MiRNA profiling in male and female chicken embryonic gonads during sexual differentiation
ABSTRACT: MicroRNAs (miRNAs) are a highly conserved class of small RNAs which function in a sequence-specific manner to post-transcriptionally regulate expression of target genes. Tissue-specific miRNA expression studies have discovered numerous functions for miRNAs in various aspects of embryonic development, but a role for miRNAs in gonadal development and sex differentiation has not yet been reported. Using the chicken embryo as a vertebrate model, differential miRNA expression between male and female embryonic gonads, was analysed at three developmental stages (embryonic days (E) 5.5, E6.5 and E9.5), using custom-designed 4x2K CombiMatrix miRNA microarray. The aims of this study were to: 1-identify miRNAs differentialy expressed by sex; 2-identify sex-specific miRNAs; 3-analyse global changes in miRNA up-regulation in male versus female gonads before, during and after the histological onset of sexual differentiation. This study provides a basis for establishing whetehr miRNAs are involved in either initiating or regulating vertebrate gonadal sex differentiation. Keywords: miRNA, sex comparison, developmental stage comparison. Overall design: miRNA samples from male and female embryonic chicken gonads from three developmental stages: embryonic day (E) 5.5 (Hamilton & Hamburger (HH) stage 27-28), E6.5 (HH stage 29-30) & E9.5 (HH stage 35-36). Samples are listed with biological replicates used for analysis in brackets following: 1 - Male E5.5 (5); 2 - Female E5.5 (4); 3 - Male E6.5 (5); 4 - Female E6.5 (3); 5 - Male E9.5 (4); 6 - Female E9.5 (4).
Project description:MicroRNAs (miRNAs) are a highly conserved class of small RNAs which function in a sequence-specific manner to post-transcriptionally regulate expression of target genes. Tissue-specific miRNA expression studies have discovered numerous functions for miRNAs in various aspects of embryonic development, but a role for miRNAs in gonadal development and sex differentiation has not yet been reported. Using the chicken embryo as a vertebrate model, differential miRNA expression between male and female embryonic gonads, was analysed at three developmental stages (embryonic days (E) 5.5, E6.5 and E9.5), using custom-designed 4x2K CombiMatrix miRNA microarray. The aims of this study were to: 1-identify miRNAs differentialy expressed by sex; 2-identify sex-specific miRNAs; 3-analyse global changes in miRNA up-regulation in male versus female gonads before, during and after the histological onset of sexual differentiation. This study provides a basis for establishing whetehr miRNAs are involved in either initiating or regulating vertebrate gonadal sex differentiation. Keywords: miRNA, sex comparison, developmental stage comparison. miRNA samples from male and female embryonic chicken gonads from three developmental stages: embryonic day (E) 5.5 (Hamilton & Hamburger (HH) stage 27-28), E6.5 (HH stage 29-30) & E9.5 (HH stage 35-36). Samples are listed with biological replicates used for analysis in brackets following: 1 - Male E5.5 (5); 2 - Female E5.5 (4); 3 - Male E6.5 (5); 4 - Female E6.5 (3); 5 - Male E9.5 (4); 6 - Female E9.5 (4).
Project description:MicroRNAs are a highly conserved class of small RNAs that function in a sequence-specific manner to posttranscriptionally regulate gene expression. Tissue-specific miRNA expression studies have discovered numerous functions for miRNAs in various aspects of embryogenesis, but a role for miRNAs in gonadal development and sex differentiation has not yet been reported. Using the chicken embryo as a model, microarrays were used to profile the expression of chicken miRNAs prior to, during, and after the time of gonadal sex differentiation (Embryonic Day 5.5 [E5.5], E6.5, and E9.5). Sexually dimorphic miRNAs were identified, and the expression patterns of several were subjected to further validation by in situ hybridization and Northern blot analysis. Expression of one chicken miRNA, MIR202*, was observed to be sexually dimorphic, with upregulation in the developing testis from the onset of sexual differentiation. Additional data from deep sequencing of male and female embryonic gonad RNA samples also indicated upregulation of MIR202* in male gonads. These findings provide the first evidence of sexually dimorphic miRNA expression during vertebrate gonadal sex differentiation and suggest that MIR202* may function in regulating testicular development.
Project description:MiRNAs bear an increasing number of functions throughout development and in the aging adult. Here we address their role in establishing sexually dimorphic traits and sexual identity in male and female Drosophila. Our survey of miRNA populations in each sex identifies sets of miRNAs differentially expressed in male and female tissues across various stages of development. The pervasive sex-biased expression of miRNAs generally increases with the complexity and sexual dimorphism of tissues, gonads revealing the most striking biases. We find that the male-specific regulation of the X chromosome is relevant to miRNA expression on two levels. First, in the male gonad, testis-biased miRNAs tend to reside on the X chromosome. Second, in the soma, X-linked miRNAs do not systematically rely on dosage compensation. We set out to address the importance of a sex-biased expression of miRNAs in establishing sexually dimorphic traits. Our study of the conserved let-7-C miRNA cluster controlled by the sex-biased hormone ecdysone places let-7 as a primary modulator of the sex-determination hierarchy. Flies with modified let-7 levels present doublesex-related phenotypes and express sex-determination genes normally restricted to the opposite sex. In testes and ovaries, alterations of the ecdysone-induced let-7 result in aberrant gonadal somatic cell behavior and non-cell-autonomous defects in early germline differentiation. Gonadal defects as well as aberrant expression of sex-determination genes persist in aging adults under hormonal control. Together, our findings place ecdysone and let-7 as modulators of a somatic systemic signal that helps establish and sustain sexual identity in males and females and differentiation in gonads. This work establishes the foundation for a role of miRNAs in sexual dimorphism and demonstrates that similar to vertebrate hormonal control of cellular sexual identity exists in Drosophila.
Project description:The marine medaka (Oryzias melastigma) has been increasingly used as a fish model for detecting environmental stresses and chemical contaminants in the marine environment. Recent mammalian studies have shown that environmental stresses can alter the expression profiles of microRNAs (miRNAs), leading to transgenerational effects. Here, we use high-throughput Illumina RNA sequencing (RNA-Seq) for miRNA transcriptome analysis of brain, liver, and gonads from sexually mature male and female marine medaka. A total of 128,883,806 filtered sequence reads were generated from six small RNA libraries, identifying a total of 2,125,663 non-redundant sequences. These sequences were aligned and annotated to known animal miRNAs (miRBase) using the BLAST method. A total of 223 distinct miRNA types were identified, with the greatest number expressed in brain tissue. Our data suggested that 55 miRNA types from 34 families are common to all tested tissues, while some of the miRNAs are tissue-enriched or sex-enriched. Quantitative real-time PCR analysis further demonstrated that let-7a, miR-122, and miR-9-3p were downregulated in hypoxic female medaka, while miR-2184 was specifically upregulated in the testis of hypoxic male fish. This is the first study to identify miRNAs in O. melastigma using small RNA deep sequencing technology. Because miRNA expression is highly conserved between marine medaka and other vertebrates, marine medaka may serve as a good model for studies on the functional roles of miRNAs in hypoxia stress response and signaling in marine fish.
Project description:DMRT1 encodes a conserved transcription factor with an essential role in gonadal function. In the chicken, DMRT1 in located on the Z sex chromosome and is currently the best candidate master regulator of avian gonadal sex differentiation. We previously showed that knockdown of DMRT1 expression during the period of sexual differentiation induces feminisation of male embryonic chicken gonads. This gene is therefore necessary for proper testis development in the chicken. However, whether it is sufficient to induce testicular differentiation has remained unresolved. We show here that over-expression of DMRT1 induces male pathway genes and antagonises the female pathway in embryonic chicken gonads. Ectopic DMRT1 expression in female gonads induces localised SOX9 and AMH expression. It also induces expression of the recently identified Z-linked male factor, Hemogen (HEMGN). Masculinised gonads show evidence of cord-like structures and retarded female-type cortical development. Furthermore, expression of the critical feminising enzyme, aromatase, is reduced in the presence of over-expressed DMRT1. These data indicate that DMRT1 is an essential sex-linked regulator of gonadal differentiation in avians, and that it likely acts via a dosage mechanism established through the lack of global Z dosage compensation in birds.
Project description:Estrogens play a key role in sexual differentiation of both the gonads and external traits in birds. The production of estrogen occurs via a well-characterised steroidogenic pathway, which is a multi-step process involving several enzymes, including cytochrome P450 aromatase. In chicken embryos, the aromatase gene (CYP19A1) is expressed female-specifically from the time of gonadal sex differentiation. To further explore the role of aromatase in sex determination, we ectopically delivered this enzyme using the retroviral vector RCASBP in ovo. Aromatase overexpression in male chicken embryos induced gonadal sex-reversal characterised by an enlargement of the left gonad and development of ovarian structures such as a thickened outer cortex and medulla with lacunae. In addition, the expression of key male gonad developmental genes (DMRT1, SOX9 and Anti-Müllerian hormone (AMH)) was suppressed, and the distribution of germ cells in sex-reversed males followed the female pattern. The detection of SCP3 protein in late stage sex-reversed male embryonic gonads indicated that these genetically male germ cells had entered meiosis, a process that normally only occurs in female embryonic germ cells. This work shows for the first time that the addition of aromatase into a developing male embryo is sufficient to direct ovarian development, suggesting that male gonads have the complete capacity to develop as ovaries if provided with aromatase.
Project description:In mammals, the supporting cell lineage in an embryonic gonad communicates the sex-determining decision to various sexually dimorphic cell types in the developing embryo, including the germ cells. However, the molecular nature of the sex-determining signals that pass from the supporting cells to the germ cells is not well understood. We have identified a conserved transmembrane protein, Sdmg1, owing to its male-specific expression in mouse embryonic gonads. Sdmg1 is expressed in the Sertoli cells of embryonic testes from 12.5 dpc, and in granulosa cells of growing follicles in adult ovaries. In Sertoli cells, Sdmg1 is localised to endosomes, and knock-down of Sdmg1 in Sertoli cell lines causes mis-localisation of the secretory SNARE Stx2 and defects in membrane trafficking. Upregulation of Sdmg1 appears to be part of a larger programme of changes to membrane trafficking pathways in embryonic Sertoli cells, and perturbing secretion in male embryonic gonads in organ culture causes male-to-female germ cell sex reversal. These data suggest that changes that occur in the cell biology of embryonic Sertoli cells may facilitate the communication of male sex-determining decisions to the germ cells during embryonic development.
Project description:Sex of birds is genetically determined through inheritance of the ZW sex chromosomes (ZZ males and ZW females). Although the mechanisms of avian sex determination remains unknown, the genetic sex is experimentally reversible by in ovo exposure to exogenous estrogens (ZZ-male feminization) or aromatase inhibitors (ZW-female masculinization). Expression of various testis- and ovary-specific marker genes during the normal and reversed gonadal sex differentiation in chicken embryos has been extensively studied, but the roles of sex-specific epigenetic marks in sex differentiation are unknown. In this study, we show that a 170-nt region in the promoter of CYP19A1/aromatase, a key gene required for ovarian estrogen biosynthesis and feminization of chicken embryonic gonads, contains highly quantitative, nucleotide base-level epigenetic marks that reflect phenotypic gonadal sex differentiation. We developed a protocol to feminize ZZ-male chicken embryonic gonads in a highly quantitative manner by direct injection of emulsified ethynylestradiol into yolk at various developmental stages. Taking advantage of this experimental sex reversal model, we show that the epigenetic sex marks in the CYP19A1/aromatase promoter involving DNA methylation and histone lysine methylation are feminized significantly but only partially in sex-converted gonads even when morphological and transcriptional marks of sex differentiation show complete feminization, being indistinguishable from gonads of normal ZW females. Our study suggests that the epigenetic sex of chicken embryonic gonads is more stable than the morphologically or transcriptionally characterized sex differentiation, suggesting the importance of the nucleotide base-level epigenetic sex in gonadal sex differentiation.
Project description:Cytochrome P450 Family 19 SubFamily A member 1 (CYP19A1) gene encodes an aromatase which regulates the sexual differentiation in vertebrates by initiating and maintaining 17?-Estradiol (E2) synthesis. Here, we described the spatiotemporal expression pattern of CYP19A1 and its functional role in the embryonic gonad development in amphoteric chickens (Gallus gallus). Results showed that CYP19A1 exhibited a sexually dimorphic expression pattern in female gonads early at embryonic day 5.5 (HH 28) and robustly expressed within the cytoplasm in ovarian medullas. Most importantly, we induced the gonadal sex reversal by ectopically delivering the aromatase inhibitor (AI) or estradiol (E2) into chicken embryos. To further explore the role of CYP19A1 in chicken embryonic sexual differentiation, we successfully developed an effective method to deliver lentiviral particles with CYP19A1 manipulation into chicken embryos via embryonic intravascular injection. The analysis of interference and overexpression of CYP19A1 provided solid evidences that CYP19A1 is both necessary and sufficient to initiate sex differentiation toward female in chicken embryos. Collectively, this work demonstrates that CYP19A1 is a crucial sex differentiation gene in the embryonic development, which provides a foundation for understanding the mechanism of sex determination and differentiation in chickens.
Project description:In vertebrates, the primary sex-determining signals that initiate sexual development are remarkably diverse, ranging from complete genetic to environmental cues. However, no sex determination-related genes have been functionally identified in reptiles. Here, we characterized a conserved DM domain gene, Dmrt1, in Chinese soft-shelled turtle Pelodiscus sinensis (P. sinensis), which exhibits ZZ/ZW sex chromosomes. Dmrt1 exhibited early male-specific embryonic expression, preceding the onset of gonadal sex differentiation. The expression of Dmrt1 was induced in ZW embryonic gonads that were masculinized by aromatase inhibitor treatment. Dmrt1 knockdown in ZZ embryos by RNA interference resulted in male to female sex reversal, characterized by obvious feminization of gonads, significant down-regulation of testicular markers Amh and Sox9, and remarkable up-regulation of ovarian regulators, Cyp19a1 and Foxl2. Conversely, ectopic expression of Dmrt1 led to largely masculinized genetic females, production of Amh and Sox9, and a decline in Cyp19a1 and Foxl2. These findings demonstrate that Dmrt1 is both necessary and sufficient to initiate testicular development, thereby acting as an upstream regulator of the male pathway in P. sinensis.