Molecular cloning and characterization of amh,dax1 and cyp19a1a genes and their response to 17α-methyltestosterone in Pengze crucian carp

The proteins encoded by amh, dax1 and cyp19a1a play important roles in gonad differentiation. Their functions have been far less studied in teleosts. In this study, the full-length cDNAs of amh, dax1 and cyp19a1a were cloned and characterized in a triploid gynogenic fish, the Pengze crucian carp. Their expression profilings in juvenile development, adult tissues and juveniles exposed to 100 ng/L 17α-methyltestosterone (MT) were investigated. Results showed that their putative proteins shared high identities to their counterparts in cyprinid fish species, respectively. The tissue distribution results indicated that amh and cyp19a1a were predominantly expressed in the ovary and dax1 was dominantly expressed in the liver. Gene profiling in the developmental stages showed that all the three target genes had a consistent highest expression at 48 days post hatching (dph). The period of 48 dph appeared to be a key time during the process of the gonad development of Pengze crucian carp. 100 ng/L MT significantly increased the mRNA expression of amh at 2- and 4-week exposures and enhanced dax1 and cyp19a1a at 6-week exposure. The present study indicated that MT could influence the gonad development in Pengze crucian carp by disturbing sex-differentiation associated gene expression. Furthermore, the present study will be of great significance to broaden the understanding of molecular mechanisms of the physiological processes of reproduction in fish.     

Germ line control of female sex determination in zebrafish

A major transition during development of the gonad is commitment from an undifferentiated “bi-potential” state to ovary or testis fate. In mammals, the oogonia of the developing ovary are known to be important for folliculogenesis. An additional role in promoting ovary fate or female sex determination has been suggested, however it remains unclear how the germ line might regulate this process. Here we show that the germ line is required for the ovary versus testis fate choice in zebrafish. When the germ line is absent, the gonad adopts testis fate. These germ line deficient testes have normal somatic structures indicating that the germ line influences fate determination of surrounding somatic tissues. In germ line deficient animals the expression of the ovary specific gene cyp19a1a fails to be maintained whereas the testis genes sox9a and amh remain expressed. Furthermore, we observed decreased levels of the ovary specific genes cyp19a1a and foxL2 in germ line deficient animals prior to morphological sex differentiation of the gonad. We propose that the germ line has a common role in female sex determination in fish and mammals. Additionally, we show that testis specification is sufficient for masculinization of the fish pointing to a direct role of hormone signaling from the gonad in directing sex differentiation of non-gonadal tissues.     

Expression profiles of <Emphasis Type="Italic">amh</Emphasis> and <Emphasis Type="Italic">foxl2</Emphasis> in <Emphasis Type="Italic">Schizothorax kozlovi</Emphasis>, and their response to temperature during the early developmental stage

To elucidate the role of amh and foxl2 in sex differentiation of the teleost fish Schizothorax kozlovi, the full-length cDNAs were cloned from the mature testis and ovary by rapid amplification of cDNA ends (RACE), and their relative mRNA expression levels were determined by quantitative real-time polymerase chain reaction among tissues and temperature groups. The complete amh and foxl2 cDNAs of S. kozlovi were 2060 bp and 1750 bp, which encoded 568 and 306 amino acids, respectively. The amh were expressed only in gonads, while foxl2 was expressed in the gills, brain and gonads, both exhibiting relatively high tissue specificity. The amh exhibited sex-specific expression pattern in the gonads. No sex differences in the foxl2 expression were observed in the brain and gonads, but significant sex differences were found in the gills. No significant differences were found in the foxl2 expression, from the larval to the juvenile stage, and also between different temperature groups. However, significant differences were found in the expression levels of amh from the larval (12–63 days posthatching (dph)) to the juvenile stage (190 dph), and also among the \(18{^{\circ }}\hbox {C}\) and \(10{^{\circ }}\hbox {C}\) groups at 31 dph. This result suggested that amh plays an important role in male sex differentiation of S. kozlovi during the early developmental stage, but no similar effect was observed in foxl2.     

Differential expression of foxl2 and cyp19a1a mRNA during gonad developmental stages in great sturgeon Huso huso

This study aimed to determine the sex specificity and expression pattern of foxl2 and cyp19a1a genes in great sturgeon Huso huso gonads during gonadal sex differentiation and development. The results revealed that foxl2 and cyp19a1a mainly expressed in female gonads and during gonad development the foxl2 and cyp19a1a mRNA expression is required for ovarian development.     

Sex Reversal in Zebrafish fancl Mutants Is Caused by Tp53-Mediated Germ Cell Apoptosis

The molecular genetic mechanisms of sex determination are not known for most vertebrates, including zebrafish. We identified a mutation in the zebrafish fancl gene that causes homozygous mutants to develop as fertile males due to female-to-male sex reversal. Fancl is a member of the Fanconi Anemia/BRCA DNA repair pathway. Experiments showed that zebrafish fancl was expressed in developing germ cells in bipotential gonads at the critical time of sexual fate determination. Caspase-3 immunoassays revealed increased germ cell apoptosis in fancl mutants that compromised oocyte survival. In the absence of oocytes surviving through meiosis, somatic cells of mutant gonads did not maintain expression of the ovary gene cyp19a1a and did not down-regulate expression of the early testis gene amh; consequently, gonads masculinized and became testes. Remarkably, results showed that the introduction of a tp53 (p53) mutation into fancl mutants rescued the sex-reversal phenotype by reducing germ cell apoptosis and, thus, allowed fancl mutants to become fertile females. Our results show that Fancl function is not essential for spermatogonia and oogonia to become sperm or mature oocytes, but instead suggest that Fancl function is involved in the survival of developing oocytes through meiosis. This work reveals that Tp53-mediated germ cell apoptosis induces sex reversal after the mutation of a DNA–repair pathway gene by compromising the survival of oocytes and suggests the existence of an oocyte-derived signal that biases gonad fate towards the female developmental pathway and thereby controls zebrafish sex determination.     

Sex- and tissue-specific expression of P450 aromatase (cyp19a1a) in the yellowtail clownfish,Amphiprion clarkii

To investigate the role of estrogen in the gonad of yellowtail clownfish Amphiprion clarkii, we isolated cDNA encoding cytochrome P450 aromatase (Cyp19a1a) from the adult ovary. The full-length cDNA of clownfish cyp19a1a is 1928-bp long and encodes 520 amino acids. Real-time quantitative RT-PCR analysis showed that cyp19a1a was expressed mainly in the ovary of female-phase fish. In situ hybridization and immunohistochemical observations showed that positive signals were restricted to the ovarian follicle of the female-phase fish. In contrast, Cyp19a1a signal was not detected in the ambisexual gonad of the male-phase fish. These findings suggest that Cyp19a1a is involved in oogenesis in the female-phase fish, but not in the ambisexual gonad of male-phase fish.     

黄河鲤性别决定时间和相关基因表达研究

鱼类性别决定和分化机制极为复杂,通过性腺组织切片鉴定得出黄河鲤从未分化性腺发育为Ⅱ期精巢、卵巢的时间为受精后第40天到第80天。选取一些可能参与黄河鲤性别决定分化相关的基因（amh、ar、cyp19a、cyp19b、dax1、dmrt1、er、foxl2、nobox、sox9a、sox9b、zp2）进行实时荧光定量PCR分析各个基因在受精后40d、45d、50d、55d、65d和80d的表达情况。结果显示性别决定相关基因在50d都有高表达,推测45-50 d为性别决定的关键时间。ar、amh、dax1、dmrt1、sox9a、sox9b六个基因在80d雄性表达量升高,且雄性明显高于雌性,推测这些基因参与精巢分化发育过程。cyp19a、cyp19b、foxl2、nobox、zp2五个基因在80d雌性表达升高,且高于雄性,推测其可能参与卵巢分化发育。     

Genome-wide mapping of DNA methylation in Nile Tilapia

Cytosine DNA methylation is crucial for gene regulation and maintenance of genome stability. However, the detailed nile tilapia methylome remains uncharacterized. In this study, we present the first high-resolution methylome of tilapia gonad generated using methylated DNA immunoprecipitation (MeDIP) and high-throughput sequencing. In the ovary, 265 and 56 methylation peaks were identified in the genebody and promoter region of 145 genes, respectively. In the testis, 293 and 80 methylation peaks were identified in the genebody and promoter region of 144 genes. Furthermore, 8 and 49 genes showed differentially higher and lower promoter-region methylation rates, respectively, in the ovary relative to those of the testis. Quantitative PCR results revealed that the expression level of fibroblast growth factor 16 (fgf16), sialidase-3-like, fibroblast growth factor 20, aromatase (cyp19a), estrogen receptor, and gonadotropin receptor II precursor were negatively correlated to their methylation levels in the ovary and testis. The methylated levels of cyp19a and fgf16 were validated by bisulfite sequencing PCR technology, and the results were consistent with the MeDIP results. Thus, apart from generating the first methylation map, this study produced a candidate gene repository that provides additional options to explore the relationship between DNA methylation and sex differentiation or maintenance.     

Identification and functional analysis of foxl2 and nodal in sea cucumber,Apostichopus japonicus

Sea cucumber (Apostichopus japonicus) is an important mariculture species in China. To date, the mechanisms of sex determination and differentiation in sea cucumber remain unclear. Identifying sex-specific molecular markers is an effective method for revealing the genetic basis of sex determination and sex differentiation. In this study, foxl2 and nodal homologous genes were identified in A. japonicus. Foxl2 exhibited dynamic and sexually dimorphic expression patterns in the gonads, with prominent expression in the ovaries and minimal expression in the testis according to real-time quantitative PCR (RT-qPCR) study. As nodal was specifically expressed in the ovary, it could serve as an ovary-specific marker in sea cucumber. Additionally, knockdown of foxl2 or nodal using RNA interference (RNAi) led to the down-regulation of piwi, germ cell-less, and dmrt1, suggesting that foxl2 and nodal may play important roles in gonad maintenance of sea cucumber. Overall, this study adds to our understanding of the roles of foxl2 and nodal in the gonadal development of A. japonicus, which provides further insight into the mechanisms of sea cucumber sex determination and differentiation.