| All oganisms with sexual reproducation undergo meiosis in order to produce male and female gametes. Therefore, the meiotic mechanism is one of the most basic biological issues. Meiosis is also an important, intensively studied and most difficult area in aquaculture, cell biology, genetics, physiology, developmetal biology and molecular biology.In recent years, great progress has been achieved in the initiation of meiosis in mammals and birds. Retinoic Acid (RA) was found to play decisive roles in the initiation of meiosis, and Stra8 (Stimulated by Retinoic Acid gene 8), which is specificly expressed in germ cells while transforming from mitosis to meiosis, play key roles in the RA signal pathway. Up to now. Stra8 was only reported in mammals and birds. Though RA was also found to play a crucial role in the meiosis of amphibians, Stra8 was not isolated from it. Very little is known about the molecular mechanism governing meiosis in fish.In the present study, the orthologous gene of Stra8 was identified from three species of fish (channel catfish, rainbow trout and salmon) using bioinformatics. Stra8 (sc-Stra8) was cloned from Southern catfish(Silurus meridionalis) by RT-PCR and RACE; tissue distribution analysis was performed by RT-PCR. The recombinant protein of sc-Stra8 was expressed with Pcold I in E.coli, purified and the antibody was produced in mouse. Luciferase assays were performed to study the effects of estrogen and transcription factors, including Sf1, Foxl2. Dmrt1 and Wt1. on the transcriptional activity of sc-Stra8. The results are as follows. The cloned sc-Stra8 cDNA sequences include 163 bp 5'-UTR (untranslated region),456 bp 3'-UTR, and an ORF (open reading frame) of 987 bp, encoding a polypeptide of 328 aa. Sequence alignment showed very low similarity between sc-Stra8 and the Stra8 of mammals and birds,32.0% and 27.4%, respectively. The low conservation of Stra8 suggested their differences in function.Tissue distribution analysis showed that sc-Stra8 is expressed specificly in gonads, suggesting it possible participation in the initiation of meiosis. No apparent sexual dimorphism was detected between the testis and ovary of the adult catfish. This result was different from the mammalian Stra8 which was found to be specifically expressed in the testis of the adult mouse.The sc-Stra8 recombinant protein with His-tag at the N-terminal was obtained by prokaryotic expression. The molecular weight of sc-Stra8 recombinant protein was 43KD (kilodalton). The recombinant protein was purified and the antibody was prepared in mouse. Because of the low expression of sc-Stra8, its mRNA signal was not detected by in situ hybridization. Therefore, antibody preparation provides a way to study the cell type of Stra8.The 1.4 kb promoter of sc-Stra8 was cloned by genome walking, and it was subcloned into pGL3 promoter-luciferase reporter vector. Meanwhile, Sf1, Foxl2, Dmrt1 (two alternatively spliced variants Dmrt1a and Dmrt1b), Wt1 and three estrogen receptor (ERs) were subcloned into pcDNA3.1 vector. Luciferase assays were performed in CHO cells. Dmrtla was found to enhance the transcription of sc-Stra8; while Dmrt1b suppressed the transcription of sc-Stra8 in a dose-dependent manner. The other transcription factors tested also showed little suppression effect on the sc-Stra8, suggesting that some unknown factors maybe responsible for sc-Stra8 expression.In summary, the orthologous genes of Stra8 of higher vertebrates were isolated from the EST sequences of three fish species and it was cloned from Southern catfish. This is for the first time where Stra8 was isolated in any lower vertebrates. Tissue distribution analysis showed that Stra8 was expressed in gonads exclusively, suggesting its possible involvement in the initiation of meiosis as reported in higher vertebrates. Promoter analysis demonstrated that in various transcriptional factors tested, Stra8 was mainly regulated by Dmrt1, similar to that found in mammals.
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