ABSTRACT
BACKGROUND: Feminization of animals derived from areas polluted by endocrine disrupting chemicals (EDCs) has been observed in all classes of vertebrates. However, feminization of artificially reared offspring by feeding of specific living organisms has never been reported. METHODS: Different food (including Limnodilus spp collected from the wild) and time treatment were applied to southern catfish. In addition, EDCs in Limnodilus spp., an annelid worm collected from wild contaminated small streams, was detected by LC-MS (Liquid chromatography-mass spectrometry). Serum estradiol-17ß and vitellogenin (VTG) levels and gonadal Sf1, Dmrt1, Foxl2, Cyp19a1a expression levels in the catfish were measured through Estradiol/VTG EIA Kit and real-time PCR. RESULTS: Here we report that feeding of Limnodilus spp. resulted in complete feminization of southern catfish, which has a 1:1 sex ratio in wild conditions. Furthermore, HPLC analysis showed that the extraction of Limnodilus spp. contained EDCs, including bisphenol A (BPA), diethylstilbestrol (DES), 4-tert-octylphenol (4-t-OP) and 4-nonylphenol (4-NP), which were further confirmed by LC-MS. Feeding southern catfish using commercial diets sprayed with EDCs cocktail also resulted in 100% female, whereas the control fish displayed approximate 1:1 sex ratio. Limnodilus spp. fed fish displayed similar serum estradiol-17ß and VTG levels and gonadal Sf1, Dmrt1, Foxl2, Cyp19a1a expression levels to those of female control. CONCLUSION: These results demonstrated that EDCs in Limnodilus spp. cause southern catfish feminization by affecting aromatase expression and endogenous estrogen level. This is the first report showing that feeding of any living organism resulted in complete feminization of a vertebrate.
Subject(s)
Annelida/chemistry , Catfishes , Endocrine Disruptors/analysis , Feminization/chemically induced , Water Pollution, Chemical/adverse effects , Animals , Chromatography, Liquid , Endocrine Disruptors/adverse effects , Female , Gonads/metabolism , Gonads/pathology , Male , Mass Spectrometry , Sex Differentiation , Vitellogenins/bloodABSTRACT
Transcription activator-like effector nucleases (TALENs) are a powerful approach for targeted genome editing and have been proved to be effective in several organisms. In this study, we reported that TALENs can induce somatic mutations in Nile tilapia, an important species for worldwide aquaculture, with reliably high efficiency. Six pairs of TALENs were constructed to target genes related to sex differentiation, including dmrt1, foxl2, cyp19a1a, gsdf, igf3, and nrob1b, and all resulted in indel mutations with maximum efficiencies of up to 81% at the targeted loci. Effects of dmrt1 and foxl2 mutation on gonadal phenotype, sex differentiation, and related gene expression were analyzed by histology, immunohistochemistry, and real-time PCR. In Dmrt1-deficient testes, phenotypes of significant testicular regression, including deformed efferent ducts, degenerated spermatogonia or even a complete loss of germ cells, and proliferation of steroidogenic cells, were observed. In addition, disruption of Dmrt1 in XY fish resulted in increased foxl2 and cyp19a1a expression and serum estradiol-17ß and 11-ketotestosterone levels. On the contrary, deficiency of Foxl2 in XX fish exhibited varying degrees of oocyte degeneration and significantly decreased aromatase gene expression and serum estradiol-17ß levels. Some Foxl2-deficient fish even exhibited complete sex reversal with high expression of Dmrt1 and Cyp11b2. Furthermore, disruption of Cyp19a1a in XX fish led to partial sex reversal with Dmrt1 and Cyp11b2 expression. Taken together, our data demonstrated that TALENs are an effective tool for targeted gene editing in tilapia genome. Foxl2 and Dmrt1 play antagonistic roles in sex differentiation in Nile tilapia via regulating cyp19a1a expression and estrogen production.
Subject(s)
Estrogens/metabolism , Forkhead Transcription Factors/metabolism , Gene Expression Regulation, Developmental/physiology , Sex Differentiation/physiology , Tilapia/growth & development , Transcription Factors/metabolism , Animals , Base Sequence , Deoxyribonucleases/genetics , Deoxyribonucleases/metabolism , Estrogens/genetics , Fish Proteins/genetics , Fish Proteins/metabolism , Forkhead Transcription Factors/genetics , Gene Deletion , Gene Expression Regulation, Enzymologic , Molecular Sequence Data , Mutation , Tilapia/embryology , Tilapia/metabolism , Transcription Factors/geneticsABSTRACT
Fibroblast growth factors (FGFs) have been proved to participate in a wide variety of processes, including growth, differentiation, cell proliferation, migration, sex determination and sex differentiation. The roles of FGF9/16/20 subfamily members in the gonadal development of teleost fish have not yet been reported. Three FGFs (16, 20a and 20b) of the FGF9/16/20 subfamily were cloned from the Nile tilapia by RT-PCR and RACE. Phylogenetic, bioinformatic and syntenic analyses demonstrated that these cloned FGFs are genuine FGF16, 20a and 20b. Our analyses further supported the non-existence of FGF9 ortholog and the existence of two FGF20 paralogs in teleost genomes. Tissue distribution analysis by RT-PCR demonstrated that FGF16 was expressed in a wide range of tissues including the testis and ovary, FGF20b in the brain, pituitary, intestine and ovary, but not in the testis, while FGF20a in the brain, pituitary and spleen, but not in the gonad. These results were consistent with the Northern blot analysis. The expression profiles of FGF16 and FGF20b during normal and sex reversed gonadal development were investigated by real-time PCR. Both showed much higher expression in the XX ovary and 17 beta-estradiol induced XY ovary compared with the XY testis and fadrozole and tamoxifen induced XX testis, with the highest in both sexes at 120 dah. Strong signals of FGF16 and FGF20b were detected in phase II oocytes by in situ hybridization. These data suggest that FGF9/16/20 subfamily is involved in the early oocyte development of the female.
