| FOXL2is one of the forkhead box transcription factor family members, whichhas been detected in many species. However, studies in depth and systematicness onfoxl2only have been carried out in higher mammals, and revealed that FOXL2isinvolved in ovarian determination and/or differentiation as well as functionalmaintenance. Until now, limited data were reported on foxl2genes in invertebrates,and no data indicated the gene is female-related gene in invertebrate.In this study, scallop Chlamys farreri, a commercially important aquatic productwas chosen as the expereimental animal. The full-length cDNA sequences of the C.farreri foxl2(Cf-foxl2) was cloned and its sequence characteristics was analysed.Furthermore, RT-PCR was used to detected the spatial expression in different tissuesof adult at proliferative stage; real-time PCR and in situ hybridization were employedto analyse the expression pattern in C. farreri during ontogenesis and gonadicreproductive cycle. Moreover, effect of exogenous estradiol-17β on the Cf-foxl2expression level was detected. Finally, Cf-folx2function in mollusks was probedprimarily.The full-length sequence of Cf-foxl2cDNA has1824bp with an open readingframe of1107bp encoding369amino acid residues containing the conserved domainforkhead box, a putative NLS sequences was characterized by RRRRRMRR and thethree-dimensional protein structure was predicted, containing three α-helices and twowing-like structure.RT–PCR showed that Cf-foxl2is specifically expressed in ovary of theproliferative stage and a weakly expression in hepatopancreas, but is not detected inother organs including the testis. The result showed primarily that the Cf-foxl2expresses in a sexually dimorphic pattern. Using quantitative real-time PCR, it was found that Cf-foxl2mRNA is amaternal product and the highest expression during the development is detected inD-shaped larvae in which the larval structure is rapidly established, and then itdeclines significantly. Until the sex of the juvenile is differentiated, the expression ofCf-foxl2is increased significantly. In situ hybridization revealed that Cf-foxl2mRNAare evenly distributed at various stages before trochophore, then concentrate in theventral surface near the mouth concave and exists symmetrically in trochophore.Intensity of the signals enhances significantly in D-shaped larvae, focuses on thevisceral mass and the mantle margin, after that the signals are weakened to disappear.When the shell height reaches to9mm, male and female can be distinguished basedon the histological structure, the signals are located in the cytoplasm of follicle cellsand germ cells in the ovary, but no signal is detected in testis. The result waspresumed that Cf-foxl2is the early marker gene of the ovary phenotype formation inscallop, and participates in regulation of ovary differentiation.Using quantitative real-time PCR, the Cf-foxl2mRNA expression was comparedbetween the ovary and the testis during the reproductive cycle. The result showed thatthe expression level in the ovary is significantly higher than that in the testis at thesame stage, and Cf-foxl2presents a significant sexually dimorphic expression pattern,such as the highest expression is detected in the ovary of proliferative stage, about62-fold higher than that in the testis; in ovary, about2-fold higher in that ofproliferative stage than in that of growing and mature stages. The expression pattern issimilar to that in mouse (Mus musculus) and medaka (Oryzias latipes). In situhybridization of adult scallop revealed that Cf-foxl2mRNA is located in the same locias that of the juvenile, but weak signals are also detected in testis except in thespermatozoa. Interestingly, Signals of sense probes are presented in the same loci asthat of antisense probes in the adult ovary and testis, which is different from the resultin the embryos, the larvae and the juveniles. It was hypothesized that antisense RNAof Cf-foxl2might be present in adult scallop, and might act in vivo as a factor toinvolve in the regulation of Cf-foxl2in the adult scallop.By injecting estradiol-17β into adductor muscle of the scallop, the effect of the estradiol-17β on Cf-foxl2mRNA expression level is detected. The result showed theCf-foxl2mRNA expression levels in ovary and testis are significantly increased. Theresult implied that estradiol-17β can promote the Cf-folx2expression at mRNA leveland a feedback relationship may exist.In summary, the forkhead box of Cf-foxl2is high conservative with that in otherspecies. Its expression pattern during the development indicated that Cf-foxl2ismaternal gene and is the early marker gene in the formation of ovary phenotype. AndCf-foxl2presents a sexually dimorphic expression pattern in adult scallop which isconserved with vertebrate. Exogenous estradiol-17β can promote the Cf-foxl2RNAsynthesis. More extensive studies should be performed to conclude the function ofCf-foxl2in ovary and oogenesis of the scallop. |
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