|
内容記述 |
In nonmammalian vertebrates, estradiol-17β a major estrogen in all vertebrates, is responsible for the growth of oocytes, vitellogenesis. The cytochrome P-450 aromatase (P-450arom) is an important steroidogenic enzyme which converts testosterone to estradiol- 17β. In contrast to well-known physiological functions of estradiol-17β, very little is known about the molecular mechanisms of P-450arom activation in ovarian follicles during oocyte growth.<br /> The medaka, Oryzias latipes, under the conditions of 26℃, 14 hours light and 10 hours dark, usually spawn within 1 hour of the onset of light for several consecutive days. The majority of vitellogenesis and meiotic maturation of individual oocytes occurs within 48 hours; germinal vesicle breakdown and ovulation being completed at 6 and 1 hour before spawning, respectively. Thus, medaka follicles provide an excellent model for understanding the hormonal regulation of follicular growth and maturation. Previous studies in our laboratory involving incubations of isolated medaka ovarian follicles with gonadotropins, forskolin and dbcAMP which are known to raise the cellular level of cAMP, and/or actinomycin D suggest that P-450arom activity is<br />transcriptionally regulated. The aim of this study is to elucidate the regulatory mechanism of time-specific P-450arom transcription responsible for estradil-17β production by medaka ovarian follicles during active vitellogenesis.<br /> Promoter analysis of the medaka P-450arom gene identified putative orphan nuclear receptor binding sites (Ad4-1, Ad4-2). To clone orphan nuclear receptors from medaka ovarian follicle, RT-PCR was performed using degenerate primers designed against highly conserved amino acid sequences in the DNA binding region of orphan nuclear receptors. Seven distinct fragments encoding putative RXR, Ad4BP/SF-1, TAK1, PPARα, PPARγ, COUP-TF and Rev-erb were amplified. RNase protection assays using cRNA probes prepared against these seven fragments were used to investigate the expression profile of each fragment in medaka ovarian follicles during various stages of oogenesis; total RNAs from follicles collected at 35, 29, 23, 11 and 5 hours before the onset of light as well as the previtellogenic stage were used. The results showed that only the Ad4BP/SF-1 homologue gave a clear expression profile which correlated well with P-450arom expression.<br /> Based upon these data, an attempt was made to alone a full length cDNA enconding the Ad4BP/SF-1 homologue by library screening and 5' RACE. The 1,458 bp open reading frame of this cDNA is predicted to encode a 486 amino acid polypeptide. Sequence analysis showed high homology of this cDNA clone to Ad4BP/sf-1 and Ftz-F1s from various species. The DNA binding region, FTZ-F1 box, and AF-2 domain showed almost 100% homology with Ad4BP/SF-1 and LRH-1/FTF. Therefore, we designated the medaka clone as medaka Ftz-F1 cDNA (mdFtz-F1).<br /> There are two groups of FTZ-F1 family proteins. One, the Ad4BP/SF-1 group, is mainly expressed in steroidogenic tissues. The other is the LRH-1/FTF group which is expressed in liver. Despite a close similarity of the primary structure of mdFTZ-F1 to that of either FTZ-F1 group, tissue distribution analysis indicating that mdFtz-F1 transcripts are highly expressed in ovary and testis with weak signals in brain, spleen and kidney, suggest that mdFTZ-F1 functions similar to Ad4BP/SFI.<br /> To investigate whether mdFTZ-F1 can specifically bind to putative orphan nuclear receptor binding sites of P-450arom gene, get shift assays were performed. mdFTZ-F1 translated in reticulocyte lysate bound the Ad4-1 and Ad4-2 sites. The band disappeared in the presence of 50 times excess cold competitor, demonstrating the ability of mdFTZ-F1 to specifically bind to the Ad4-1 and Ad4-2 sites on the P-450arom promoter.<br /> A series of transfection experiments were performed to investigate the mdFTZ-F1 function the P-45Oarom promoter in living cells. In these experiments, P-450arom promoter-reporter constructs having the P-450arom promoter region upstream of firefly luciferase and an mdFtz-F1 expression vector which is driven by the SV40 promoter in CV- 1 mammalian cells were used. Luciferase activity increased when the mdFtz-F1 expression vector were co-transfected. He also prepared deletion reporter constructs to identify the essential region for the transcriptional activation by mdFTZ-F1. Luciferase activity dropped when the Ad4-1 and Ad4-2 sites were deleted. To further investigate the importance of the Ad4-1 and Ad4-2 sites, these sites were mutated such that mdFTZ-F1 could not bind to them. Single or double mutation of the Ad4-1 and Ad4-2 sites caused decreased reporter activity. These results suggest that the P-450arom promoter can be positively regulated by specific binding of mdFTZ-F1 to the Ad4-1 and Ad4-2 sites.<br /> The expression profile of mdFtz-F1 transcripts during oogenesis was analyzed by RNase protection assay. The expression profile of mdFtz-F1 correlated with the P-450arom expression profile which also supports the hypothesis that mdFTZ-F1 may regulate P-450arom.<br /> To investigate whether there is specific binding activity to the Ad4-1 and Ad4-2 in medaka ovarian follicles, get shift assays using nuclear extracts prepared from medaka ovarian follicles at the time (23 hours before the onset of light) when the transcript level of mdFtz-F1 peaks were performed. Three bands appeared when the nuclear extracts were incubated with the radio-labeled Ad4-1 or Ad4-2. One of these bands appeared at the same position as the band which appeared when reticulocyte-expressed mdFtz-F1 was incubated with Ad4-1 or Ad4-2. These three bands disappeared simultaneously in the presence of 50 times excess of cold competitor. When a nuclear extract from medaka ovarian follicles at the time of oocyte maturation was incubated with the radio-labeled Ad4-1 or Ad4-2, no specific band appeared. These results suggest that there is specific binding activity to the Ad4-1 and Ad4-2 in vivo at the time of vitellogenesis. Furthermore, this binding activity disappears concomitant with oocyte maturation.<br /> In summary, the results of this study suggest a potential role of mdFTZ-F1 on the time-specific transcriptional regulation of P-450arom in medaka ovarian follicles. This is the first investigation with respect to the transcriptional regulation of P-450arom in ovarian follicles during oogenesis. Further study of other transcription factors which synergistically act with mdFTZ-F1 or modifications of mdFTZ-F1 such as phosphorylation will reveal the molecular detail of the intracellular signaling pathways which regulate P-450arom transcription in response to gonadotropin. |
要旨・審査要旨 / Abstract, Screening Result (377.8 kB)
