Variation in the TGF-β signaling pathway is emerging as an important mechanism by which gonadal sex determination is controlled in teleosts. Here we show that amhy, a Y-specific duplicate of the anti-Müllerian hormone (amh) gene, induces male sex determination in Nile tilapia. amhy is a tandem duplicate located immediately downstream of amhΔ-y on the Y chromosome. The coding sequence of amhy was identical to the X-linked amh (amh) except a missense SNP (C/T) which changes an amino acid (Ser/Leu92) in the N-terminal region. amhy lacks 5608 bp of promoter sequence that is found in the X-linked amh homolog. The amhΔ-y contains several insertions and deletions in the promoter region, and even a 5 bp insertion in exonVI that results in a premature stop codon and thus a truncated protein product lacking the TGF-β binding domain. Both amhy and amhΔ-y expression is restricted to XY gonads from 5 days after hatching (dah) onwards. CRISPR/Cas9 knockout of amhy in XY fish resulted in male to female sex reversal, while mutation of amhΔ-y alone could not. In contrast, overexpression of Amhy in XX fish, using a fosmid transgene that carries the amhy/amhΔ-y haplotype or a vector containing amhy ORF under the control of CMV promoter, resulted in female to male sex reversal, while overexpression of AmhΔ-y alone in XX fish could not. Knockout of the anti-Müllerian hormone receptor type II (amhrII) in XY fish also resulted in 100% complete male to female sex reversal. Taken together, these results strongly suggest that the duplicated amhy with a missense SNP is the candidate sex determining gene and amhy/amhrII signal is essential for male sex determination in Nile tilapia. These findings highlight the conserved roles of TGF-β signaling pathway in fish sex determination.
Objectives: We performed a national cross-sectional survey to determine the epidemiologic characteristics of patients with sepsis in ICU in China. Design: A cross-section survey study. Setting: Forty-four hospitals in mainland China from December 1, 2015, to January 31, 2016. Patients: All septic patients diagnosed according sepsis-1 criteria admitted to participating ICU. Interventions: None. Measurements and Main Results: We recorded demographic, physiologic, and microbiological data with follow-up for 90 days or death, if sooner. The frequency of sepsis and 90-day mortality rate were computed, and the relationship with gross domestic product determined. Multivariate logistic regression analysis was used to determine risk factors for 90-day mortality in patients with sepsis. Two-thousand three-hundred twenty-two patients with sepsis were included in the analysis, of whom 786 patients (33.9%) had hospital-acquired sepsis. The most common infection site was the lung (68.2%), followed by abdomen (26.6%) and bloodstream (7.8%). The frequency of sepsis in the ICU was 20.6 cases per 100 ICU admissions (95% CI, 15.8–25.4) with a 90-day mortality of 35.5%. The proportion of sepsis, severe sepsis, and septic shock were 3.10%, 43.6%, and 53.3% with a 90-day mortality of 2.78%, 17.69%, and 51.94%, respectively. Older age, low body weight, higher Sequential Organ Failure Assessment score, the number of systemic inflammatory response syndrome criteria, comorbid with heart failure, hematologic cancer, immunosuppression, higher level of lactate, infection site (pneumonia and bloodstream) were associated with 90-day mortality. Conclusions: Sepsis affects a fifth of patients admitted to ICUs in mainland China with a 90-day mortality rate of 35.5%. Our findings indicate that a large burden of sepsis, and we need to focus on sepsis as a quality improvement target in China given the high mortality. In addition, further studies are needed to delineate the epidemiology of sepsis outside the ICU.
Studies of gene function in non-model animals have been limited by the approaches available for eliminating gene function. The CRISPR/Cas9 (clustered regularly interspaced short palindromic repeats/CRISPR associated) system has recently become a powerful tool for targeted genome editing. Here, we report the use of the CRISPR/Cas9 system to disrupt selected genes, including nanos2, nanos3, dmrt1, and foxl2, with efficiencies as high as 95%. In addition, mutations in dmrt1 and foxl2 induced by CRISPR/Cas9 were efficiently transmitted through the germline to F 1 . Obvious phenotypes were observed in the G0 generation after mutation of germ cell or somatic cell-specific genes. For example, loss of Nanos2 and Nanos3 in XY and XX fish resulted in germ cell-deficient gonads as demonstrated by GFP labeling and Vasa staining, respectively, while masculinization of somatic cells in both XY and XX gonads was demonstrated by Dmrt1 and Cyp11b2 immunohistochemistry and by up-regulation of serum androgen levels. Our data demonstrate that targeted, heritable gene editing can be achieved in tilapia, providing a convenient and effective approach for generating loss-of-function mutants. Furthermore, our study shows the utility of the CRISPR/Cas9 system for genetic engineering in non-model species like tilapia and potentially in many other teleost species. R ECENTLY, a simple and efficient genome editing technology, type II CRISPR/Cas9, has been developed based on the Streptococcus pyogenes clustered regularly interspaced short palindromic repeats (CRISPR)-associated protein (Cas9) adaptive immune system. It requires three components for effective DNA cleavage: the nuclease Cas9, a targeting CRISPR RNA (crRNA), and an additional transactivating crRNA (tracrRNA) (Gasiunas et al. 2012;Jinek et al. 2012;Cho et al. 2013;Cong et al. 2013;Hwang et al. 2013;Mali et al. 2013). Further improvement of the system was achieved by fusing the crRNA and tracrRNA to form a single guide RNA (gRNA) that is sufficient to direct Cas9-mediated target cleavage (Hwang et al. 2013). Importantly, previous studies performed in vitro (Jinek et al. 2012), in bacteria , and in human cells (Cong et al. 2013) have shown that Cas9-mediated cleavage can be abolished by single mismatch at the gRNA-target site interface, particularly in the last 10-12 nucleotides located in the 39 end of the 20-nt gRNA targeting region. Compared to the other two engineered nuclease genome-editing technologies, zinc-finger nucleases (ZFNs) (Urnov et al. 2005;Doyon et al. 2008) and transcription activator-like effector nucleases (TALENs) (Huang et al. 2011;Sander et al. 2011;Tesson et al. 2011), the CRISPR/Cas9 system is substantially less expensive and much easier to program for editing new target sites. This new approach has been widely used for genome engineering in model animals, including Caenorhabditis elegans (Dickinson et al. 2013;Friedland et al. 2013;Tzur et al. 2013), Drosophila (Bassett et al. 2013;Ren et al. 2013;Yu et al. 2013), zebrafish (Chang et al. 2013Hrusc...
Four pairs of XX and XY gonads from Nile tilapia were sequenced at four developmental stages, 5, 30, 90, and 180 days after hatching (dah) using Illumina HiseqTM technology. This produced 28 Gb sequences, which were mapped to 21,334 genes. Of these, 259 genes were found to be specifically expressed in XY gonads, and 69 were found to be specific to XX gonads. Totally, 187 XX- and 1,358 XY-enhanced genes were identified, and 2,978 genes were found to be co-expressed in XX and XY gonads. Almost all steroidogenic enzymes, including cyp19a1a, were up-regulated in XX gonads at 5 dah; but in XY gonads these enzymes, including cyp11b2, were significantly up-regulated at 90 dah, indicating that, at a time critical to sex determination, the XX fish produced estrogen and the XY fish did not produce androgens. The most pronounced expression of steroidogenic enzyme genes was observed at 30 and 90 dah for XX and XY gonads, corresponding to the initiation of germ cell meiosis in the female and male gonads, respectively. Both estrogen and androgen receptors were found to be expressed in XX gonads, but only estrogen receptors were expressed in XY gonads at 5 dah. This could explain why exogenous steroid treatment induced XX and XY sex reversal. The XX-enhanced expression of cyp19a1a and cyp19a1b at all stages suggests an important role for estrogen in female sex determination and maintenance of phenotypic sex. This work is the largest collection of gonadal transcriptome data in tilapia and lays the foundation for future studies into the molecular mechanisms of sex determination and maintenance of phenotypic sex in non-model teleosts.
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