| Gobiocypris rarus, a tiny fish in China, had not been reported on its mechanism of sex determination and sex differentiation. To address the role of Foxl2 in sex determination in the Gobiocypris rarus, we clone the full length of Foxl2 cDNA from the Gobiocypris rarus ovary by RACE. The full-length cDNA of Foxl2 is 1700bp, which comprises a 221bp 5’ untranslated region (UTR), a 558bp 3’-UTR; a 921bp open reading frame (ORF) encoding a 306 amino acids peptide, and contains a typical DNA binding domain (FH-domain). Phylogenetic analysis with other species shows that the FH-domain of Gobiocypris rarus FOXL2 is highly conserved and the C-terminal region is more conserved than the N-terminal region. In addition, similar to the FOXL2s of other non-mammals, Gobiocypris rarus FOXL2 contains neither the 14-polyalanine tract nor the glycine-raline repeats. Semi-quantitative RT-PCR is employed to reveal the spacial expression pattern of Foxl2 in Gobiocypris rarus:it is expressed at a high level in the eyes, brain, gill and gonad and at a very low level in liver, intestines and muscle. Importantly, the expression level in the ovary is significantly higher than that in testis. This expression pattern indicates that Foxl2 is possibly involved in the brain-pituitary-gonad axis. We use Real-Time PCR to detect the Foxl2 expression level in Gobiocypris rarus treated with 2-methyl-testosterone (MT) and estradiol (E2). In the E2 treated group, Foxl2 was up-regulated, while the expression of Foxl2 was down-regulated in the MT treated group. This result suggests that the expression of Foxl2 is regulated in a feedback pattern by downstream sex hormones.Maternal mRNAs, which are expressed in oocytes, play an important role in the success of early embryonic development. Zygote arrest 1(Zar1) and Zar1-like (Zar1L) are the oocyte-specific maternal genes that function at the oocyte-to-embryo transition in mouse. Zar1 and Zar1L are conserved in evolution, but the expression patterns of Zar1 and Zar1L are diverse in animal kingdom. In this experiment, the full length of Zar1L cDNA was cloned from the zebrafish ovary by RACE. The expression patterns of Zar1 and Zar1L in different tissues, developing oocytes and embryogenesis were checked by RT-PCR. The full-length cDNA of Zar1L is 1146bp, which comprises a 20bp 5’ untranslated region (UTR), a 190bp 3’-UTR; a 936bp open reading frame (ORF) encoding a 311 amino acids peptide. Structurally, the Zar1L protein contains a atypical PHD motif (plant homeo domain) and two C4 type zinc finger in the C-terminal, and showing 74% homolgy to known counterparts from mammals and birds. In gene structure, Zar1 and Zar1L have four exons, each of the two genes exons are relatively similar in size. It shows that zebrafish Zar1 and Zar1L gene belong to two different genes, and it may be the ancestors of the same source gene duplication and evolution. Zar1 and Zar1L are expressed at a similar pattern, and both of them are ovary specific. The expressions of Zar1 and Zar1L did not change during oogenesis. Both Zar1 and Zar1L mRNA are maternal deposited, and the expression levels of both genes were high in the early embryos till gastrulation. A significant decrease of Zar1L occurred at gastrula, while Zar1 decreased apparently at neurula. However, both of Zar1 and Zar1L are expressed in the whole process of embryonic development till hatching. The consistency of expression level of Zebrafish Zar1 and Zar1L, seems to imply that the function of the two genes are basically the same.The results suggest that Zar1 and Zar1L are maternal factors for mid-blastula transition, and Zar1 and Zar1L may have other important roles during embryonic development. |
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