Generation of Antibodies Against DMRT1 and DMRT4 of Oreochromis aurea and Analysis of Their Expression Profile in Oreochromis aurea Tissues

a.
Fishery College, Nanjing Agricultural University, Wuxi 214081, China
b.
Key Open Laboratory for Genetic Breeding of Aquatic Animals and Aquaculture Biology, Ministry of Agriculture, Freshwater Fishery Research Center, Chinese Academy of Fishery Sciences, Wuxi 214081, China

More Information

Corresponding author: E-mail address: wutt@ffrc.cn (Tingting Wu)

摘要

- /
- /
- /
- /
Abstract: Sex determination is composed of somatic and germ-line sex differentiation hierarchies whose interaction is poorly understood. A single gene known to control somatic sex determination, the DM-domain containing (Doublesex/Mab-3 DNA-binding motif) gene, is highly conserved across species. Vertebrate DMRT1 (DM-related transcription factor 1) expression occurs predominantly in the testis. Here, however, isolated two distinct DM-domain cDNA from Oreochromis aurea ovary and testis have been named DMRT4 (DM-related transcription factor 4) and DMRT1 by BLAST, respectively. Despite high homology in the DM-domain, there is little similarity outside the DM-domain. To better understand the structure, function, and possible roles of DMRT4 and DMRT1 as potential candidates for sex differentiation and sex determination, the intact regions encoding DMRT4 and DMRT1 obtained by PCR were sub-cloned into the vector pMAL-c2x and introduced into the Escherichia coli TB1 cell for efficient fusion expression. After purification and cleavage, DMRT4 and DMRT1 proteins were used to immunize adult rabbits following standard protocols. Consequently, it was found by using Western blot analysis that polyclonal antibodies against DMRT4 and DMRT1 had high specificity. The relative expression levels of DMRT4 and DMRT1 mRNA were determined by fluorescent Real-time RT-PCR in female and male Oreochromis aurea with β-actin as the internal standard. DMRT1 was expressed only in testis, whereas DMRT4 was over expressed in the ovary, but in both female and male, a slight expression in the brain was also detected. Statistical analysis showed that in the brain, mean DMRT4 mRNA levels in female were significantly higher than in male. Meanwhile, the expression of DMRT4 and DMRT1 protein was also analyzed using the purified antibodies through Western blot and immunohistochemistry. It was found that DMRT4 was exclusively expressed in the ovary and DMRT1 in the testis. Study on DMRT4 and DMRT1 expression facilitated the elucidation of their roles and the understanding of sex differentiation of fish.
- DMRT1 /
- DMRT4 /
- prokaryotic expression /
- polyclonal antibody /
- expression profile

HTML全文

参考文献(40)

[1]	Marin, I, Baker, et al. The evolutionary dynamics of sex determination Science, 281 (1998),pp. 1990-1994
[2]	Kuwabara, PE Developmental genetics of Caenorhabditis elegans sex determination Current Topics in Developmental Biology, 41 (1999),pp. 99-132
[3]	Hodgkin, JA Sex, cell death, and the genome of C. elegans Cell, 98 (1999),pp. 277-280
[4]	Sinclair, AH, Berta, et al. A gene from the human sex-determining region encodes a protein with homology to a conserved DNA-binding motif Nature, 346 (1990),pp. 240-244
[5]	Hawkins, JR Mutational analysis of SRY in XY females Human Mutation, 2 (1993),pp. 347-350
[6]	Burtis, KC, Baker, et al. Drosophila doublesex gene controls somatic sexual differentiation by producing alternatively spliced mRNAs encoding related sexspecific polypeptides Cell, 56 (1989),pp. 997-1010
[7]	Shen, MM, Hodgkin, et al. Mab-3, a gene required for sex-specific yolk protein expression and a male-specific lineage in C. elegans Cell, 54 (1988),pp. 1019-1031
[8]	Smith, CA, MeCLive, et al. Conservation of a sex-determining gene Nature, 402 (1999),pp. 601-602
[9]	Raymond, CS, Shamu, et al. Evidence for evolutionary conservation of sex-determining genes Nature, 391 (1998),pp. 691-695
[10]	Raymond, CS, Parker, et al. A region of human chromosome 9p required for testis development contains two genes related to known sexual regulators Human Moecular Genetics, 8 (1999),pp. 989-996
[11]	Raymond, CS, Murphy, et al. DMRT1, a gene related to worm and fly sexual regulators, is required for mammalian testis differentiation Genes Development, 14 (2000),pp. 2587-2595
[12]	Guan, GJ, Kobayashi, et al. Sexually dimorphic expression of types of DM (Doublesex-Mab-3)domain genes in a teleost fish, the tilapia (Oreochromis niloticus) Biochemal and Biophysical Research. Communication, 272 (2000),pp. 662-666
[13]	Ellegren, H Hens cocks and avian sex determination: A quest for genes on Z or W. EMBO Reports, 2 (2001),pp. 192-196
[14]	Ren, I, Cheng, et al. Evolutionary conservation of Dmrt gene family in amphibians reptiles and birds Chinese Science Bulletin, 46 (2001),pp. 1992-1996
[15]	Nanda, I, Shan, et al. 300 million years of conserved synteny between chicken Z and human chromosome 9 Natural Genetics, 21 (1999),pp. 258-259
[16]	Nanda, L, Kondo, et al. A duplicated copy of DMRT1 in the sex-determining region of the Y chromosome of the medaka, Oryzias latipes Proc Natl Acad Sci USA, 99 (2002),pp. 11778-11783
[17]	Devlin, RH, Nagahama, et al. Sex determination and sex differentiation in fish: an overview of genetic, physiological, and environmental influences Aquaculture, 208 (2002),pp. 191-364
[18]	Kondo, M, Froschauer, et al. Molecular cloning and characterization of DMRT genes from the medaka Oryzias and the platyfish Xiphophorus maculatus Gene, 295 (2002),pp. 213-222
[19]	Koopman, P, Loffler, et al. Sex determination: The fishy tale of DMRT1 Current Biology, 13 (2003),pp. 177-179
[20]	Kobayashi, T, Matsuda, et al. Two DM domain genes, DMY and DMRT1, involved in testicular differentiation and development in the medaka, Oryzias latipes Developmental Dynamics, 231 (2004),pp. 518-526
[21]	Shui, Y, Yu, et al. Cloning of four members of giant panda Dmrt genes Acta Genetica Sinica., 31 (2004),pp. 468-473
[22]	Flejter, WL, Fergastad, et al. A gene involved in XY sex reversal is located on chromosome 9, distal to marker D9S1779 Human Genetics, 63 (1998),pp. 794-802
[23]	Sakamoto, T, Danzmann, et al. A microsatellite linkage map of rainbow trout characterized by large sex-specific differences in recombination rates Genetics, 155 (2000),pp. 1331-1345
[24]	Raymond, CS, Kettlewell, et al. Expression of DMRT1 in the genital ridge of mouse and chicken embryos suggests a role in vertebrate sexual development Developmental Biology, 215 (1999),pp. 208-220
[25]	Grandi, AD, Calvari, et al. The expression pattern of a mouse doublesex-related gene is consistent with a role in gonadal differentiation Mechanics of Development, 90 (2000),pp. 323-326
[26]	Moniot, B, Berta, et al. Male specific expression suggests role of DMRT1 in human sex determination Mechanics of Development, 91 (2000),pp. 323-325
[27]	Kim, S, Kettlewell, et al. Sexually dimorphic expression of multiple doublesex-related genes in the embryonic mouse gonad. Gene Express Patterns, 3 (2003),pp. 77-82
[28]	Kettlewell, JR, Raymond, et al. Temperature-dependent expression of turtle DMRT1 prior to sexual differentiation Genetics, 26 (2000),pp. 174-178
[29]	Marchand, O, Govoroun, et al. DMRT1 expression during gonadal differentiation and spermatogenesis in the rainbow trout, Orrcorhynchus mykiss Biochemical Biophysics Acta, 1493 (2000),pp. 180-187
[30]	Ohmuro-Matsuyama, Y, Matsuda, et al. Expression of DMY and DMRT1 in Various Tissues of the Medaka(Oryzias Latipes) Zoological Science, 20 (2003),pp. 1395-1398
[31]	Zhao, QG, Zhou, et al. Generation of mouse FANCL antibody and analysis of FANCL protein expression profile in mouse tissues Acta Genetica Sinica, 33 (2006),pp. 49-55
[32]	Sambrook, J, Fritsch, et al.
[33]	Biolabs. pMALTMprotein fusion and purification system. New England, Inc.2001.
[34]	Kobayashi, T, Chang, et al. Fish 3f-hydroxysteroid dehydrogenase/D5- D4 isomerase: Antibody production and their use for the immunohistochemical detection of fish steroidogenic tissures Zoological Science, 13 (1996),pp. 909-914
[35]	Kobayashi, T, Nakamura, et al. Immunolocalization of steroidegenic enzymes (P450scc, P450c17, P450arom, and 3f-HSD) in immature and mature testes of rainbow trout (Oncorhynchus mykiss) Cell Tissue Research, 292 (1998),pp. 573-578
[36]	Kobayashi, T, Kajiura-Kobayashi, et al. A novel stage-specific antigen is expressed only in early stages of spermatogonia in Japanese eel, Anguila japonica testis Molecular Reproductive Development, 51 (1998),pp. 355-361
[37]	Erdman, SE, Burtis, et al. The Drosophila doublesex proteins share a novel zinc finger related DNA binding domain EMBO J, 12 (1993),pp. 527-535
[38]	Erdman, SE, Chen, et al. Funtional and genetic characterization of the oligomerization and DNA binding properties of the Drosophila doublesex proteins Genetics, 144 (1996),pp. 1639-1652
[39]	Yamaguchi, A, Lee, et al. Expression of the DMRT gene and its roles in early gonadal development of the Japanese pufferfish Takifugu rubripes. Comparative Biochemistry and Physiology, Part D: Genomics and Proteomics, 1 (2006),pp. 59-68
[40]	Brunner, B, Hornung, et al. Genomic organization and expression of the Doublesex-related gene cluster in vertebrates and detection of putative regulatory regions for DMRT1 Genomics, 77 (2001),pp. 8-17