecular Approaches Detect Early Signals of Programmed Cell Death in Hippolyte inermis Leach.

The protandric shrimp Hippolyte inermis is the only known marine invertebrate whose sex determination is strongly influenced by the composition of its food. In H. inermis, a sex reversal is triggered by the ingestion of diatoms of the genus Cocconeis associated with leaves of the seagrass Posidonia oceanica. These diatoms contain compounds that promote programmed cell death (PCD) in H. inermis and also in human cancer cells. Transcriptomic analyses suggested that ferroptosis is the primary trigger of the shrimp's sex reversal, leading to the rapid destruction of the androgen gland (AG) followed by a chain of apoptotic events transforming the testes into ovaries. Here, we propose a molecular approach to detect the effects of compounds stimulating the PCD. An RNA extraction method, suitable for young shrimp post-larvae (five days after metamorphosis; PL5 stage), was established. In addition, six genes involved in apoptosis, four involved in ferroptosis, and seven involved in the AG switch were mined from the transcriptome, and their expression levels were followed using real-time qPCR in PL5 fed on Cocconeis spp., compared to PL5 fed on a basic control feed. Our molecular approach, which detected early signals of sex reversal, represents a powerful instrument for investigating physiological progression and patterns of PCD in marine invertebrates. It exemplifies the physiological changes that may start a few days after the settlement of post-larvae and determine the life destiny of an individual.

Gsdf is not indispensable for male differentiation in the medaka species Oryzias hubbsi.

Sex determination mechanisms differ widely among vertebrates, particularly in fish species, where diverse sex chromosomes and sex-determining genes have evolved. However, the sex-differentiation pathways activated by these sex-determining genes appear to be conserved. Gonadal soma-derived growth factor (Gsdf) is one of the genes conserved across teleost fish, especially in medaka fishes of the genus Oryzias, and is implicated in testis differentiation and germ cell proliferation. However, its role in sex differentiation remains unclear. In this study, we investigated Gsdf function in Oryzias hubbsi, a species with a ZW sex-determination system. We confirmed its male-dominant expression, as in other species. However, histological analyses revealed no male-to-female sex reversal in Gsdf-knockout fish, contrary to findings in other medaka species. Genetic sex determination remained intact without Gsdf function, indicating a Gsdf-independent sex-differentiation pathway in O. hubbsi. Instead, Gsdf loss led to germ cell overproliferation in both sexes and accelerated onset of meiosis in testes, suggesting a role in germ cell proliferation. Notably, the feminizing effect of germ cells observed in O. latipes was absent, suggesting diverse germ cell-somatic cell relationships in Oryzias gonad development. Our study highlights species-specific variations in the molecular pathways governing sex determination and differentiation, emphasizing the need for further exploration to elucidate the complexities of sexual development.

DNA Methylation Mediates Sperm Quality via piwil1 and piwil2 Regulation in Japanese Flounder (Paralichthys olivaceus).

DNA methylation is an important way to regulate gene expression in eukaryotes. In order to reveal the role of DNA methylation in the regulation of germ cell-specific piwi gene expression during spermatogenesis of Japanese flounder (Paralichthys olivaceus), the expression profiles of piwil1 (piwi-like 1) and piwil2 (piwi-like 2) genes in the gonads of female, male, and sex-reversed pseudo-male P. olivaceus were analyzed, and the dynamic of DNA methylation was investigated. As a result, piwil1 and piwil2 genes were highly expressed in the testis of both male and pseudo-male P. olivaceus, with significant variation among male individuals. The DNA methylation levels in the promoter regions of both piwil1 and piwil2 were negatively correlated with their expression levels, which may contribute to the transcriptional regulation of piwi genes during spermatogenesis. There was also sperm quality variation among male P. olivaceus, and the sperm curvilinear velocity was positively correlated with the expression of both piwil1 and piwil2 genes. These results indicated that the DNA methylation in piwil1 and piwil2 promoter regions may affect the initiation of piwi gene transcription, thereby regulating gene expression and further affecting the spermatogenesis process and gamete quality in P. olivaceus.

Molecular and Physiological Effects of 17α-methyltestosterone on Sex Differentiation of Black Rockfish, Sebastes schlegelii.

It is widely known that all-female fish production holds economic value for aquaculture. Sebastes schlegelii, a preeminent economic species, exhibits a sex dimorphism, with females surpassing males in growth. In this regard, achieving all-female black rockfish production could significantly enhance breeding profitability. In this study, we utilized the widely used male sex-regulating hormone, 17α-methyltestosterone (MT) at three different concentrations (20, 40, and 60 ppm), to produce pseudomales of S. schlegelii for subsequent all-female offspring breeding. Long-term MT administration severely inhibits the growth of S. schlegelii, while short term had no significant impact. Histological analysis confirmed sex reversal at all MT concentrations; however, both medium and higher MT concentrations impaired testis development. MT also influenced sex steroid hormone levels in pseudomales, suppressing E2 while increasing T and 11-KT levels. In addition, a transcriptome analysis revealed that MT down-regulated ovarian-related genes (cyp19a1a and foxl2) while up-regulating male-related genes (amh) in pseudomales. Furthermore, MT modulated the TGF-ß signaling and steroid hormone biosynthesis pathways, indicating its crucial role in S. schlegelii sex differentiation. Therefore, the current study provides a method for achieving sexual reversal using MT in S. schlegelii and offers an initial insight into the underlying mechanism of sexual reversal in this species.

Molecular cytogenetic screening of sex chromosome abnormalities in young horse populations.

BACKGROUND: Chromosomal abnormalities occur in the equine population at a rate of approximately 2%. The use of molecular cytogenetic techniques allows a more accurate identification of chromosomal abnormalities, especially those with a low rate of abnormal metaphases, demonstrating that the actual incidence in equine populations is higher. OBJECTIVES: Estimation of the number of carriers of karyotypic abnormalities in a sample from a population of young horses of various breeds, using molecular cytogenetic techniques. STUDY DESIGN: Cross-sectional. METHODS: Venous blood samples were collected from 500 young horses representing 5 breeds (Purebred Arabian, Hucul, Polish primitive horse [Konik], Malopolska, Coldblood, Silesian). Chromosomes and DNA were obtained from blood lymphocytes and evaluated by fluorescence in situ hybridisation (FISH) and PCR, using probes and markers for the sex chromosomes and select autosomes. RESULTS: Nineteen horses, 18 mares and 1 stallion, were diagnosed with different chromosomal abnormalities: 17 cases of mosaic forms of sex chromosome aneuploidies with a very low incidence (0.6%-4.7%), one case of a SRY-negative 64,XY sex reversal mare, and one mare with X-autosome translocation. The percentage of sex chromosomal aberrations was established as 3.8% in the whole population, 6.08% in females and 0.49% in males. MAIN LIMITATIONS: Limited sample size, confined to horses from Poland. CONCLUSIONS: The rate of sex chromosomal abnormalities we identified was almost double that reported in previous population studies that used classical chromosome staining techniques. FISH allowed the detection of aneuploid cell lines which had a very low incidence. The FISH technique is a faster and more precise method for karyotype examination; however, it is usually focused on only one or two chromosomes while banding karyotyping includes the entire chromosome set.

Integrative analysis of miRNA-mRNA expression in the brain during high temperature-induced masculinization of female Nile tilapia (Oreochromis niloticus).

Temperature is one of the most important non-genetic sex differentiation factors for fish. The technique of high temperature-induced sex reversal is commonly used in Nile tilapia (Oreochromis niloticus) culture, although the molecular regulatory mechanisms involved in this process remain unclear. The brain is an essential organ for the regulation of neural signals involved in germ cell differentiation and gonad development. To investigate the regulatory roles of miRNAs-mRNAs in the conversion of female to male Nile tilapia gender under high-temperature stress, we compared RNA-Seq data from brain tissues between a control group (28 °C) and a high temperature-treated group (36 °C). The result showed that a total of 123,432,984 miRNA valid reads, 288,202,524 mRNA clean reads, 1128 miRNAs, and 32,918 mRNAs were obtained. Among them, there were 222 significant differentially expressed miRNAs (DE miRNAs) and 810 differentially expressed mRNAs (DE mRNAs) between the two groups. Eight DE miRNAs and eight DE mRNAs were randomly selected, and their expression patterns were validated by qRT-PCR. The miRNA-mRNA co-expression network demonstrated that 40 DE miRNAs targeted 136 protein-coding genes. Functional enrichment analysis demonstrated that these genes were involved in several gonadal differentiation pathways, including the oocyte meiosis signaling pathway, progesterone-mediated oocyte maturation signaling pathway, cell cycle signaling pathway and GnRH signaling pathway. Then, an interaction network was constructed for 8 miRNAs (mir-137-5p, let-7d, mir-1388-5p, mir-124-4-5p, mir-1306, mir-99, mir-130b and mir-21) and 10 mRNAs (smc1al, itpr2, mapk1, ints8, cpeb1b, bub1, fbxo5, mmp14b, cdk1 and hrasb) involved in the oocyte meiosis signaling pathway. These findings provide novel information about the mechanisms underlying miRNA-mediated sex reversal in female Nile tilapia.

Case report: A severe clinical phenotype of pontocerebellar hypoplasia type 7 with compound heterozygous variants of TOE1.

Pontocerebellar Hypoplasia (PCH) is a rare autosomal recessive hereditary neurological degenerative disease. To elaborate upon the clinical phenotypes of PCH and explore the correlation between TOE1 gene mutations and clinical phenotype, we analyze the clinical and genetic features of a Chinese infant afflicted with pontocerebellar dysplasia accompanied by gender reversal with bioinformatics methods. The main clinical features of this infant with TOE1 gene mutation included progressive lateral ventricle widening, hydrocephalus, severe postnatal growth retardation, and hypotonia, and simultaneously being accompanied by 46, XY female sex reversal. Whole exome sequencing revealed a compound heterozygous mutation in the TOE1 gene (c.299T > G, c.1414T > G), with the protein homology modeling-generated structure predicting a pathogenic variation, which is closely related to the clinical manifestations in the patient. The new mutation sites, c.299T > G and c.1414T > G, in the TOE1 gene are pathogenic variants of pontocerebellar hypoplasia type 7.

Inefficient Sox9 upregulation and absence of Rspo1 repression lead to sex reversal in the B6.XYTIR mouse gonad†.

Sry on the Y-chromosome upregulates Sox9, which in turn upregulates a set of genes such as Fgf9 to initiate testicular differentiation in the XY gonad. In the absence of Sry expression, genes such as Rspo1, Foxl2, and Runx1 support ovarian differentiation in the XX gonad. These two pathways antagonize each other to ensure the development of only one gonadal sex in normal development. In the B6.YTIR mouse, carrying the YTIR-chromosome on the B6 genetic background, Sry is expressed in a comparable manner with that in the B6.XY mouse, yet, only ovaries or ovotestes develop. We asked how testicular and ovarian differentiation pathways interact to determine the gonadal sex in the B6.YTIR mouse. Our results showed that (1) transcript levels of Sox9 were much lower than in B6.XY gonads while those of Rspo1 and Runx1 were as high as B6.XX gonads at 11.5 and 12.5 days postcoitum. (2) FOXL2-positive cells appeared in mosaic with SOX9-positive cells at 12.5 days postcoitum. (3) SOX9-positive cells formed testis cords in the central area while those disappeared to leave only FOXL2-positive cells in the poles or the entire area at 13.5 days postcoitum. (4) No difference was found at transcript levels of all genes between the left and right gonads up to 12.5 days postcoitum, although ovotestes developed much more frequently on the left than the right at 13.5 days postcoitum. These results suggest that inefficient Sox9 upregulation and the absence of Rspo1 repression prevent testicular differentiation in the B6.YTIR gonad.

Characterization of Two Gonadal Genes, zar1 and wt1b, in Hermaphroditic Fish Asian Seabass (Lates calcarifer).

Zygote arrest-1 (Zar1) and Wilms' tumor 1 (Wt1) play an important role in oogenesis, with the latter also involved in testicular development and gender differentiation. Here, Lczar1 and Lcwt1b were identified in Asian seabass (Lates calcarifer), a hermaphrodite fish, as the valuable model for studying sex differentiation. The cloned cDNA fragments of Lczar1 were 1192 bp, encoding 336 amino acids, and contained a zinc-binding domain, while those of Lcwt1b cDNA were 1521 bp, encoding a peptide of 423 amino acids with a Zn finger domain belonging to Wt1b family. RT-qPCR analysis showed that Lczar1 mRNA was exclusively expressed in the ovary, while Lcwt1b mRNA was majorly expressed in the gonads in a higher amount in the testis than in the ovary. In situ hybridization results showed that Lczar1 mRNA was mainly concentrated in oogonia and oocytes at early stages in the ovary, but were undetectable in the testis. Lcwt1b mRNA was localized not only in gonadal somatic cells (the testis and ovary), but also in female and male germ cells in the early developmental stages, such as those of previtellogenic oocytes, spermatogonia, spermatocytes and spermatids. These results indicated that Lczar1 and Lcwt1b possibly play roles in gonadal development. Therefore, the findings of this study will provide a basis for clarifying the mechanism of Lczar1 and Lcwt1b in regulating germ cell development and the sex reversal of Asian seabass and even other hermaphroditic species.

The role of DNA methylation reprogramming during sex determination and sex reversal in the Pacific oyster Crassostrea gigas.

DNA methylation is instrumental in vertebrate sex reversal. However, the mechanism of DNA methylation regulation regarding sex reversal in invertebrates is unclear. In this study, we used whole genome bisulfite sequencing (WGBS) to map single-base resolution methylation profiles of the Pacific oyster, including female-to-male (FMa-to-FMb) and male-to-female (MFa-to-MFb) sex reversal, as well as sex non-reversed males and females (MMa-to-MMb and FFa-to-FFb). The results showed that global DNA methylation levels increase during female-to-male sex reversals, with a particular increase in the proportion of high methylation levels (mCGs >0.75) and a decrease in the proportion of intermediate methylation levels (0.25 < mCGs <0.75). This increase in DNA methylation was mainly associated with the elevated expression of DNA methylase genes. Genome-wide methylation patterns of females were accurately remodeled to those of males after sex reversal, while the opposite was true for the male-to-female reversal. Those findings directly indicate that alterations in DNA methylation play a significant role in sex reversal in Pacific oysters. Comparative analysis of the DNA methylomes of pre- and post- sex reversal gonadal tissues (FMb-vs-FMa or MFb-vs-MFa) revealed that differentially methylated genes were mainly involved in the biological processes of sex determination or gonadal development. However critical genes such as Dmrt1, Foxl2 and Sox-like, which are involved in the putative sex determination pathway in Pacific oysters, showed almost an absence of methylation modifications, varying greatly from vertebrates. Additionally, comparative analysis of the DNA methylomes of sexual reversal and sex non-reversal (FMa-vs-FFa or MFa-vs-MMa) revealed that heat shock protein genes, such as Hsp68-like and Hsp70B, were important for the occurrence of sex reversal. These findings shed light on the epigenetic mechanisms underlying the maintenance of gonadal plasticity and the reversal of organ architecture in oysters.

Bidirectional sex-change behavior and physiological aspects in the Gorgeous goby Lythrypnus pulchellus (Gobiidae).

The Gorgeous goby Lythrypnus pulchellus shows extreme sexual plasticity with the bidirectional sex-change ability socially controlled in adults. Therefore, this study describes how the hierarchical status affects hormone synthesis through newborn hormone waste products in water and tests the influence of body size and social dominance establishment in sex reversal duration and direction. The associated changes in behavior and hormone levels are described under laboratory conditions in male-male and female-female pairs of similar and different body sizes, recording the changes until spawning. The status establishment occurred in a relatively shorter time period in male and female pairs of different sizes (1-3 days) compared to those of similar size (3-5 days), but the earlier one did not significantly affect the overall time of sex change (verified by pair spawning). The changes in gonads, hormones, and papilla occurred in sex-changer individuals, but the first one was observed in behavior. Courtship started at 3-5 days in male pairs and from 2 h to 1 day in female pairs of both groups of different and similar sizes. Hormones did not gradually move in the new sexual phenotype direction during the sex-change time course. Nonetheless, estradiol regulated sex change and 11-ketotestosterone enabled bidirectional sex change and was modulated by agonistic interactions. Cortisol is associated with status and gonadal sex change. In general, similar mechanisms underlie sex change in both directions with a temporal change sequence in phases. These results shed new light on sex-change mechanisms. Further studies should be performed to determine whether these localized changes exist in the steroid hormone synthesis along the brain-pituitary gonad axis during social and bidirectional sex changes in L. pulchellus.

Has recombination changed during the recent evolution of the guppy Y chromosome?

Genome sequencing and genetic mapping of molecular markers have demonstrated nearly complete Y-linkage across much of the guppy (Poecilia reticulata) XY chromosome pair. Predominant Y-linkage of factors controlling visible male-specific coloration traits also suggested that these polymorphisms are sexually antagonistic (SA). However, occasional exchanges with the X are detected, and recombination patterns also appear to differ between natural guppy populations, suggesting ongoing evolution of recombination suppression under selection created by partially sex-linked SA polymorphisms. We used molecular markers to directly estimate genetic maps in sires from 4 guppy populations. The maps are very similar, suggesting that their crossover patterns have not recently changed. Our maps are consistent with population genomic results showing that variants within the terminal 5 Mb of the 26.5 Mb sex chromosome, chromosome 12, are most clearly associated with the maleness factor, albeit incompletely. We also confirmed occasional crossovers proximal to the male-determining region, defining a second, rarely recombining, pseudo-autosomal region, PAR2. This fish species may therefore have no completely male-specific region (MSY) more extensive than the male-determining factor. The positions of the few crossover events suggest a location for the male-determining factor within a physically small repetitive region. A sex-reversed XX male had few crossovers in PAR2, suggesting that this region's low crossover rate depends on the phenotypic, not the genetic, sex. Thus, rare individuals whose phenotypic and genetic sexes differ, and/or occasional PAR2 crossovers in males can explain the failure to detect fully Y-linked variants.