Activation of NF

Downloaded from http://www.jbc.org/ at Murdoch University (CAUL) on March 7, 2016 sox9 , and , ‡1 dead end B activation , ␬ ) Department ¶ cyp19a1a , foxl2 NOVEMBER 2, 2012 Department of ʈ sox9 , Marie Karlsson ) (7–10). Nr5a or steroido- ), anti-Müllerian hormone ‡1 ␣ fig ( sox9 ␣ nuclear receptor subfamily 5, group ). Earlier, neither cytogenetic studies b), VOLUME 287•NUMBER 45• and ), forkhead box protein L2 ( , Jarno Koskinen b ‡3 Danio rerio ¶1,2 Department of Animal Science and Breeding, and ** cyp19a1a leads to lack of its transcription and development of and factor in germ line (11). In mice, homozygous mutation of Sox9 binding site , ), cytochrome P450, family 19, subfamily A, polypeptide 4 (7, 10). In mammals, SOX9 is involved in the regulation of ) THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 287, NO. 45, pp. 37926–37938, November 2, 2012 nr5a1 a Amh © 2012 by The American Society for Biochemistry and Molecular Biology, Inc. Published in the U.S.A. The molecular mechanism of sex determination is unknown Zebrafish gonadal differentiation starts with the formation of On the other hand, a number of candidate genes with poten- apoptosis protein; DOC, sodium deoxycholate; NAI, NF- inhibitor; qRT-PCR, quantitative RT-PCR; I, intermediate;CF, CM, control control females. male; The abbreviations used are: AMH, anti-Müllerian hormone; IAP, inhibitor of amh dnd 4 in zebrafish ( zebrafish and represents an important step toward theunderstanding complete of the complicated process of sexin differentiation this species and possibly other cyprinid teleosts as well. in pseudohermaphrodites (12), whereasgene mutations result in in sex reversal in the (13). XY campomelic dysplasia patients a juvenile ovarian structure that eitherries or matures transforms into into adult testes ova- (14, 15).process The testis has transformation been suggested to depend on apoptosis (16). This genic factor-1 controls theamh expression of Amh A( (1, 2) nor comparative analysis ofthe recombination two rates sexes between (3) nor an array-basedto genome screen the (4) identification have of led sexgenetic chromosomes. Recently, linkage analysis of mapsassociated have with sex revealed determination on the fourindicating chromosomes presence (5, polygenic 6), sex of determination in regions zebrafish (4–7). tial role in sexual developmentSry-related have HMG been identified, box including gene( 9 ( 1a and b ( ( Published, JBC Papers in Press, September 17, 2012, DOI 10.1074/jbc.M112.386284 , and Per-Erik Olsson , Rajini Sreenivasan ** B-me- ʈ §1 ␬ ¶ B in zebrafish ␬ B pathways and ␬ , László Orbán § B Protein Prevents the Transition from B using NAI resulted in a ␬ B-regulated genes. To con- , Scott A. Ochsner ␬ ␬ ‡1 B in gonadal differentiation of B-induced anti-apoptotic effects, ␬ ␬ S □ B activation inhibitor (NAI). Sodium activated the NF- * ␬ B is a key regulator of anti-apoptotic processes and plays a role in gonad formation in mammals. ␬ , Neil J. McKenna supplemental Tables 1–3. B activation leads to female-biased sex differentiation in zebrafish. NF- Department of Molecular and Cellular Biology, Baylor College of Medicine, Houston, Texas 77030, Unraveling the regulation of apoptotic processes during gonadal transformation will facilitate understanding the ‡1 , Hazem Khalaf Anti-apoptotic signaling during the juvenile ovary stage is needed for the maintenance of oocytes in zebrafish. § ␬ BorNF- ‡1 ␬ —Final version full access. NF- B is activated at an early stage of development and ␬ Department of Biology, Örebro Life Science Center, School of Science and Technology, Örebro University, SE-701 82 ‡ JOURNAL OF BIOLOGICAL CHEMISTRY Escherichia coli ) gene expression. Because steroidogenic factor-1 and Background: molecular mechanism of zebrafish sex differentiation. Conclusion: Significance: Results: ry’s Institute of Medical Research, Monash Medical Centre,3168, Clayton, Victoria Australia. 46-19-303566; E-mail: [email protected]. edge Foundation, Sweden, and ÖrebroAgri-Food University and (to Veterinary P. Authority E.-O.) asoratory, well and Singapore as by (to Temasek the L. Life O.). Sciences Lab- under the accession number E-MEXP-3249. Testis differentiation in zebrafish involves juvenile ovary to This article contains Author’s Choice Author’s Choice This research was financed by the Swedish Research Council, the Knowl- Present address: Molecular Genetics and Development Division, Prince Hen- To whom correspondence should be addressed. Tel.: 46-19-301244; Fax: These authors have contributed equally. Amh S in Zebrafish Juvenile Ovary to Testis and Promotes Ovarian Development gonad differentiation. Exposure ofkilled juvenile zebrafish to heat- decrease in females from 45for to 20%. an This essential study provides role proof of NF- Jesper Karlsson 37926 2 3 □ 1 * resulted in anMicroarray and increased quantitative ratio real-time-PCR analysisshowed of of elevated expression gonads females of NF- from 30 to 85%. diated induction of anti-apoptotic genesplay could, a therefore, role also instudy zebrafish was gonad to differentiation. examine The the aim potential of role this of NF- Ajay Pradhan From the Received for publication, June 20, 2012, and in revised form, September 14, 2012 Activation of NF- firm the involvement of NF- has been shown to interact with steroidogenicmals, factor-1 in leading mam- to( the suppression ofAmh anti-Müllerian are hormone important for proper testis development, NF- Biological Sciences, National University of Singapore, SingaporeGeorgikon 117543, Faculty, and University of Pannonia, Keszthely, H-8360 Hungary of Reproductive Genomics, Strategic Research Program, Temasek Life Sciences Laboratory, Singapore 117604, deoxycholate treatment mimicked the effect ofteria heat-killed and bac- resulted in an increased proportionto of 45%, females whereas from the 25 inhibition of NF- The full set of microarray expression data has been deposited in ArrayExpress Örebro, Sweden, zebrafish were treatedinducer of with NF- sodium deoxycholate, a known testis transformation initiated by an apoptotic wave. Theular molec- regulation of this transformation process is notstood. fully NF- under- NF-␬B Activation Causes Female Bias in Zebrafish was further supported by a recent study where mutations in the EXPERIMENTAL PROCEDURES Fanconi anemia, complementation group L (fancl) gene led to Breeding—Adult zebrafish were maintained in a recirculat- germ cell apoptosis and consequently activated the testis devel- ing system (Aquaneering) with a 14-h light/10-h dark cycle. opment pathway (17). Introducing mutations in the tumor pro- The fish were fed twice a day with newly hatched Artemia tein p53 (tp53) gene counteracted the effect of the fancl muta- salina nauplii and commercial flake food (Tetrarubin). The tions (17), suggesting a role for apoptosis in this process. In line male and female brooders were kept in separate aquaria at with this, germ cell numbers are also important for ovarian 26–27 °C, and they were allowed to breed once a week. The fish development and complete absence of germ cells results in handling procedures were approved by the Swedish Ethical development of sterile males (9, 18). Committee in Linköping (Permit 32-10). Nuclear factor ␬-light-chain-enhancer of activated B cells Preparation of Heat-killed Bacteria—E. coli MG1655 was (NF-␬B) is involved in regulation of inflammation, apoptosis, grown on Luria-Bertani (LB) agar and incubated at 37 °C over- cell growth, and differentiation and can be activated by various night. One colony was inoculated into 10 ml of LB broth and physical and chemical factors (19). NF-␬B is a protein complex incubated on a shaker (200 rpm) at 37 °C overnight. The bacte- composed of homo- or heterodimers of five members of the Rel ria were then centrifuged and washed with 5 ml of phosphate- family including NF-␬B1 (p50), NF-␬B2 (p52), Rel A (p65), Rel buffered saline (Sigma). The bacterial pellet was resuspended in B, and c-Rel. These proteins are capable of binding one another 2 ml of PBS and killed by heating to 70 °C for 1 h. To ensure Downloaded from with different binding specificities resulting in different DNA 100% bacteria death, 10 ␮l of the heat-killed suspension was binding properties. The p65/p50 dimer is the most abundant plated and incubated overnight at 37 °C. heterodimeric form present in cells and is involved in transcrip- NF␬B-pGL4 Plasmid Construction—The pGL4 plasmid with tion activation of a multitude of genes (19, 20). NF-␬B subunits promoter-less Luciferase gene and neomycin selection marker ␬ are generally sequestered in the cytoplasm by the inhibitor pro- was purchased from Promega, and the NF B cis-element insert http://www.jbc.org/ ␬ tein I␬B. Numerous factors including UV irradiation, stress, was obtained from commercially available pNF B-Luc plasmid ␬ cytokine, and free radicals can promote I␬B degradation of the (BD Biosciences). The pGL4 luciferase plasmid and the pNF B- I␬B/NF-␬B complex, allowing the translocation of NF-␬Bto Luc plasmid were cut with HindIII and KpnI (Fermentas) and the nucleus resulting in subsequent induction of the transcrip- gel-purified by using Wizard SV Gel and PCR Clean-Up System (Promega). The NF␬B cis-element and the pGL4 luciferase tion of its target genes (20–22). at Murdoch University (CAUL) on March 7, 2016 NF-␬B activation blocks apoptotic processes and promotes plasmid were ligated using T4 DNA ligase (Fermentas). cell survival by interacting with the inhibitor of apoptosis pro- Cell Culture and Development of Stably

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