New insights into the all-testis differentiation in zebrafish with compromised endogenous androgen and estrogen synthesis.

The regulatory mechanism of gonadal sex differentiation, which is complex and regulated by multiple factors, remains poorly understood in teleosts. Recently, we have shown that compromised androgen and estrogen synthesis with increased progestin leads to all-male differentiation with proper testis development and spermatogenesis in cytochrome P450 17a1 (cyp17a1)-/- zebrafish. In the present study, the phenotypes of female-biased sex ratio were positively correlated with higher Fanconi anemia complementation group L (fancl) expression in the gonads of doublesex and mab-3 related transcription factor 1 (dmrt1)-/- and cyp17a1-/-;dmrt1-/- fish. The additional depletion of fancl in cyp17a1-/-;dmrt1-/- zebrafish reversed the gonadal sex differentiation from all-ovary to all-testis (in cyp17a1-/-;dmrt1-/-;fancl-/- fish). Luciferase assay revealed a synergistic inhibitory effect of Dmrt1 and androgen signaling on fancl transcription. Furthermore, an interaction between Fancl and the apoptotic factor Tumour protein p53 (Tp53) was found in vitro. The interaction between Fancl and Tp53 was observed via the WD repeat domain (WDR) and C-terminal domain (CTD) of Fancl and the DNA binding domain (DBD) of Tp53, leading to the K48-linked polyubiquitination degradation of Tp53 activated by the ubiquitin ligase, Fancl. Our results show that testis fate in cyp17a1-/- fish is determined by Dmrt1, which is thought to stabilize Tp53 by inhibiting fancl transcription during the critical stage of sexual fate determination in zebrafish.

Use of All-Male cyp17a1-Deficient Zebrafish (Danio rerio) for Evaluation of Environmental Estrogens.

Natural and synthetic environmental estrogens (EEs) are widespread and have received extensive attention. Our previous studies demonstrated that depletion of the cytochrome P450 17a1 gene (cyp17a1) leads to all-testis differentiation phenotype in zebrafish and common carp. In the present study, cyp17a1-deficient zebrafish with defective estrogen biosynthesis were used for the evaluation of EEs, as assessed by monitoring vitellogenin (vtg) expression. A rapid and sensitive assessment procedure was established with the 3-day administration of estradiol (E2), followed by examination of the transcriptional expression of vtgs in our cyp17a1-deficient fish. Compared with the control fish, a higher E2-mediated vtg upregulation observed in cyp17a1-deficient zebrafish exposed to 0.1 µg/L E2 is known to be estrogen receptor-dependent and likely due to impaired in vivo estrogen biosynthesis. The more responsive vtg expression in cyp17a1-deficient zebrafish was observed when exposed to 200 and 2000 µg/L bisphenol A (BPA) and perfluoro-1-octanesulfonate (PFOS). The estrogenic potentials of E2, BPA, and PFOS were compared and assessed by the feminization effect on ovarian differentiation in cyp17a1-deficient zebrafish from 18 to 50 days postfertilization, based on which a higher sensitivity of E2 in ovarian differentiation than BPA and PFOS was concluded. Collectively, through the higher sensitivity to EEs and the capacity to distinguish chemicals with different estrogenic potentials exhibited by the all-male cyp17a1-deficient zebrafish with impaired estrogen biosynthesis, we demonstrated that they can be used as an excellent in vivo model for the evaluation of EEs. Environ Toxicol Chem 2024;43:1062-1074. © 2024 SETAC.

Androgen signaling inhibits de novo lipogenesis to alleviate lipid deposition in zebrafish.

Testosterone is closely associated with lipid metabolism and known to affect body fat composition and muscle mass in males. However, the mechanisms by which testosterone acts on lipid metabolism are not yet fully understood, especially in teleosts. In this study, cyp17a1-/- zebrafish ( Danio rerio) exhibited excessive visceral adipose tissue (VAT), lipid content, and up-regulated expression and activity of hepatic de novo lipogenesis (DNL) enzymes. The assay for transposase accessible chromatin with sequencing (ATAC-seq) results demonstrated that chromatin accessibility of DNL genes was increased in cyp17a1-/- fish compared to cyp17a1+/+ male fish, including stearoyl-CoA desaturase ( scd) and fatty acid synthase ( fasn). Androgen response element (ARE) motifs in the androgen signaling pathway were significantly enriched in cyp17a1+/+ male fish but not in cyp17a1-/- fish. Both androgen receptor ( ar)-/- and wild-type (WT) zebrafish administered with Ar antagonist flutamide displayed excessive visceral adipose tissue, lipid content, and up-regulated expression and activity of hepatic de novo lipogenesis enzymes. The Ar agonist BMS-564929 reduced the content of VAT and lipid content, and down-regulated acetyl-CoA carboxylase a ( acaca), fasn, and scd expression. Mechanistically, the rescue effect of testosterone on cyp17a1-/- fish in terms of phenotypes was abolished when ar was additionally depleted. Collectively, these findings reveal that testosterone inhibits lipid deposition by down-regulating DNL genes via Ar in zebrafish, thus expanding our understanding of the relationship between testosterone and lipid metabolism in teleosts.

Estrogen Signaling Inhibits the Expression of anti-Müllerian hormone (amh) and gonadal-soma-derived factor (gsdf) during the Critical Time of Sexual Fate Determination in Zebrafish.

The mechanism of fish gonadal sex differentiation is complex and regulated by multiple factors. It has been widely known that proper steroidogenesis in Leydig cells and sex-related genes in Sertoli cells play important roles in gonadal sex differentiation. In teleosts, the precise interaction of these signals during the sexual fate determination remains elusive, especially their effect on the bi-potential gonad during the critical stage of sexual fate determination. Recently, all-testis phenotypes have been observed in the cyp17a1-deficient zebrafish and common carp, as well as in cyp19a1a-deficient zebrafish. By mating cyp17a1-deficient fish with transgenic zebrafish Tg(piwil1:EGFP-nanos3UTR), germ cells in the gonads were labelled with enhanced green fluorescent protein (EGFP). We classified the cyp17a1-deficient zebrafish and their control siblings into primordial germ cell (PGC)-rich and -less groups according to the fluorescence area of the EGFP labelling. Intriguingly, the EGFP-labelled bi-potential gonads in cyp17a1+/+ fish from the PGC-rich group were significantly larger than those of the cyp17a1-/- fish at 23 days post-fertilization (dpf). Based on the transcriptome analysis, we observed that the cyp17a1-deficient fish of the PGC-rich group displayed a significantly upregulated expression of amh and gsdf compared to that of control fish. Likewise, the upregulated expressions of amh and gsdf were observed in cyp19a1a-deficient fish as examined at 23 dpf. This upregulation of amh and gsdf could be repressed by treatment with an exogenous supplement of estradiol. Moreover, tamoxifen, an effective antagonist of both estrogen receptor α and ß (ERα and Erß), upregulates the expression of amh and gsdf in wild-type (WT) fish. Using the cyp17a1- and cyp19a1a-deficient zebrafish, we provide evidence to show that the upregulated expression of amh and gsdf due to the compromised estrogen signaling probably determines their sexual fate towards testis differentiation. Collectively, our data suggest that estrogen signaling inhibits the expression of amh and gsdf during the critical time of sexual fate determination, which may broaden the scope of sex steroid hormones in regulating gonadal sex differentiation in fish.

Approved immune checkpoint inhibitors in hepatocellular carcinoma: a large-scale meta-analysis and systematic review.

A meta-analysis was performed to assess the benefits and safety profile of approved immune checkpoint inhibitors in hepatocellular carcinoma patients. Eligible studies were searched from Cochrane, Embase, and PubMed databases based on a well-established strategy. Following the exclusion of ineligible studies, 12 studies were included in this meta-analysis. Compared with control group, immune checkpoint inhibitors were associated with improved ORR (OR 3.03, 95% CI 2.26-4.05, P < 0.00001), SD (OR 0.77, 95% CI 0.62-0.95, P = 0.02), OS (HR 0.75, 95% CI 0.68-0.83, P < 0.00001), and PFS (HR 0.74, 95% CI 0.63-0.87, P < 0.0003). However, no significant differences were observed in DCR (OR 1.33, 95% CI 0.97-1.81, P = 0.07), PD (OR 0.90, 95% CI 0.67-1.21, P = 0.48), and all caused any-grade adverse events (OR 1.22, 95% CI 0.62-2.39, P = 0. 57), all caused ≥ grade 3 adverse events (OR 1.10, 95% CI 0.97-1.25, P = 0.14), treatment-related any-grade adverse events (OR 1.13, 95% CI 0.55-2.32, P = 0.73), and treatment-related ≥ grade 3 events (OR 0.82, 95% CI 0.34-1.97, P = 0.65) between the two groups. After subgroup analysis conducted, patients in the immune checkpoint inhibitor group compared with targeted drug group showed significant improvements in OS (HR 0.74, 95% CI 0.66-0.84, P < 0.00001) and PFS (HR 0.75, 95% CI 0.61-0.91, P = 0.004). Immune checkpoint inhibitors have demonstrated peculiar benefits in the treatment of HCC with an acceptable safety profile. Compared to targeted drugs, immune checkpoint inhibitors still offer advantages in the treatment of hepatocellular carcinoma. However, there is still considerable room for further improvement.

Correlation between single nucleotide polymorphisms of folate metabolism genes and ethnic distribution in pregnant women.

This retrospective study aims to identify the single nucleotide polymorphisms (SNPs) of 5,10-methylenetetrahydrofolate reductase (MTHFR) (C677T, A1298C), methionine synthase reductase (MTRR) (A66G) and ethnic distribution characteristics in pregnant women, and to explore the risk correlation with folate metabolism. The demographic data of 8735 pregnant women aged 15 to 47 years were retrospectively analyzed, and peripheral blood samples were collected and tested. Reverse transcription-quantitative polymerase chain reaction was applied to determine the genotype and allele frequency of MTHFR C677T, A1298C and MTRR A66G in blood samples. Sperman correlation analysis, univariate and multivariate logistic regression analysis were used to verify the correlation between SNPs of MTHFR (C677T, A1298C), MTRR (A66G), different ethnic groups and the susceptibility and risk levels of folate metabolism. The relative risk of the SNPs was further determined by calculating the odds ratio (OR) at a 95% confidence interval (CI). The average age of 8735 pregnant women was 28.87 ± 4.20 years old. The evaluation of risk levels for folate metabolism was relative high, including 2296 cases with low risk, 3971 cases with medium risk, and 752 cases with high risk. Among the MTHFR C677T locus, the CC genotype had the highest frequency, MTHFR A1298C locus had the highest frequency of the AA genotype, and MTRR A66G locus had the highest frequency of the AA genotype. The frequency distribution of SNPs in different ethnic groups revealed that the frequency of CT genotype among the MTHFR C677T locus, AA genotype among the MTHFR A1298C locus and the MTRR A66G locus was the highest in Han, Buyi, Miao and Dong ethnic groups. The results of logistic regression analysis showed that the Han, Buyi, Miao and other ethnic groups (including Yi, Bai, Zhuang, Chuanqing) had the possibility of increasing the risk levels of folate metabolism. The CC genotype of MTHFR C677T (adjusted OR = 2.46, 95% CI: 2.14-2.84, P < .001) and the AG genotype of MTRR A66G (adjusted OR = 1.89, 95% CI: 1.61-2.22, P < .001) were significantly related to the risk levels of folate metabolism, which is an independent risk factor for the susceptibility of folate metabolism.

Enhanced insulin activity achieved in VDRa/b ablation zebrafish.

Introduction: 1α,25-dihydroxyvitamin D3 (1α,25[OH]2VD3) is a hormone known for its key roles in calcium absorption and nutrient metabolism. In teleost fishes, 1α,25(OH)2VD3 insufficiency causes impaired glucose metabolism and lipid oxidation. However, the cascade and mechanisms of 1α,25(OH)2VD3 and the vitamin d receptor (VDR) signaling are unclear. Results: In this study, two genes (vdra and vdrb) encoding paralogs of VDRs were genetically knocked out in zebrafish. Growth retardation and accumulated visceral adipose tissue have been observed in vdra -/-;vdrb -/- deficient line. In the liver elevated accumulation of triglycerides and suppressed lipid oxidation were detected. Morover significantly elevated 1α,25(OH)2VD3 levels were detected in vdra-/-;vdrb-/- zebrafish due to cyp24a1 transcription repression. Furthermore VDRs ablation Enhanced insulin signaling including elevated insulin/insra trancriptional levels, glycolysis, lipogenesis and promoted AKT/mTOR activity. Discussion: In conclusion, our present studies provides a zebrafish model with an elevated 1α,25(OH)2VD3 levels in vivo. The 1α,25(OH)2VD3/VDRs signaling promote lipid oxidation activity. However 1α,25(OH)2VD3 activity of regulation of glucose homeostasis through Insulin/Insr was independent of nuclear VDRs in teleosts.

Effect of Bushen Antai recipe on pyroptosis mechanism of subclinical hypothyroidism decidual cells in early pregnancy.

Background: Subclinical hypothyroidism (SH) is common during pregnancy. It is not clear whether decidual cells in SH undergo pyroptosis during pregnancy. This study aimed to investigate the possible mechanism of Bushen Antai recipe (BAR) in the treatment of SH in early pregnancy and the relationship between SH during pregnancy and decidual cell pyroptosis through a rat model. Methods: A total of 60 female rats were divided into control group, model group, levothyroxine (L-T4) group, low-dose BAR group (6 g/kg), medium-dose BAR group (12 g/kg), and high-dose BAR group (24 g/kg). The control group underwent pseudothyroidectomy, while the remaining groups established nonpregnant SH rat models. Except for the blank control group, rats were successfully established with SH models during pregnancy. The control group and the model group were treated with saline or BAR. The animals were sacrificed 12 hours after the last administration. The levels of serum thyroid-stimulating hormone (TSH), free thyroxine (FT4), interleukin-1ß (IL-1ß), and interleukin-18 (IL-18) were detected by enzyme-linked immunosorbent assay (ELISA). Western blot was used to detect the expression of decidual nucleotide-binding oligomerization domain-like receptor protein 3 (NLRP3) inflammasome, Caspase-1, and porin family proteins. Results: There was no significant difference in serum FT4 among groups (P>0.05). Compared with the control group, serum TSH, IL-1ß, IL-18, and NLRP3, Caspase-1, and gasdermin D (GSDMD) proteins in the decidua of the model group were significantly increased (P<0.05). Compared with the model group, the L-T4 group and the high-dose BAR group could significantly decrease the levels of serum TSH, IL-1ß, IL-18, and NLRP3, Caspase-1, and GSDMD in decidual tissue (P<0.05). The medium dose of BAR could significantly decrease the levels of TSH, NLRP3, Caspase-1, and GSDMD (P<0.05), and the low dose group of BAR significantly decreased the levels of TSH, NLRP3, and GSDMD (P<0.05). Among them, the high-dose group of BAR had the best reducing effect on IL-18, NLRP3, Caspase-1, and GSDMD. Conclusions: The decidual cells of SH rats in early pregnancy underwent pyroptosis with a high inflammatory response. BAR could improve TSH level in SH during pregnancy, inhibit decidual cell pyroptosis, and reduce the expression of inflammatory factors.

Domestication of farmed fish via the attenuation of stress responses mediated by the hypothalamus-pituitary-inter-renal endocrine axis.

Human-directed domestication of terrestrial animals traditionally requires thousands of years for breeding. The most prominent behavioral features of domesticated animals include reduced aggression and enhanced tameness relative to their wild forebears, and such behaviors improve the social tolerance of domestic animals toward both humans and crowds of their own species. These behavioral responses are primarily mediated by the hypothalamic-pituitary-adrenal (inter-renal in fish) (HPA/I) endocrine axis, which is involved in the rapid conversion of neuronal-derived perceptual information into hormonal signals. Over recent decades, growing evidence implicating the attenuation of the HPA/I axis during the domestication of animals have been identified through comprehensive genomic analyses of the paleogenomic datasets of wild progenitors and their domestic congeners. Compared with that of terrestrial animals, domestication of most farmed fish species remains at early stages. The present review focuses on the application of HPI signaling attenuation to accelerate the domestication and genetic breeding of farmed fish. We anticipate that deeper understanding of HPI signaling and its implementation in the domestication of farmed fish will benefit genetic breeding to meet the global demands of the aquaculture industry.

Sexual dimorphic effects of igf1 deficiency on metabolism in zebrafish.

Insulin-like growth factor 1 (IGF1) is an essential effector of the growth hormone (GH)/IGF1 axis for somatic growth regulation in mammals. However, its functions have not been thoroughly investigated in zebrafish in vivo. In this study, the igf1-deficient zebrafish model was developed using the CRISPR/Cas9 technique. In this study all the results were performed on both male and female animals. The growth of both male and female igf1-deficient zebrafish were reduced. The igf1 deficiency leads to significant complementary up-regulation of transcriptional expression levels of insulin, igf2 and igf3. This suggested that igf2 and igf3 may act with redundant functions. While the upregulation of gh1 expression can only be detected in igf1-deficient females. At the same time, significant growth retardation, fatty liver, reduced activated levels of ribosomal S6 (S6) are seen only in igf1-deficient males. On the other hand, significant hyperglycemia, elevated transcriptional expression levels of phosphenolpyruvate carboxykinase (pepck) and levels of phosphorylated extracellular signal-regulated kinase (ERK1/2), with additional reduced hepatic lactate/pyruvate (L/P) ratios can only observed in igf1-deficient females. Impaired glucose uptake has been recorded in the primary cultured hepatocytes from igf1-deficient females, but not males. Intriguingly, exposure to 17beta-estroadiol (E2) can partially ameliorated the defects of fatty liver and activation of AKT/mTOR signaling in igf1-deficient males. Our studies demonstrate the significant functions of IGF1 on somatic regulation in zebrafish, with asymmetric gender-related consequences. Our data thus suggest that the zebrafish IGF1 is preferentially required for the activation of AKT/mTOR signaling in male zebrafish, but glucose uptake in females.

Characterization of the Interrenal Gland and Sexual Traits Development in cyp17a2-Deficient Zebrafish.

Unlike the Cytochrome P450, family 17, subfamily A, member 1 (Cyp17a1), which possesses both 17α-hydroxylase and 17,20-lyase activities involved in the steroidogenic pathway that produces androgens and estrogens, Cytochrome P450, family 17, subfamily A, polypeptide 2 (Cyp17a2) possesses only 17α-hydroxylase activity and is known essential for the synthesis of cortisol. Besides with expressed in testes and ovaries, where the cyp17a1 is mainly expressed, cyp17a2 is also expressed in the interrenal gland in fish. Until now, the roles of cyp17a2 in fish, especially in sexual traits development and hypothalamic-pituitary-interrenal (HPI) axis, are poorly studied. To investigate the roles of Cyp17a2 in teleosts, the cyp17a2-null zebrafish was generated and analyzed by us. The significantly decreased cortisol concentration was observed both in the cyp17a2-deficient males and females at adult stage. The interrenal gland enlargement, increased pituitary proopiomelanocortin a (pomca) expression, decreased locomotion activity and response to light-stimulated stress were observed in cyp17a2-deficient fish. Intriguingly, the cyp17a2-deficient males were fertile and with normal breeding tubercles on the pectoral fin, but females were infertile, deficient in genital papilla and with decreased gonadosomatic index (GSI). The increased progesterone (P4), 17α,20ß-dihydroxy-4-pregnen-3-one (DHP) and 11-ketotestosterone (11-KT) in the cyp17a2-deficient males and females were observed. The increased concentration of testosterone (T) and estradiol (E2) was observed in cyp17a2-/- females and cyp17a2-/- males, respectively. By examining the ovaries development of cyp17a2-deficient fish at 3 months postfertilization (mpf), we observed that the oocytes were over-activated. Taken together, our findings demonstrate that Cyp17a2 is indispensable for production and physiology of cortisol, and cyp17a2-deficiency resulted in diminished cortisol but accumulated P4 and DHP, which may result in the over-activated oocytes in cyp17a2-deficient females.

Augmentation of progestin signaling rescues testis organization and spermatogenesis in zebrafish with the depletion of androgen signaling.

Disruption of androgen signaling is known to cause testicular malformation and defective spermatogenesis in zebrafish. However, knockout of cyp17a1, a key enzyme responsible for the androgen synthesis, in ar-/- male zebrafish paradoxically causes testicular hypertrophy and enhanced spermatogenesis. Because Cyp17a1 plays key roles in hydroxylation of pregnenolone and progesterone (P4), and converts 17α-hydroxypregnenolone to dehydroepiandrosterone and 17α-hydroxyprogesterone to androstenedione, we hypothesize that the unexpected phenotype in cyp17a1-/-;androgen receptor (ar)-/- zebrafish may be mediated through an augmentation of progestin/nuclear progestin receptor (nPgr) signaling. In support of this hypothesis, we show that knockout of cyp17a1 leads to accumulation of 17α,20ß-dihydroxy-4-pregnen-3-one (DHP) and P4. Further, administration of progestin, a synthetic DHP mimetic, is sufficient to rescue testicular development and spermatogenesis in ar-/- zebrafish, whereas knockout of npgr abolishes the rescue effect of cyp17a1-/- in the cyp17a1-/-;ar-/- double mutant. Analyses of the transcriptomes among the mutants with defective testicular organization and spermatogenesis (ar-/-, ar-/-;npgr-/- and cyp17a-/-;ar-/-;npgr-/-), those with normal phenotype (control and cyp17a1-/-), and rescued phenotype (cyp17a1-/-;ar-/-) reveal a common link between a downregulated expression of insl3 and its related downstream genes in cyp17a-/-;ar-/-;npgr-/- zebrafish. Taken together, our data suggest that genetic or pharmacological augmentation of the progestin/nPgr pathway is sufficient to restore testis organization and spermatogenesis in zebrafish with the depletion of androgen signaling.

Genomic polymorphisms at the crhr2 locus improve feed conversion efficiency through alleviation of hypothalamus-pituitary-interrenal axis activity in gibel carp (Carassius gibelio).

Improvement in fish feed conversion efficiency (FCE) is beneficial for sustaining global food fish supplies. Here, we show that a set of polymorphisms at locus of the corticotropin releasing hormone receptor 2 (crhr2), which is involved in hypothalamus-pituitary-interrenal (HPI) axis signaling, is associated with improved FCE in farmed allogynogenetic gibel carp strain CAS III compared with that in the wild gibel carp strain Dongting (DT). This set of polymorphisms downregulates the expression levels of crhr2 mRNA in the brain and pituitary tissues in gibel carp strain CAS III compared with those in strain DT. Furthermore, compromised HPI axis signaling is observed in gibel carp strain CAS III, such as decreased α-melanocyte stimulating hormone protein levels, plasma cortisol content, and stress responses. Moreover, enhanced activation of protein kinase B/mammalian target of rapamycin complex 1 signaling observed in the muscle tissue of strain CAS III in comparison to that in strain DT indicated elevated anabolic metabolism in strain CAS III. Thus, these studies demonstrate that the genetic markers associated with compromised HPI axis signaling, such as crhr2, are potentially useful for genetic selection toward improvement in farmed fish growth and FCE, which would reduce fishmeal consumption and thereby indirectly facilitate sustainable fisheries.

Functions of the Thyroid-Stimulating Hormone on Key Developmental Features Revealed in a Series of Zebrafish Dyshormonogenesis Models.

The hypothalamic-pituitary-thyroid (HPT) axis regulates many critical features in vertebrates. Utilizing TALENs and CRISPR/Cas9 techniques, thyroid-stimulating hormone subunit beta a (tshba), thyroglobulin (tg), and solute carrier family 16 member 2 (slc16a2) mutant zebrafish lines were generated. Among the three mutants, the earliest time point for the significantly altered T3 contents was observed in tshba mutants, which resulted in the most severe defects, including typical defects such as the retardation of inflated anterior swimming bladder (aSB), proper formation of fin ray and posterior squamation (SP), the larval-to-juvenile transition (LTJT) process, juvenile growth retardation, and mating failure. In tg mutants, which are actually compensated with an alternative splicing form, growth retardation was observed in the juvenile stage without LTJT and reproductive defects. The evident goiter phenotype was only observed in tg- and slc16a2 mutants, but not in tshba mutants. Other than goiters being observed, no other significant developmental defects were found in the slc16a2 mutants. Regarding the reproductive defects observed in tshba mutants, the defective formation of the secondary sex characteristics (SSCs) was observed, while no obvious alterations during gonad development were found. Based on our analyses, zebrafish at the 6-12 mm standard length or 16-35 days post-fertilization (dpf) should be considered to be in their LTJT phase. Using a series of zebrafish dyshormonogenesis models, this study demonstrated that the TSH function is critical for the proper promotion of zebrafish LTJT and SSC formation. In addition, the elevation of TSH levels appears to be essential for goiter appearance in zebrafish.

Rbm46, a novel germ cell-specific factor, modulates meiotic progression and spermatogenesis.

It has been suggested that many novel RNA-binding proteins (RBPs) are required for gametogenesis, but the necessity of few of these proteins has been functionally verified. Here, we identified one RBP, Rbm46, and investigated its expression pattern and role in zebrafish reproduction. We found that rbm46 is maternally provided and specifically expressed in the germ cells of gonadal tissues using in situ hybridization, reverse transcription-PCR, and quantitative real-time polymerase chain reaction (qRT-PCR). Two independent rbm46 mutant zebrafish lines were generated via the transcription activator-like effector nuclease technique. Specific disruption of rbm46 resulted in masculinization and infertility in the mutants. Although the spermatogonia appeared grossly normal in the mutants, spermatogenesis was impaired, and meiosis events were not observed. The introduction of a tp53M214K mutation could not rescue the female-to-male sex-reversal phenotype, indicating that rbm46 acts independently of the p53-dependent apoptotic pathway. RNA sequencing and qRT-PCR subsequently indicated that Rbm46 might be involved in the posttranscriptional regulation of functional genes essential for germ cell development, such as nanos3, dazl, and sycp3, during gametogenesis. Together, our results reveal for the first time the crucial role of rbm46 in regulating germ cell development in vivo through promotion of germ cell progression through meiosis prophase I.

Research on the Relationships between Psychological Problems and School Bullying and Non-Suicidal Self-Injury among Rural Primary and Middle School Students in Developing Areas of China.

(1) Purpose: To analyze the role of psychological problems in connection with school bullying and non-suicidal self-injury (NSSI) among rural primary and middle school students in developing areas of China. (2) Methods: A multi-stage, stratified, cluster random sampling method was used to select 2284 rural primary and middle school students in Jiangxi Province for study. Questionnaires regarding the health risk behaviors of children in developing areas were left behind at primary and middle schools, and they were later collected and analyzed by using the mental health diagnostic monitoring scale for Chinese primary and middle school students. Pearson correlation, logistic regression, and bootstrap tests were conducted to analyze the association between psychological problems, school bullying, and NSSI. (3) Results: The incidence of NSSI in rural primary and middle school students in Jiangxi Province was 14.84%. Compared with other children with behavioral problems, those who had experienced school bullying and had mild/severe psychological problems were more likely to have engaged in NSSI behaviors (p < 0.001). Psychological problems have a mediating effect between school bullying and NSSI, which accounted for 12.96% of the total effect. (4) Conclusion: Psychological problems are likely an effect modifier in the connection between school bullying and NSSI behaviors. Therefore, effectively targeting psychological problems in rural primary and middle school students in Jiangxi Province may help prevent and control NSSI behaviors in students who have experienced school bullying.

Hyperandrogenism in POMCa-deficient zebrafish enhances somatic growth without increasing adiposity.

The endocrine regulatory roles of the hypothalamic-pituitary-adrenocortical axis on anxiety-like behavior and metabolic status have been found throughout animal taxa. However, the precise effects of the balancing adrenal corticosteroid biosynthesis under the influence of adrenocorticotrophic hormone (ACTH), a pro-opiomelanocortin (POMC)-derived peptide, on animal energy expenditure and somatic growth remain unknown. POMC has also been identified as one of the candidate loci for polycystic ovary syndrome, which features hyperandrogenism and some prevalence of obesity in patients. Here we show that zebrafish lacking functional POMCa exhibit similar phenotypes of stress response and body weight gain but not obesity as observed in mammalian models. In contrast with the impaired anorexigenic signaling cascade of melanocyte-stimulating hormones and leptin, which are responsible for their obesity-prone weight gain observed in various pomc mutant mammals, analyses with our pomca mutant series indicate that ACTH is the key regulator for the phenotype with enhanced somatic growth without obesity in pomca-deficient zebrafish. Hypocortisolism associated with hyperandrogenism has been observed in the pomca-deficient zebrafish, with enhanced activation of mammalian target of rapamycin complex 1; reutilization of amino acids and fatty acid ß-oxidation are observed in the muscle tissue of the pomca-deficient fish. After reducing hyperandrogenism by crossing our pomca mutant fish with a cyp17a1-deficient background, the phenotype of enhanced somatic growth in pomca-deficient fish was no longer observed. Thus, our work also demonstrated that the role of POMCa in stress response seems to be conserved in vertebrates, whereas its effect on adipostasis is unique to teleosts.

Steroidogenic acute regulatory protein and luteinizing hormone are required for normal ovarian steroidogenesis and oocyte maturation in zebrafish†.

In recent studies, luteinizing hormone (LH) was reported to play important roles in oocyte maturation. However, the mechanism by which LH signaling, especially regarding the steroidogenesis process, affects oocyte maturation has not been clarified. In this study, zebrafish models with a functional deficiency in luteinizing hormone beta (Lhb) or steroidogenic acute regulatory protein (Star), an enzyme that promotes the transport of cholesterol into the inner mitochondrial membrane for maturation-induced hormone (MIH) production, were generated using transcription activator-like effector nucleases (TALENs). Similar phenotypes of the maturation-arrested oocytes in both female mutants have been observed. The levels of MIH in the oocytes of the female mutants were clearly decreased in both the lhb and star knockout zebrafish. The expression of star was dramatically down-regulated in the lhb mutant follicles and was clearly promoted by forskolin and hCG in vitro. Furthermore, treatment with the MIH precursors, pregnenolone or progesterone, as well as with MIH itself rescued the maturation-arrested oocyte phenotypes in both lhb and star mutants. The plasma levels of other steroids, including testosterone, estradiol, and cortisol, were not affected in the lhb mutants, while the levels of gonad hormones testosterone and estradiol were significantly increased in the star mutants. The cortisol levels were decreased in the star mutants. Collectively, our results confirm that LH plays important roles in the initiation of MIH synthesis from cholesterol and maintains oocyte maturation in zebrafish, as well as provide evidence that Star might act downstream of LH signaling in steroidogenesis.