Characterization, phylogeny, alternative splicing and expression of Sox30 gene

<p>Abstract</p> <p>Background</p> <p>Members of the Sox gene family isolated from both vertebrates and invertebrates have been proved to participate in a wide variety of developmental processes, including sex determination and differentiation. Among these members, <it>Sox30 </it>had been considered to exist only in mammals since its discovery, and its exact function remains unclear.</p> <p>Results</p> <p><it>Sox30 </it>cDNA was cloned from the Nile tilapia by RT-PCR and RACE. Screening of available genome and EST databases and phylogenetic analysis showed that <it>Sox30 </it>also exists in non-mammalian vertebrates and invertebrates, which was further supported by synteny analyses. Tissue expression in human, mouse and tilapia suggested that <it>Sox30 </it>was probably a gonad-specific gene, which was also supported by the fact that <it>Sox30 </it>EST sequences were obtained from gonads of the animal species. In addition, four alternatively spliced isoforms were isolated from tilapia gonad. Their temporal and spatial expression patterns during normal and sex reversed gonadal development were investigated by RT-PCR and <it>in situ </it>hybridization. Our data suggest that expressions of <it>Sox30 </it>isoforms are related to stage and phenotypic-sex, observed in the germ cells of male gonad and in somatic cells of the female gonad.</p> <p>Conclusions</p> <p><it>Sox30 </it>is not a gene only existed in mammals, but exists widely throughout the animal kingdom as supported by our bioinformatic, phylogenetic and syntenic analyses. It is very likely that <it>Sox30 </it>is expressed exclusively in gonads. Expression analyses revealed that <it>Sox30 </it>may be involved in female and male gonadal development at different stages by alternative splicing.</p

Integrated analysis of miRNA and mRNA expression profiles in tilapia gonads at an early stage of sex differentiation

MicroRNAs (miRNAs) represent a second regulatory network that has important effects on gene expression and protein translation during biological process. However, the possible role of miRNAs in the early stages of fish sex differentiation is not well understood. In this study, we carried an integrated analysis of miRNA and mRNA expression profiles to explore their possibly regulatory patterns at the critical stage of sex differentiation in tilapia. We identified 279 pre-miRNA genes in tilapia genome, which were highly conserved in other fish species. Based on small RNA library sequencing, we identified 635 mature miRNAs in tilapia gonads, in which 62 and 49 miRNAs showed higher expression in XX and XY gonads, respectively. The predicted targets of these sex-biased miRNAs (e.g., miR-9, miR-21, miR-30a, miR-96, miR-200b, miR-212 and miR-7977) included genes encoding key enzymes in steroidogenic pathways (Cyp11a1, Hsd3b, Cyp19a1a, Hsd11b) and key molecules involved in vertebrate sex differentiation (Foxl2, Amh, Star1, Sf1, Dmrt1, and Gsdf). These genes also showed sex-biased expression in tilapia gonads at 5 dah. Some miRNAs (e.g., miR-96 and miR-737) targeted multiple genes involved in steroid synthesis, suggesting a complex miRNA regulatory network during early sex differentiation in this fish. The sequence and expression patterns of most miRNAs in tilapia are conserved in fishes, indicating the basic functions of vertebrate miRNAs might share a common evolutionary origin. This comprehensive analysis of miRNA and mRNA at the early stage of molecular sex differentiation in tilapia XX and XY gonads lead to the discovery of differentially expressed miRNAs and their putative targets, which will facilitate studies of the regulatory network of molecular sex determination and differentiation in fishes.https://doi.org/10.1186/s12864-016-2636-

Genome-Wide Identification and Transcriptome-Based Expression Profiling of the Sox Gene Family in the Nile Tilapia (Oreochromis niloticus)

The Sox transcription factor family is characterized with the presence of a Sry-related high-mobility group (HMG) box and plays important roles in various biological processes in animals, including sex determination and differentiation, and the development of multiple organs. In this study, 27 Sox genes were identified in the genome of the Nile tilapia (Oreochromis niloticus), and were classified into seven groups. The members of each group of the tilapia Sox genes exhibited a relatively conserved exon-intron structure. Comparative analysis showed that the Sox gene family has undergone an expansion in tilapia and other teleost fishes following their whole genome duplication, and group K only exists in teleosts. Transcriptome-based analysis demonstrated that most of the tilapia Sox genes presented stage-specific and/or sex-dimorphic expressions during gonadal development, and six of the group B Sox genes were specifically expressed in the adult brain. Our results provide a better understanding of gene structure and spatio-temporal expression of the Sox gene family in tilapia, and will be useful for further deciphering the roles of the Sox genes during sex determination and gonadal development in teleosts

Identification and Evolution of TGF-β Signaling Pathway Members in Twenty-Four Animal Species and Expression in Tilapia

Transforming growth factor β (TGF-β) signaling controls diverse cellular processes during embryogenesis as well as in mature tissues of multicellular animals. Here we carried out a comprehensive analysis of TGF-β pathway members in 24 representative animal species. The appearance of the TGF-β pathway was intrinsically linked to the emergence of metazoan. The total number of TGF-β ligands, receptors, and smads changed slightly in all invertebrates and jawless vertebrates analyzed. In contrast, expansion of the pathway members, especially ligands, was observed in jawed vertebrates most likely due to the second round of whole genome duplication (2R) and additional rounds in teleosts. Duplications of TGFB2, TGFBR2, ACVR1, SMAD4 and SMAD6, which were resulted from 2R, were first isolated. Type II receptors may be originated from the ACVR2-like ancestor. Interestingly, AMHR2 was not identified in Chimaeriformes and Cypriniformes even though they had the ligand AMH. Based on transcriptome data, TGF-β ligands exhibited a tissue-specific expression especially in the heart and gonads. However, most receptors and smads were expressed in multiple tissues indicating they were shared by different ligands. Spatial and temporal expression profiles of 8 genes in gonads of different developmental stages provided a fundamental clue for understanding their important roles in sex determination and reproduction. Taken together, our findings provided a global insight into the phylogeny and expression patterns of the TGF-β pathway genes, and hence contribute to the greater understanding of their biological roles in the organism especially in teleosts

Gata2a Mutation Causes Progressive Microphthalmia and Blindness in Nile Tilapia

The normal development of lens fiber cells plays a critical role in lens morphogenesis and maintaining transparency. Factors involved in the development of lens fiber cells are largely unknown in vertebrates. In this study, we reported that GATA2 is essential for lens morphogenesis in Nile tilapia (Oreochromis&nbsp;niloticus). In this study, Gata2a was detected in the primary and secondary lens fiber cells, with the highest expression in primary fiber cells. gata2a homozygous mutants of tilapia were obtained using CRISPR/Cas9. Different from fetal lethality caused by Gata2/gata2a mutation in mice and zebrafish, some gata2a homozygous mutants of tilapia are viable, which provides a good model for studying the role of gata2 in non-hematopoietic organs. Our data showed that gata2a mutation caused extensive degeneration and apoptosis of primary lens fiber cells. The mutants exhibited progressive microphthalmia and blindness in adulthood. Transcriptome analysis of the eyes showed that the expression levels of almost all genes encoding crystallin were significantly down-regulated, while the expression levels of genes involved in visual perception and metal ion binding were significantly up-regulated after gata2a mutation. Altogether, our findings indicate that gata2a is required for the survival of lens fiber cells and provide insights into transcriptional regulation underlying lens morphogenesis in teleost fish

Clinical utility of Liqui-PREP™ cytology system for primary cervical cancer screening in a large urban hospital setting in China

<b>Background:</b> Liquid based cytology (LBC) has been reported to increase the sensitivity of cervical cytology, in comparison with conventional cytology Pap smear (CPS). Most LBC systems though require expensive automated devices. <b> Aims:</b> To evaluate the efficiency of a new and inexpensive LBC system - LPT cytology system. <b> Materials and Methods:</b> Cervical screening was performed on 31500 patients utilizing the LPT cytology system test from January 2006 to May 2007. A similar number (n = 31500) of CPS were performed from January 2004 to July 2006. All cytology positive patients underwent colposcopy and cervical biopsy with histopathology examination. Fifty cases positive both on cytology and biopsy were submitted to the high-risk human papillomavirus (HPV) L1 protein (HR-HPV L1) tests. <b> Results:</b> The LPT cytology system adequately preserved cellular structure for morphologic evaluation. There was a significant difference of the histology/cytology diagnosis concordant rate between that of the CPS and LPT systems [93.6 vs. 78.4&#x0025;, <i>p</i>=0.001]. The significant higher concordant rate was also seen in the low grade intraepithelial lesion (LSIL) (95.4 vs. 78.9&#x0025;, <i>p</i>=0.001) and in high grade intraepithelial lesion (HSIL) (90.2 vs. 76.1&#x0025;, p=0.001) cytology diagnosis. There was no statistical difference in rate in atypical glandular cells (AGC) (61.5 vs. 60&#x0025;) and glandular cell carcinoma (GCC) (83.3 vs. 80&#x0025;). LPT resulted in a marked increased global detection over the CPS. Nuclear expression of HPV L1 was seen in 34&#x0025; (17/50) of cases. <b> Conclusions:</b> LPT showed an increase in detection rate compared to CPS (<i>P</i> = 0.001) and a significantly higher histological versus cytological concordant referral rate

Dnmt3aa but Not Dnmt3ab Is Required for Maintenance of Gametogenesis in Nile Tilapia (Oreochromis niloticus)

Dnmt3a, a de novo methyltransferase, is essential for mammalian germ line DNA methylation. Only one Dnmt3a is identified in mammals, and homozygous mutants of Dnmt3a are lethal, while two Dnmt3a paralogs, dnmt3aa and dnmt3ab, are identified in teleosts due to the third round of genome duplication, and homozygous mutants of dnmt3aa and dnmt3ab are viable in zebrafish. The expression patterns and roles of dnmt3aa and dnmt3ab in gonadal development remain poorly understood in teleosts. In this study, we elucidated the precise expression patterns of dnmt3aa and dnmt3ab in tilapia gonads. Dnmt3aa was highly expressed in oogonia, phase I and II oocytes and granulosa cells in ovaries and spermatogonia and spermatocytes in testes, while dnmt3ab was mainly expressed in ovarian granulosa cells and testicular spermatocytes. The mutation of dnmt3aa and dnmt3ab was achieved by CRISPR/Cas9 in tilapia. Lower gonadosomatic index (GSI), increased apoptosis of oocytes and spermatocytes and significantly reduced sperm quality were observed in dnmt3aa−/− mutants, while normal gonadal development was observed in dnmt3ab−/− mutants. Consistently, the expression of apoptotic genes was significantly increased in dnmt3aa−/− mutants. In addition, the 5-methylcytosine (5-mC) level in dnmt3aa−/− gonads was decreased significantly, compared with that of dnmt3ab−/− and wild type (WT) gonads. Taken together, our results suggest that dnmt3aa, not dnmt3ab, plays important roles in maintaining gametogenesis in teleosts

Occurrence and risk assessment of triclosan in freshwater lakes in the middle Yangtze River basin (Wuhan, Central China)

Triclosan (TCS) is an endocrine disrupting chemical which is commonly used as a disinfectant in pharmaceuticals and personal care products (PPCP's). Since early 2020, the worldwide outbreak of COVID-19 has increased the use of PPCP's, so the occurrence and impact of TCS on freshwater lakes should be considered. However, little attention has been given to the effect of TCS on freshwater lakes in China. This study is the first attempt at a risk assessment focusing on the temporal and spatial occurrence of TCS in freshwater lakes in the middle Yangtze River basin. The surface water and sediments of Donghu Lake and Liangzi Lake (Wuhan, Central China) were collected from October 2020 to August 2021. The maximum concentrations of TCS were 466 ​ng/L and 239 ​ng/L in surface water, 71 ​ng/g and 25 ​ng/g (dry weight) in sediments of Donghu Lake and Liangzi Lake, respectively. Significant temporal and spatial differences of TCS were observed within and between the lakes, with the highest concentrations measured in winter. Furthermore, higher concentrations of TCS were observed in areas that are more impacted by human activities. There was a significant positive correlation between TCS and nitrogen in the surface water. A risk assessment using the risk quotient (RQ) method showed that a potentially high risk (RQ ​> ​1) was found only in surface waters from Donghu Lake, and that the sediments posed a lower risk than the surface waters. These results provide timely data on the temporal and spatial occurrence of TCS in freshwater lakes in China following the outbreak of COVID-19 and demonstrate a possible high risk of exposure to TCS for aquatic biota