{"created":"2023-06-20T13:21:11.717347+00:00","id":1312,"links":{},"metadata":{"_buckets":{"deposit":"2916044d-7311-461f-8f92-4d5528d67217"},"_deposit":{"created_by":1,"id":"1312","owners":[1],"pid":{"revision_id":0,"type":"depid","value":"1312"},"status":"published"},"_oai":{"id":"oai:ir.soken.ac.jp:00001312","sets":["2:430:27"]},"author_link":["9578","9576","9577"],"item_1_creator_2":{"attribute_name":"著者名","attribute_type":"creator","attribute_value_mlt":[{"creatorNames":[{"creatorName":"小林, 大介"}],"nameIdentifiers":[{}]}]},"item_1_creator_3":{"attribute_name":"フリガナ","attribute_type":"creator","attribute_value_mlt":[{"creatorNames":[{"creatorName":"コバヤシ, ダイスケ"}],"nameIdentifiers":[{}]}]},"item_1_date_granted_11":{"attribute_name":"学位授与年月日","attribute_value_mlt":[{"subitem_dategranted":"1996-03-21"}]},"item_1_degree_grantor_5":{"attribute_name":"学位授与機関","attribute_value_mlt":[{"subitem_degreegrantor":[{"subitem_degreegrantor_name":"総合研究大学院大学"}]}]},"item_1_degree_name_6":{"attribute_name":"学位名","attribute_value_mlt":[{"subitem_degreename":"博士（理学）"}]},"item_1_description_12":{"attribute_name":"要旨","attribute_value_mlt":[{"subitem_description":"　　　In nonmammlian vertebrates, ovarian follicle cells produce two different steroid hormones, estradiol-17β and maturation-inducing hormone(progestogens), in response to pituitary gonadotropins, which play important roles in two phases of oogenesis, vitellogenesis and oocyte maturation, respectively. Estradiol-17β promotes vitellogenesis in members of all　nonmammalian vertebrates. On the other hand, a variety of progestogens have been shown to be effective in the initiation of meiotic maturation in fish and amphibian oocytes. These ovarian steroid hormones are known to be secreted from ovarian follicles in response to pituitary gonadotropins. A distinct shift from estradiol-17β to 17α, 20β-dihydroxy-4-pregnen-3-one (17α, 20β-DP, the maturation-inducing hormone of salmonid fishes) has been reported to occur in ovarian follicles immediately prior to oocyte maturation, and seems to be a prerequisite step for growing oocytes to enter the final stage of maturation. However, the regulatory mechanism of this steroidogenic shift is not clearly understood. <br />　　　The medaka, 0ryzias latipes, under a long photopeiod (14 hours light-10 hours dark) at 26℃, spawns daily within 1 hr of the onset of light for a number of consecutive days. Under these conditions, the sequence of events leading to spawning such as vitellogenesis, oocyte maturation, and ovulation can be timed accurately. These features make medaka an ideal model in which to investigate the regulatory mechanism of the steroidogenic shift occurring in ovarian follicles prior to oocyte maturation. <br />　　　During vitellogenesis, estradiol-17βis secreted from medaka ovarian follicle cell layers and I7α , 20β-DP was identified as the most potent steroid　to induce oocyte maturation. During oocyte maturation, 20β-hydroxysteroid　dehydrogenase (20β-HSD) activity catalyzing the production of 17α, 20β-DP from 17α-hydroxyprogesterone is highin medaka ovarian follicle cell layers. Whereas 20β-HSD activity is kept undetectable during the vitellogenic stage. Instead, cytochrome P-450 aromataseactivity is high, leading to the production of estradiol-17β. However, the keyenzyme to switch this pathway is unknown. This investigation would require the precise information on steroidogenic pathway in medaka ovarian follicle cell layers. <br />　　　In this study, changes in the steroidogenic pathway in medaka ovarian follicles during vitellogenesis and oocyte maturation were investigated in vitro by incubation of follicles with several radiolabeled steroid precursors, followed by thin layer chromatography (TLC) fractionation and recrystallizaion. When vitellogenic follicles collected at 18 hours before the expected time of spawning (vitellogenic follicles) were incubated with <SUP>3</SUP>H-labeled pregnenolone,  the major metabolites were 17α-hydroxypregenenolone, 17α-hydroxyprogesterone, and androstenedione. Incubations of vitellogenic follicles with androstenedione produced testosterone and estradiol-17β. By contrast, when maturing follicles (postvitellogenic follicles undergoing maturation) collected at 10 hours before spawning were incubated with <SUP>3</SUP>H-labeled pregnenolone, the  major metabolites were 17α-hydroxypregnenolone, 17α-hydroxyprogesterone, and 17α, 20β-DP; androstenedione was not detected. Neither vitellogenic and maturing follicles produced progesterone when they were incubated with <SUP>3</SUP>H-labeled pregnenolone, suggesting that in medaka ovarian follicles both estradiol-17β and 17α, 20β-DP are synthesized by the <SUP>5</SUP>△-steroid pathway. Thus, there is a distinct shift in the steroidogenic pathway from estradiol-17β to 17α, 20β-DP production in medaka ovarian follicles, and it is suggested that the decrease in C17, 20-lyase activity is responsible for this shift. <br />　　　Next he attempted to mimic the decrease of C17, 20-lyase activity, by addition of several reagents. His experiments using PMSG (pregnant mare serum gonadotropin) demonstrated that gonadotropin is probably not directly responsible for controlling this shift. On the other hand, the phosphodiesterase inhibitor IBMX enhanced androstenedione production in incubations of vitellogenic follicles with <SUP>14</SUP>C-labeled progesterone. This may suggest that IBMX stimulates androstenedione production not by cAMP mediated signal transduction pathway alone, but by the involvement of other pathways affected by inhibition of phosphodiesterase activity. Calcium ionophore A23187 and the phorbol ester TPA (a protein kinase C activator) blocked the stimulatory actions of IBMX on androstenedione production. These findings suggest that multiple signalling pathways may participate in the regulation of ovarian steroidogenesis, and further emphasize the importance of calcium as a regulator of P-450c17 activity. <br />　　　A rapid decrease in C17, 20-lyase activity appeared to be one of the critical steps in the shift of the steroidogenic pathway during oogenesis. C17, 20-lyase activity is supported by P-450cl7 geneproduct. Interestingly, a single product of the cytochrome P-450c17 (P-450c17) gene has both l7α-hydroxylase and C17, 20-lyase activities in several species. The mechanism by which a single polypeptide regulates these two distinct activities differentially through oogenesis remains to be elucidated. To address this problem at the molecular level, he cloned P-450c17 cDNAs from medaka ovarian follicle cells. This study confirms that two types of cytochrome P-450c17 transcripts are present in medaka ovarian follicles. One type (P-450cl7L) is similar in structure to those reported in other species. The second type (P-450cl7S) is a novel type of P-450cl7cDNA which lacks exon 4. These two types of transcripts are synthesized by alternative splicing from a single copy gene. Both transcripts were abundant in medaka gonads but absent in other tissues. Throughout the course of oogenesis, both transcripts are present during vitellogenesis and disappear at maturation. This prosperity and declin are correlated with the changes in stroidogenic activities in medaka ovarian follicle cells. Expression studies demonstrate that P-450cl7L transcripts result in an enzyme with 17α-hydroxylase and C17, 20lyase activities. Expression of P-45017S result in no detectable enzyme activity.  <br />　　　His preriminary experiments showed that cytochrome b<SUB>5</SUB> stimulated P-450c17L product activities as those reported in other species. This observation indicates that cytochrome b<SUB>5</SUB> may be involved in the steroidgenesis shift in medaka ovarian follicle cells.","subitem_description_type":"Other"}]},"item_1_description_18":{"attribute_name":"フォーマット","attribute_value_mlt":[{"subitem_description":"application/pdf","subitem_description_type":"Other"}]},"item_1_description_7":{"attribute_name":"学位記番号","attribute_value_mlt":[{"subitem_description":"総研大甲第217号","subitem_description_type":"Other"}]},"item_1_select_14":{"attribute_name":"所蔵","attribute_value_mlt":[{"subitem_select_item":"有"}]},"item_1_select_8":{"attribute_name":"研究科","attribute_value_mlt":[{"subitem_select_item":"生命科学研究科"}]},"item_1_select_9":{"attribute_name":"専攻","attribute_value_mlt":[{"subitem_select_item":"X2 分子生物機構論専攻"}]},"item_1_text_10":{"attribute_name":"学位授与年度","attribute_value_mlt":[{"subitem_text_value":"1995"}]},"item_creator":{"attribute_name":"著者","attribute_type":"creator","attribute_value_mlt":[{"creatorNames":[{"creatorName":"KOBAYASHI, Daisuke","creatorNameLang":"en"}],"nameIdentifiers":[{}]}]},"item_files":{"attribute_name":"ファイル情報","attribute_type":"file","attribute_value_mlt":[{"accessrole":"open_date","date":[{"dateType":"Available","dateValue":"2016-02-17"}],"displaytype":"simple","filename":"甲217_要旨.pdf","filesize":[{"value":"319.7 kB"}],"format":"application/pdf","licensetype":"license_11","mimetype":"application/pdf","url":{"label":"要旨・審査要旨 / Abstract, Screening Result","url":"https://ir.soken.ac.jp/record/1312/files/甲217_要旨.pdf"},"version_id":"c4e987a0-7458-4c34-ba55-2a15edf633f6"},{"accessrole":"open_date","date":[{"dateType":"Available","dateValue":"2016-02-17"}],"displaytype":"simple","filename":"甲217_本文.pdf","filesize":[{"value":"9.1 MB"}],"format":"application/pdf","licensetype":"license_11","mimetype":"application/pdf","url":{"label":"本文","url":"https://ir.soken.ac.jp/record/1312/files/甲217_本文.pdf"},"version_id":"f53c7b4c-d7e6-44e8-8698-f1c8d70be987"}]},"item_language":{"attribute_name":"言語","attribute_value_mlt":[{"subitem_language":"eng"}]},"item_resource_type":{"attribute_name":"資源タイプ","attribute_value_mlt":[{"resourcetype":"thesis","resourceuri":"http://purl.org/coar/resource_type/c_46ec"}]},"item_title":"Ovarian Cytochrome P-450 17α-Hydroxylase/C17, 20-Lyase of the Medaka (Oryzias latipes) : Structure,Activity and Function","item_titles":{"attribute_name":"タイトル","attribute_value_mlt":[{"subitem_title":"Ovarian Cytochrome P-450 17α-Hydroxylase/C17, 20-Lyase of the Medaka (Oryzias latipes) : Structure,Activity and Function"},{"subitem_title":"Ovarian Cytochrome P-450 17α-Hydroxylase/C17, 20-Lyase of the Medaka (Oryzias latipes) : Structure,Activity and Function","subitem_title_language":"en"}]},"item_type_id":"1","owner":"1","path":["27"],"pubdate":{"attribute_name":"公開日","attribute_value":"2010-02-22"},"publish_date":"2010-02-22","publish_status":"0","recid":"1312","relation_version_is_last":true,"title":["Ovarian Cytochrome P-450 17α-Hydroxylase/C17, 20-Lyase of the Medaka (Oryzias latipes) : Structure,Activity and Function"],"weko_creator_id":"1","weko_shared_id":1},"updated":"2023-06-20T14:45:55.017740+00:00"}