| Sexual development is a fundamental process of life, which is a regulatory cascade of spatial-temporal expression of multiple genes. It is also a promising and interesting model for study of organogenisis. Although several mechanisms have been employed for explanation of sex determination/differentiation, including genotypic, temperature-dependent, and behavior-dependent sex determination, molecular and evolutionary mechanisms are still not completely understood.The rice field eel, Monopterus albus, one of fresh-water fishes, has a characteristic of natural sexual reversal from female via intersex into male during its life. Medaka, Oryzias latipes, is a small, egg-laying freshwater fish, which has XY chromosomes. Therefore these two species have been used as ideal models for study of sex determination and differentiation, which will be informative to elucidate mechanisms of human sex determination/differentiation, and to provide theoretic bases for prevention and treatment of sex-related diseases. This paper presents first discovery of alternative splicing events occurred in gonads: P450cl7(CYP17) gene in rice field eel, and DMY and Dmrtl in medaka.1 Alternative splicing of P450c17 in rice field eel-cloning, gene structure and expression patternsSMART technique was used to synthesize the cDNA from different gonads in rice field eel, and four different isoforms of the CYP17 genes (GenBank numbers: AY224681 - AY224684), CYP17-al (3312bp) CYP17-a2 (1934bp) CYP17-b (1676bp ) CYP17-c (1121 bp ), were obtained by using RACE strategy, which were generated in gonads by alternative splicing and polyadenylation. Alternative splicing events of all these isoforms occurred in 3' regions, which encoded three different sizes (517 aa, 512 aa and 159 aa) of proteins. Furthermore, P450 domain is highly conserved, especially near to 5' regions.Basis on sequences of four different CYP17 isforms, specific primers were designed to carry out expression patterns. RT-PCR results indicate mainly expression in ovary, testis and ovotestis of these isoforms, and little expression of CYP17-c also in brain, but not in other tissues, such as heart, liver and kidney, which can identify in gonads, and are closely related to sexual development.Northern blot analysis was used to investigate differences in expression among the three forms of gonads. Total RNA(10^g, respectively) extracted from ovary, testis and ovotestis, which were separated in 1.0% sugar gel, probes labeled by [ a -32p] dCTP who is product of 3'-RACE in CYP17-a2 of rice field eel including conserved P450 domain, were prepared in the course of Northern blotting using ULTRAhyb solution (Ambion) under 42 C and more than 16hrs. Dominant expression of the 3.3 kb form (CYP17-a1) was observed in testis, less in ovary, and at low levels in ovotestis, arid another band of 1.7 kb (CYP17-b) was also observed. A very faint band was detected between the bands of 1.7 and 3.3 kb (CYP17-a2), while CYP17-C was not detected by Northern blot analysis.In order to gain insight into the role of the CYP17 gene in sex differentiation in this species, we analyzed the gene expression patterns in the three forms of gonads by in situ hybridization to gonad sections. Antisense and sense RNA probes were prepared separately from a region includingP450 domain of CYP17-a2 (1.6 kb) of the rice field eel and labeled with digoxigenin-UTP, using SP6 or T7 RNA polymerase separately (SP6 for production of sense probe, T7 for antisense probe). Gonads tissues were cryosectioned and the sections were immediately hybridized (42C) and hybridization signals were detected by NBT/BCIP system according to the manufacturer's instructions (Boehringer). It shows its specific expression in germinal lamellae, the gonadal epithelium of the gonads, but not in developing germs. In addition, strong signal of testis is detected, then ovary, and very low in ovotestis, which are consistent with the results of Northern blotting.Neighbour joint (NJ) method is employed in the course of Phylogenetic analysis by using clustalx (...
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