Disruption of Zar1 leads to arrested oogenesis by regulating polyadenylation via Cpeb1 in tilapia (Oreochromis niloticus).

Oogenesis is a complex process regulated by precise coordination of multiple factors, including maternal genes. Zygote arrest 1 (zar1) has been identified as an ovary-specific maternal gene that is vital for oocyte-to-embryo transition and oogenesis in mouse and zebrafish. However, its function in other species remains to be elucidated. In the present study, zar1 was identified with conserved C-terminal zinc finger domains in Nile tilapia. zar1 was highly expressed in the ovary and specifically expressed in phase I and II oocytes. Disruption of zar1 led to the failed transition from oogonia to phase I oocytes, with somatic cell apoptosis. Down-regulation and failed polyadenylation of figla, gdf9, bmp15 and wee2 mRNAs were observed in the ovaries of zar1-/- fish. Cpeb1, a gene essential for polyadenylation that interacts with Zar1, was down-regulated in zar1-/- fish. Moreover, decreased levels of serum estrogen and increased levels of androgen were observed in zar1-/- fish. Taken together, zar1 seems to be essential for tilapia oogenesis by regulating polyadenylation and estrogen synthesis. Our study shows that Zar1 has different molecular functions during gonadal development by the similar signaling pathway in different species.

Expression and tissue distribution analysis of vimentin and transthyretin proteins associated with coat colors in sheep (Ovis aries).

OBJECTIVE: Pigment production and distribution are controlled through multiple proteins, resulting in different coat color phenotypes of sheep. METHODS: The expression distribution of vimentin (VIM) and transthyretin (TTR) in white and black sheep skins was detected by liquid chromatography-electrospray ionization tandem MS (LC-ESI-MS/MS), gene ontology (GO) statistics, immunohistochemistry, Western blot, and quantitative real time polymerase chain reaction (qRT-PCR) to evaluate their role in the coat color formation of sheep. RESULTS: LC-ESI-MS/MS results showed VIM and TTR proteins in white and black skin tissues of sheep. Meanwhile, GO functional annotation analysis suggested that VIM and TTR proteins were mainly concentrated in cellular components and biological process, respectively. Further research confirmed that VIM and TTR proteins were expressed at significantly higher levels in black sheep skins than in white sheep skins by Western blot, respectively. Immunohistochemistry notably detected VIM and TTR in hair follicle, dermal papilla, and outer root sheath of white and black sheep skins. qRT-PCR results also revealed that the expression of VIM and TTR mRNAs was higher in black sheep skins than in white sheep skins. CONCLUSION: The expression of VIM and TTR were higher in black sheep skins than in white sheep skins and the transcription and translation were unanimous in this study. VIM and TTR proteins were expressed in hair follicles of white and black sheep skins. These results suggested that VIM and TTR were involved in the coat color formation of sheep.

Exposure to bisphenol A induces neurotoxicity associated with synaptic and cytoskeletal dysfunction in neuro-2a cells.

Bisphenol A (BPA) has been reported to injure the developing and adult brain. However, the underlying mechanism still remains elusive. This study used neuro-2a cells as a cellular model to investigate the neurotoxic effects of BPA. Microtubule-associated protein 2 (MAP2) and tau protein maintain microtubule normal function and promote the normal development of the nervous system. Synaptophysin (SYP) and drebrin (Dbn) proteins are involved in regulating synaptic plasticity. Cells were exposed to the minimum essential medium (MEM), 0.01% (v/v) DMSO, and 150 µM BPA for 12, 24, or 36 h. Morphological analysis revealed that the cells in the BPA-treated groups shrank and collapsed compared with those in the control groups. CCK-8 and lactate dehydrogenase assay (LDH) assays showed that the mortality of neuro-2a cells increased as the BPA treatment time was prolonged. Ultrastructural analysis further revealed that cells demonstrated nucleolar swelling, dissolution of nuclear and mitochondrial membranes, and partial mitochondrial condensation following exposure to BPA. BPA also decreased the relative protein expression levels of MAP2, tau, and Dbn. Interestingly, the relative protein expression levels of SYP increased. These results indicated that BPA inhibited the proliferation and disrupted cytoskeleton and synaptic integrity of neuro-2a cells.

Exposure to fluoride induces apoptosis in the liver, kidney, and heart of Xenopus laevis by regulating the Caspase-8/3 signaling pathway.

Fluoride compounds are abundant and widely distributed in the environment at various concentrations, which can seriously injure the human body. In this study, we aim to evaluate the effects of excessive fluoride exposure on the liver, kidney, and heart tissues of healthy female Xenopus laevis by administering NaF (0, 100, and 200 mg/L) in drinking water for 90 days. The expression level of procaspase-8, cleaved-caspase-8, and procaspase-3 proteins were determined by Western blot. Compared with the control group, the group exposed to NaF exhibited expression levels of procaspase-8, cleaved-caspase-8, and procaspase-3 proteins that were considerably upregulated at a concentration of 200 mg/L in the liver and kidney. The cleaved-caspase-8 protein expression in the group exposed to a high concentration of NaF was lower than that in the control group in heart. Histopathological results by hematoxylin and eosin staining showed that excessive NaF exposure caused necrosis of hepatocytes and vacuolization degeneration. Granular degeneration and necrosis in renal tubular epithelial cells were also observed. Moreover, hypertrophy of myocardial cells, atrophy of myocardial fibers and disorder of myocardial fibers were detected. These results demonstrated that NaF-induced apoptosis and the mediated death receptor pathway activation ultimately damaged the liver and kidney tissues. This finding offers a fresh perspective on the effects of F-induced apoptosis in X. laevis.

Genome-Wide Identification and Characterization of the BRD Family in Nile Tilapia (Oreochromis niloticus).

The bromodomain (BRD) proteins specifically recognize the N-acetyllysine motifs, which is a key event in the reading process of epigenetic marks. BRDs are evolutionarily highly conserved. Over recent years, BRDs attracted great interest because of their important roles in biological processes. However, the genome-wide identification of this family was not carried out in many animal groups, in particular, in teleosts. Moreover, the expression patterns were not reported for any of the members in this family, and the role of the BRD family was not extensively studied in fish reproduction. In this study, we identified 16 to 120 BRD genes in 24 representative species. BRDs expanded significantly in vertebrates. Phylogenetic analysis showed that the BRD family was divided into eight subfamilies (I-VIII). Transcriptome analysis showed that BRDs in Nile tilapia (Oreochromis niloticus) exhibited different expression patterns in different tissues, suggesting that these genes may play different roles in growth and development. Gonadal transcriptome analysis showed that most of the BRDs display sexually dimorphic expression in the gonads at 90 and 180 dah (days after hatching), including 21 testis-dominated genes (brdt, brd4a and brd2b, etc.), and nine ovary-dominated genes (brd3b, brd2a and kat2a, etc.). Consistent with transcriptomic data, the results of qRT-PCR and fluorescence in situ hybridization showed that brdt expression was higher in the testis than in the ovary, suggesting its critical role in the spermatogenesis of the tilapia. Male fish treated with JQ1 (BET subfamily inhibitor) displayed abnormal spermatogenesis. The numbers of germ cells were reduced, and the expression of steroidogenic enzyme genes was downregulated, while the expression of apoptosis-promoting genes was elevated in the testis tissue of treated fish. Our data provide insights into the evolution and expression of BRD genes, which is helpful for understanding their critical roles in sex differentiation and gonadal development in teleosts.

miR-133b targets tagln2 and functions in tilapia oogenesis.

microRNAs (miRNAs) are important components of non-coding RNAs that participate in diverse life activities by regulating gene expression at the post transcriptional level through base complementary pairing with 3'UTRs of target mRNAs. miR-133b is a member of the miR-133 family, which play important roles in muscle differentiation and tumorigenesis. Recently, miR-133b was reported to affect estrogen synthesis by targeting foxl2 in mouse, while its role in fish reproduction remains to be elucidated. In the present study, we isolated the complete sequence of miR-133b, which was highly expressed in tilapia ovary at 30 and 90 dah (days after hatching) and subsequently decreased at 120 to 150 dah by qPCR. Interestingly, only a few oogonia were remained in the antagomir-133b treated tilapia ovary, while phase I and II oocytes were observed in the ovaries of the control group. Unexpectedly, the expression of foxl2 and cyp19a1a, as well as estradiol levels in serum were increased in the treated group. Furthermore, tagln2, an important factor for oogenesis, was predicted as the target gene of miR-133b, which was confirmed by dual luciferase reporter vector experiments. miR-133b and tagln2 were co-expressed in tilapia ovaries. Taken together, miR-133b may be involved in the early oogenesis of tilapia by regulating tagln2 expression. This study enriches the understanding of miR-133b function during oogenesis and lays a foundation for further study of the regulatory network during oogenesis.

Peach allergen Pru p 1 content is generally low in fruit but with large variation in different varieties.

BACKGROUND: Pru p 1 is a major allergen in peach and nectarine, and the different content in varieties may affect the degree of allergic reactions. This study aimed to quantify Pru p 1 levels in representative peach varieties and select hypoallergenic Pru p 1 varieties. METHODS: To obtain monoclonal and polyclonal antibodies, mice and rabbits, respectively, were immunized with recombinant Pru p 1.01 and Pru p 1.02. The Pru p 1 levels in fruits from 83 representative peach varieties was quantified by sandwich enzyme-linked immunosorbent assay (sELISA). nPru p 1 was obtained through specific monoclonal antibody affinity purification and confirmed by Western blot and mass spectrometry. The variable Pru p 1 content of selected varieties was evaluated by Western blot and the expression level of encoding Pru p 1 genes by quantitative polymerase chain reaction. RESULTS: A sELISA method with monoclonal and polyclonal antibodies was built for quantifying Pru p 1 levels in peach. Pru p 1 was mainly concentrated in the peel (0.20-73.44 µg/g, fresh weight), being very low in the pulp (0.05-9.62 µg/g) and not detected in wild peach. For the 78 peach and nectarine varieties, Pru p 1 content varied widely from 0.12 to 6.45 µg/g in whole fruit. We verified that natural Pru p 1 is composed of 1.01 and 1.02 isoallergens, and the Pru p 1 expression level and Pru p 1 band intensity in the immunoblots were in agreement with protein quantity determined by ELISA for some tested varieties. In some cases, the reduced levels of Pru p 1 did not coincide with low Pru p 3 in the same variety in whole fruit, while some ancient wild peach and nectarines contained low levels of both allergens, and late-ripening yellow flesh varieties were usually highly allergenic. CONCLUSION: Pru p 1 content is generally low in peach compared to Pru p 3. Several hypoallergenic Pru p 1 and Pru p 3 varieties, "Zi Xue Tao," "Wu Yue Xian," and "May Fire," were identified, which could be useful in trials for peach allergy patients.

Salvianolic acid B protects against sepsis-induced liver injury via activation of SIRT1/PGC-1α signaling.

Liver injury occurs frequently during sepsis, which leads to high mortality and morbidity. A previous study has suggested that salvianolic acid B (SalB) is protective against sepsis-induced lung injury. However, whether SalB is able to protect against sepsis-induced liver injury remains unclear. The present study aimed to investigate the effects of SalB on sepsis-induced liver injury and its potential underlying mechanisms. Sepsis was induced in mice using a cecal ligation and puncture (CLP) method. The mice were treated with SalB (30 mg/kg intraperitoneally) at 0.5, 2 and 8 h after CLP induction. Pathological alterations of the liver were assessed using hematoxylin and eosin staining. The serum levels of alanine transaminase (ALT), aspartate aminotransferase (AST), tumor necrosis factor (TNF)-α and interleukin (IL)-6 were measured. The hepatic mRNA levels of TNF-α, IL-6, Bax and Bcl-2 were also detected. The results suggested that treatment with SalB ameliorated sepsis-induced liver injury in the mice, as supported by the mitigated pathologic changes and lowered serum aminotransferase levels. SalB also decreased the levels of the inflammatory cytokines TNF-α and IL-6 in the serum and the liver of the CLP model mice. In addition, SalB significantly downregulated Bax expression and upregulated Bcl-2 expression, and upregulated the expression levels of SIRT1 and PGC-1α. However, when sirtuin 1 (SIRT1) small interfering RNA was co-administered with SalB, the protective effects of SalB were attenuated and the expression levels of SIRT1 and PGC-1α were reduced. In summary, these results indicate that SalB mitigates sepsis-induced liver injury via reduction of the inflammatory response and hepatic apoptosis, and the underlying mechanism may be associated with the activation of SIRT1/PGC-1α signaling.