Genome-Wide Identification, Sequence Alignment, and Transcription of Five Sex-Related Genes in Largemouth Bass (Micropterus Salmoides).

BACKGROUND: Largemouth bass (Micropterus Salmoides) is an economically important fish species in China. Most research has focused on its growth, disease resistance, and nutrition improvement. However, the sex-determining genes in largemouth bass are still unclear. The transforming growth factor-beta (TGF-ß) gene family, including amh, amhr2 and gsdf, plays an important role in the sex determination and differentiation of various fishes. These genes are potentially involved in sex determination in largemouth bass. METHODS: We performed a systematic analysis of 5 sex-related genes (amh, amhr2, gsdf, cyp19a1, foxl2) in largemouth bass using sequence alignment, collinearity analysis, transcriptome, and quantitative real-time polymerase chain reaction (qRT-PCR). This included a detailed assessment of their sequences, gene structures, evolutionary traits, and gene transcription patterns in various tissues including gonads, and at different developmental stages. RESULTS: Comparative genomics revealed that the 5 sex-related genes were highly conserved in various fish genomes. These genes did not replicate, mutate or lose in largemouth bass. However, some were duplicated (amh, amhr2 and gsdf), mutated (gsdf) or lost (amhr2) in other fishes. Some genes (e.g., gsdf) showed significant differences in genomic sequence between males and females, which may contribute to sex determination and sex differentiation in these fishes. qRT-PCR was applied to quantify transcription profiling of the 5 genes during gonadal development and in the adult largemouth bass. Interestingly, amh, amhr2 and gsdf were predominantly expressed in the testis, while cyp19a1 and foxl2 were mainly transcribed in the ovary. All 5 sex-related genes were differentially expressed in the testes and ovaries from the 56th day post-fertilization (dpf). We therefore speculate that male/female differentiation in the largemouth bass may begin at this critical time-point. Examination of the transcriptome data also allowed us to screen out several more sex-related candidate genes. CONCLUSIONS: Our results provide a valuable genetic resource for investigating the physiological functions of these 5 sex-related genes in sex determination and gonadal differentiation, as well as in the control of gonad stability in adult largemouth bass.

A threshold dosage of estrogen for male-to-female sex reversal in the Glandirana rugosa frog.

Steroid hormones play very important roles in gonadal differentiation in many vertebrate species. Previously, we have determined a threshold dosage of testosterone (T) to induce female-to-male sex reversal in Glandirana rugosa frogs. Genetic females formed a mixture of testis and ovary, the so-called ovotestis, when tadpoles of G. rugosa were reared in water containing the dosage of T, which enabled us to detect primary changes in the histology of the masculinizing gonads. In this study, we determined a threshold dosage of estradiol-17ß (E2) to cause male-to-female sex reversal in this frog. We observed first signs of histological changes in the ovotestes, when tadpoles were reared in water containing the dosage of E2. Ovotestes were significantly larger than wild-type testes in size. By E2 treatment, male germ cells degenerated in the feminizing testis leading to their final disappearance. In parallel, oocytes appeared in the medulla of the ovotestis and later in the cortex as well. Quantitative polymerase chain reaction analysis revealed that the expression of sex-related genes involved in testis formation was significantly decreased in the ovotestis. In addition, immuno-positive signals of CYP17 that is involved in testis differentiation in this frog disappeared in the medulla first and then in the cortex. These results suggested that oocytes expanded in the feminizing gonad (ovary) contemporaneously with male germ cell disappearance. Primary changes in the histology of the gonads during male-to-female sex reversal occurred in the medulla and later in the cortex. This direction was opposite to that observed during female-to-male sex reversal in the G. rugosa frog.

QTL Mapping and Marker Identification for Sex-Determining: Indicating XY Sex Determination System in the Swimming Crab (Portunus trituberculatus).

Sex determination is an important area of research, which has always had an intriguing aspect in evolutionary and developmental biology. Quantitative trait locus (QTL) mapping for sex will be helpful in clarifying the sex determination system. In this study, the sex QTL mapping of the swimming crab (Portunus trituberculatus) was performed based on a high-density linkage map, and a highly significant QTL specifically mapped on a single linkage group (LG) was firstly identified (LG24, LOD > 14). Twenty markers in the QTL region showed significant associations with sex by association analysis, of which heterogametic genotypes in males supported the XY sex determination mechanism. Two sex-specific markers at the family level were identified via segregation distortion analysis, which were known to be the most closely linked to the sex of P. trituberculatus. Based on sex marker sequences (Marker3840, Marker20320, and Marker10494), three potential sex-related genes were identified, and the quantitative real-time PCR results suggested that these genes were important in spermatogenesis or sex characteristics in males. Our results will contribute to the fine-mapping of sex-determining genes and clarify the sex determination mechanism of P. trituberculatus.

Overall and allele-specific expression of the SMC1A gene in female Cornelia de Lange syndrome patients and healthy controls.

Cornelia de Lange syndrome (CdLS) is a rare multisystem disorder characterized by facial dysmorphisms, limb anomalies, and growth and cognitive deficits. Mutations in genes encoding subunits (SMC1A, SMC3, RAD21) or regulators (NIPBL, HDAC8) of the cohesin complex account for approximately 65% of clinically diagnosed CdLS cases. The SMC1A gene (Xp11.22), responsible for 5% of CdLS cases, partially escapes X chromosome inactivation in humans and the allele on the inactive X chromosome is variably expressed. In this study, we evaluated overall and allele-specific SMC1A expression. Real-time PCR analysis conducted on 17 controls showed that SMC1A expression in females is 50% higher than in males. Immunoblotting experiments confirmed a 44% higher protein level in healthy females than in males, and showed no significant differences in SMC1A protein levels between controls and patients. Pyrosequencing was used to assess the reciprocal level of allelic expression in six female carriers of different SMC1A mutations and 15 controls who were heterozygous at a polymorphic transcribed SMC1A locus. The two alleles were expressed at a 1:1 ratio in the control group and at a 2:1 ratio in favor of the wild type allele in the test group. Since a dominant negative effect is considered the pathogenic mechanism in SMC1A-defective female patients, the level of allelic preferential expression might be one of the factors contributing to the wide phenotypic variability observed in these patients. An extension of this study to a larger cohort containing mild to borderline cases could enhance our understanding of the clinical spectrum of SMC1A-linked CdLS.

CpG methylation of dmrt1 and cyp19a promoters in relation to their sexual dimorphic expression in the Japanese flounder Paralichthys olivaceus.

To better understand the effects of DNA methylation on the expression patterns of dmrt1 (Doublesex and Mab-3-related transcription factor 1) and cyp19a (Cytochrome P450 19a) in the Japanese flounder Paralichthys olivaceus, quantitative expressions, cellular distributions and cytosine-p-guanine (CpG) methylation patterns of these two genes in the gonads were analysed. The results showed that P. olivaceus dmrt1 expression was 70 times higher in the testis than in the ovary (P < 0·05). Its mRNA was detected clearly in spermatocytes and Sertoli cells of the testis, but weakly in the ovary. Paralichthys olivaceus cyp19a expression was 40 times higher in the ovary than in the testis (P < 0·01). Its mRNA was detected clearly in follicular cells of the ovary, but weakly in spermatocytes of the testis. The dmrt1 promoter CpGs were not methylated in the testis, whereas 57·69% were methylated in the ovary. For the cyp19a promoter CpGs, 97·5% were methylated in the testis and 73·33% were methylated in the ovary. These findings demonstrate that P. olivaceus dmrt1 and cyp19a are sex-related genes with sexual dimorphic expression, CpG methylation levels of the two genes are consistent with their expression quantities, and this epigenetic modification can influence the differential expression of genes in the gonads of P. olivaceus.