The RNA-binding protein Adad1 is necessary for germ cell maintenance and meiosis in zebrafish.

The double stranded RNA binding protein Adad1 (adenosine deaminase domain containing 1) is a member of the adenosine deaminase acting on RNAs (Adar) protein family with germ cell-specific expression. In mice, Adad1 is necessary for sperm differentiation, however its function outside of mammals has not been investigated. Here, through an N-ethyl-N-nitrosourea (ENU) based forward genetic screen, we identified an adad1 mutant zebrafish line that develops as sterile males. Further histological examination revealed complete lack of germ cells in adult mutant fish, however germ cells populated the gonad, proliferated, and entered meiosis in larval and juvenile fish. Although meiosis was initiated in adad1 mutant testes, the spermatocytes failed to progress beyond the zygotene stage. Thus, Adad1 is essential for meiosis and germline maintenance in zebrafish. We tested if spermatogonial stem cells were affected using nanos2 RNA FISH and a label retaining cell (LRC) assay, and found that the mutant testes had fewer LRCs and nanos2-expressing cells compared to wild-type siblings, suggesting that failure to maintain the spermatogonial stem cells resulted in germ cell loss by adulthood. To identify potential molecular processes regulated by Adad1, we sequenced bulk mRNA from mutants and wild-type testes and found mis-regulation of genes involved in RNA stability and modification, pointing to a potential broader role in post-transcriptional regulation. Our findings suggest that the RNA regulatory protein Adad1 is required for fertility through regulation of spermatogonial stem cell maintenance in zebrafish.

The Zebrafish Meiotic Cohesin Complex Protein Smc1b Is Required for Key Events in Meiotic Prophase I.

The eukaryotic structural maintenance of chromosomes (SMC) proteins are involved in key processes of chromosome structure and dynamics. SMC1ß was identified as a component of the meiotic cohesin complex in vertebrates, which aids in keeping sister chromatids together prior to segregation in meiosis II and is involved in association of homologous chromosomes in meiosis I. The role of SMC1ß in meiosis has primarily been studied in mice, where mutant male and female mice are infertile due to germ cell arrest at pachytene and metaphase II stages, respectively. Here, we investigate the function of zebrafish Smc1b to understand the role of this protein more broadly in vertebrates. We found that zebrafish smc1b is necessary for fertility and has important roles in meiosis, yet has no other apparent roles in development. Therefore, smc1b functions primarily in meiosis in both fish and mammals. In zebrafish, we showed that smc1b mutant spermatocytes initiated telomere clustering in leptotene, but failed to complete this process and progress into zygotene. Furthermore, mutant spermatocytes displayed a complete failure of synapsis between homologous chromosomes and homolog pairing only occurred at chromosome ends. Interestingly, meiotic DNA double strand breaks occurred in the absence of Smc1b despite failed pairing and synapsis. Overall, our findings point to an essential role of Smc1b in the leptotene to zygotene transition during zebrafish spermatogenesis. In addition, ovarian follicles failed to form in smc1b mutants, suggesting an essential role in female meiosis as well. Our results indicate that there are some key differences in Smc1b requirement in meiosis among vertebrates: while Smc1b is not required for homolog pairing and synapsis in mice, it is essential for these processes in zebrafish.

Arsenic and lead in foods: a potential threat to human health in Bangladesh.

The non-carcinogenic and carcinogenic risk of arsenic and lead to adults and children via daily dietary intake of food composites in Bangladesh was estimated. The target hazard quotients (THQs), hazard index (HI) and target carcinogenic risk (TR) were calculated to evaluate the non-carcinogenic and carcinogenic health risk from arsenic and lead. Most of the individual food composites contain a considerable amount of arsenic and lead. The highest mean concentrations of arsenic were found in cereals (0.254 mg kg⁻¹ fw) and vegetables (0.250 mg kg⁻¹ fw), and lead in vegetables (0.714 mg kg⁻¹ fw) and fish (0.326 mg kg⁻¹ fw). The results showed the highest THQs of arsenic in cereals and lead in vegetables for both adults and children which exceeded the safe limit (> 1) indicating that cereals and vegetables are the main food items contributing to the potential health risk. The estimated TR from ingesting dietary arsenic and lead from most of the foods exceeded 10⁻6, indicating carcinogenic risks for all adult people of the study area.

Glucocorticoid/glucocorticoid receptor inhibition of surfactant protein-A (SP-A) gene expression in lung type II cells is mediated by repressive changes in histone modification at the SP-A promoter.

Surfactant protein-A (SP-A) gene expression in human fetal lung type II cells is stimulated by cAMP and IL-1 and is inhibited by glucocorticoids. cAMP/IL-1 stimulation of SP-A expression is mediated by increased binding of thyroid transcription factor-1 and nuclear factor (NF)-kappaB to the TTF-1-binding element (TBE) in the SP-A promoter. This is associated with decreased expression of histone deacetylases (HDACs), increased recruitment of coactivators, and enhanced acetylation of histone H3 (K9,14) at the TBE. In the present study, endogenous glucocorticoid receptor (GR) was found to interact with thyroid transcription factor-1 and NF-kappaB p65 at the TBE. GR knockdown enhanced SP-A expression in type II cells cultured in serum-free medium, suggesting a ligand-independent inhibitory role of endogenous GR. Furthermore, use of chromatin immunoprecipitation revealed that dexamethasone (Dex) treatment of fetal lung type II cells increased recruitment of endogenous GR and HDACs-1 and -2 and blocked cAMP-induced binding of inhibitor of kappaB kinase-alpha (IKKalpha) to the TBE region. Accordingly, Dex reduced basal and blocked cAMP-stimulated levels of acetylated (K9,14) and phosphorylated (S10) histone H3 at the TBE. Dex also increased TBE binding of dimethylated histone H3 (K9) and of heterochromatin protein 1alpha. Thus, Dex increases interaction of GR with the complex of proteins at the TBE. This facilitates recruitment of HDACs and causes a local decline in basal and cAMP-induced histone H3 phosphorylation and acetylation and an associated increase in H3-K9 dimethylation and binding of heterochromatin protein 1alpha. Collectively, these events may culminate in the closing of chromatin structure surrounding the SP-A gene and inhibition of its transcription.

Permissive effects of oxygen on cyclic AMP and interleukin-1 stimulation of surfactant protein A gene expression are mediated by epigenetic mechanisms.

Surfactant protein A (SP-A) is important for immune defense within the alveolus. Cyclic AMP (cAMP) stimulation of SP-A expression in lung type II cells is O(2) dependent and mediated by increased phosphorylation and binding of thyroid transcription factor 1 (TTF-1) to an upstream response element (TTF-1-binding element [TBE]). Interleukin-1 (IL-1) stimulation of SP-A expression is mediated by NF-kappaB (p65/p50) activation and increased binding to the TBE. In this study, we found that O(2) also was permissive for IL-1 induction of SP-A expression and for cAMP and IL-1 stimulation of type II cell nuclear protein binding to the TBE. Using chromatin immunoprecipitation, we observed that when type II cells were cultured in 20% O(2), cAMP and IL-1 stimulated the recruitment of TTF-1, p65, CBP, and steroid receptor coactivator 1 to the TBE region of the SP-A promoter and increased local acetylation of histone H3; these effects were prevented by hypoxia. Hypoxia markedly reduced global levels of CBP and acetylated histone H3 and increased the expression of histone deacetylases. Furthermore, hypoxia caused a global increase in histone H3 dimethylated on Lys9 and increased the association of dimethyl histone H3 with the SP-A promoter. These results, together with findings that the histone deacetylase inhibitor trichostatin A and the methyltransferase inhibitor 5'-deoxy(5'-methylthio)adenosine markedly enhanced SP-A expression in lung type II cells, suggest that increased O(2) availability to type II cells late in gestation causes epigenetic changes that permit access of TTF-1 and NF-kappaB to the SP-A promoter. The binding of these transcription factors facilitates the recruitment of coactivators, resulting in the further opening of the chromatin structure and activation of SP-A transcription.

Potential role of nuclear factor kappaB and reactive oxygen species in cAMP and cytokine regulation of surfactant protein-A gene expression in lung type II cells.

The human surfactant protein-A2 (hSP-A2) gene is developmentally regulated, expressed in type II pneumonocytes, and induced by cAMP. cAMP induction of hSP-A2 expression is O2 dependent and mediated by increased phosphorylation, DNA binding, and transcriptional activation of thyroid transcription factor-1 (TTF-1). The TTF-1-binding element (TBE) at -175 bp contains a reverse-oriented nuclear factor-kappaB (NF-kappaB) binding site. IL-1 increased SP-A expression in lung type II cells and had additive stimulatory effects with cAMP. Nuclear extracts from cAMP- or IL-1-treated type II cells manifested increased binding to NF-kappaB consensus and TBE probes; cAMP and IL-1 had additive effects. Competitive and antibody supershift EMSA revealed that NF-kappaB and TTF-1 interact with TBE. IL-1 treatment of type II cells caused rapid (1 h) increases in nuclear levels of NF-kappaB (p50 and p65) and in binding to NF-kappaB and TBE probes; nuclear levels of TTF-1 were unaffected. Bt2cAMP increased binding to NF-kappaB and TBE probes more slowly; no changes in nuclear levels of p50, p65, or TTF-1 were evident, suggesting that IL-1 and cAMP act by different mechanisms. A role for endogenous NF-kappaB in cAMP and IL-1 regulation of SP-A was suggested by findings that dominant-negative forms of inhibitor of kappaB reduced binding of type II cell nuclear proteins to TBE and inhibited SP-A expression. In cotransfection assays, NF-kappaB and TTF-1 cooperatively interacted at TBE to stimulate SP-A promoter activity; this was further enhanced by IL-1. In coimmunoprecipitation assays using type II cell nuclear extracts, TTF-1 was found to interact with p65 in vivo. Finally, antioxidant inhibitors of NF-kappaB reduced type II cell nuclear protein binding to TBE and blocked stimulatory effects of cAMP on SP-A expression. This provides intriguing evidence that permissive effects of O2/reactive oxygen species on cAMP regulation of SP-A expression may be mediated by cooperative interactions of TTF-1 and NF-kappaB at the TBE.