Identification and Characterization of Cryptochrome4 in the Ovary of Western Clawed Frog Xenopus tropicalis

Abstract

CRY proteins can be classified into several groups based on their phylogenetic relationships, and they function as a photoreceptor, a photolyase, and/or a transcriptional repressor of the circadian clock. In order to elucidate the expression profile and functional diversity of CRYs in vertebrates, we focused on XtCRY4, a member of the uncharacterized cryptochrome family CRY4 in Xenopus tropicalis. XtCRY4 cDNA was isolated by RT-PCR, and a phylogenetic analysis of deduced sequence of XtCRY4 suggested that the vertebrate Cry4 genes evolved at much higher evolutionary rates than mammalian-type Cry genes, such as the CRY1 and CRY2 circadian clock molecules. A transcriptional assay was performed to examine the transcriptional regulatory function as circadian repressor, and XtCRY4 had marginal effects on the transactivation of XtCLOCK/XtBMAL1 via E-box element. In situ hybridization and quantitative RT-PCR was performed to detect mRNA expression in native tissues. Quantitative RT-PCR revealed that XtCry4 mRNA was highly transcribed in the ovary. In situ hybridization showed the presence of XtCry4 transcripts in the oocytes, testis, renal tubules, the visual photoreceptors, and the retinal ganglion cells. A specific antiserum to XtCRY4 was developed to detect endogeneous expression of XtCRY4 protein in the ovary. The expression level was estimated by immunoblot analysis, and this is the first detection and estimation of endogenous expression of CRY protein in the ovary. These results suggest that X. tropicalis ovary may respond to blue-light by using XtCRY4.