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Identification of the α Subunits of InhibitoryG-Proteins Coupled to 17α, 20β-Dihydroxy-4- pregnen-3-one Receptors in Oocytes of Medaka (Oryzias latipes)
https://ir.soken.ac.jp/records/1321
https://ir.soken.ac.jp/records/132172ed191e-cf31-4843-b7a1-670d4af8ee9c
| 名前 / ファイル | ライセンス | アクション |
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要旨・審査要旨 / Abstract, Screening Result (356.7 kB)
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本文 (8.6 MB)
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| Item type | 学位論文 / Thesis or Dissertation(1) | |||||
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| 公開日 | 2010-02-22 | |||||
| タイトル | ||||||
| タイトル | Identification of the α Subunits of InhibitoryG-Proteins Coupled to 17α, 20β-Dihydroxy-4- pregnen-3-one Receptors in Oocytes of Medaka (Oryzias latipes) | |||||
| タイトル | ||||||
| タイトル | Identification of the α Subunits of InhibitoryG-Proteins Coupled to 17α, 20β-Dihydroxy-4- pregnen-3-one Receptors in Oocytes of Medaka (Oryzias latipes) | |||||
| 言語 | en | |||||
| 言語 | ||||||
| 言語 | eng | |||||
| 資源タイプ | ||||||
| 資源タイプ識別子 | http://purl.org/coar/resource_type/c_46ec | |||||
| 資源タイプ | thesis | |||||
| 著者名 |
大場, 裕一
× 大場, 裕一 |
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| フリガナ |
オオバ, ユウイチ
× オオバ, ユウイチ |
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| 著者 |
OBA, Yuichi
× OBA, Yuichi |
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| 学位授与機関 | ||||||
| 学位授与機関名 | 総合研究大学院大学 | |||||
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| 学位名 | 博士（理学） | |||||
| 学位記番号 | ||||||
| 内容記述タイプ | Other | |||||
| 内容記述 | 総研大甲第276号 | |||||
| 研究科 | ||||||
| 値 | 生命科学研究科 | |||||
| 専攻 | ||||||
| 値 | X2 分子生物機構論専攻 | |||||
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| 学位授与年月日 | 1997-03-24 | |||||
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| 値 | 1996 | |||||
| 要旨 | ||||||
| 内容記述タイプ | Other | |||||
| 内容記述 | Fully grown oocytes of vertebrates are arrested at prophase of the first meiotic division and must progress to the second meiotic metaphase before fertilization is possible. The resumption of meiosis is induced by the action of maturation-inducing hormone (MIH) which is secreted from ovarian follicles under the influence of a pituitary gonadotropin (luteinizing hormone, GTH II in fishes). Among vertebrates, MIHs have been most extensively investigated in several species of fishes and amphibians. In these animals, the MIH is a steroidal substance, and its major function is known to induce resumption of meiosis (final meiotic maturation). 17α, 20β-Dihydroxy-4-pregnen-3-one (17α, 20β-DP) was identified, for the first time in any vertebrate, as MIH of amago salmon (Oncorhynchus rhodurus). In the most widely investigated group, the anuran amphibians, MIH is generally considered to be progesterone.<br /> The most fundamental insight into hormone action that has emerged from the study of oocyte maturation is the identification of a new type of steroid hormone action that involves a plasma membrane receptor. Unfortunately, efforts to purify and characterize MIH receptors have been difficult and unsuccessful, largely due to relatively low ligand affinities and specificity of 17α, 20β-DP and progesterone. A specific 17α, 20β-DP binding to plasma membranes prepared from defolliculated oocytes of rainbow trout was identified and charactenzed. Recently, it has been reported that pertussis toxin (PT)-sensitive inhibitory G-proteins (Gi) is involved in the signal transduction of 17α, 20β-DP during meiotic maturation of rainbow trout oocytes. However, the details of the receptors and their signaling pathway are poorly understood. Therefore, the present study was designed to further investigate the involvement of G-proteins in the signal transduction pathway during steroid hormone-induced oocyte maturation. To this end, I chose the medaka (Oryzias latipes) as an experimental animal. Medaka, under a photoperiod of 14 hr light and 10 hr dark at 26℃, usually spawns daily within 1 hr of the onset of light for a number of consecutive days. By this method, various phases of oocyte maturation can be timed accurately. This orderly spawning allows us to collect oocytes at predictable stages, which makes medaka an ideal model for the study of hormonal regulation of oocyte growth and maturation. 17α, 20β-DP was shown to be a major naturally occurring MIH in medaka. Rainbow trout (Oncorhynchus mykiss) was also used for comparison.<br /> In chapter I, I examined changes in Giα subunit protein contents and 17α, 20β-DP binding (receptor) activity during naturally occurring oocyte maturation in both rainbow trout and media. The antibody AS/7 specific for Giα1, 2 subunits immunodetected Giα subunit proteins (40 kDa) in medaka and rainbow trout plasma membranes from postvitellogenic oocytes. The α subunit of G-proteins detected in medaka oocyte membranes has properties characteristic of a member of the Gi family. This protein was shown to be a substrate for PT. Time course changes in the amount of the 40 kDa band were examined using media membrane preparations obtained during various periods (every 6 hr from 41 hr prior to spawning to ovulation) of oocyte growth and maturation. Gi protein was maximal in immature oocytes (23 hr prior to spawning), decreased in oocytes undergoing GVBD and was not detected in ovulated eggs. A similar decrease in Gi protein contents also occurs in membrane fractions of oocytes undergoing GVBD and ovulated eggs in rainbow trout.<br /> Changes in 17α, 20β-DP binding (receptor) activity in oocyte plasrha membranes were examined using medaka oocytes collected in the same time course as that used for Gi protein content measurements. 17α, 20β-DP binding activity was barely detectable at 35 hr prior to spawning. The binding activity increased significantly to reach a maximal level at 23 hr prior to spawning, followed by a rapid drop during oocyte maturation and ovulation. The maturational competence was also determined by counting percentage GVBD. The percentage of GVBD was only 5% at 38 hr prior to spawning, but increased to 48% at 29 hr prior to spawning, and reached 100% at 23 hr prior to spawning. At 17 hr prior to spawning, oocytes underwent spontaneous maturation in the absence of 17α, 20β-DP. The increases in the percentage of GVBD and 17α, 20β-DP binding activity occurred concomitantly.<br /> In chapter II I, I focus on the characterization of Giα subunits expressed in medaka postvitellogenic oocytes as candidates of signal transducer of the 17α, 20β-DP receptor. Cloning and sequencing of full-length cDNAs encoding medaka Giα subunits were also canted out. In addition, I also investigated the expression of Gs in medaka oocytes. I first used reverse transcription-polymerase chain reaction (RT-PCR) to amplify α subunits of G-proteins using total RNA from intact ovarian follicles as a template. Five different PCR products were obtained. Sequence analyses indicate that these clones include three subtypes of Giα (Giαa, Giαb and Giαc) and two subtypes of Gsα (Gsαd and Gsαe). Full length cDNA clones for Giαa and Giαc were isolated from a medaka ovarian follicle cDNA library. Predicted amino acid sequences of Giαa and Giαc exhibited significant homology with Giα1 and Giα2 of other species, respectively. Both Giαa and Giαc possessed a specific Cys residue in the C-terminal region which was the site for ADP-ribosylation by PT. A method was then developed to extract medaka oocyte RNA without contamination of follicle cell RNA. Using this method, I demonstrated that oocytes expressed both Giαa and Giαc, but not Giαb. Goα, another G-protein which is ADP-ribosylated by PT, was not expressed in oocytes, although it was expressed in brain tissue. The results presented here suggest that the 17α, 20β-DP-dependent stimulation of oocyte maturation is mediated through the G-protein α subunits, Giαa and/or Giαc.<br /> In chapter III , the coupling of 17α, 20β-DP receptors and G-protein α subunits in medaka oocyte plasma membranes was investigated. Three kinds of antisera (A2, C4, and M1) against peptides corresponding to the unique internal sequences of medaka Gia subtypes, Giαa and Giαc, were produced. Antisera A2 and C4 recognized Giαa and Giαc, respectively, while antiserum M1 recognized both subtypes of Giα. Immunoblotting using these antisera showed that medaka oocytes express two Giα, Giαa and Giαc, concomitantly. The results also indicated that Giαa behaved a little larger than Giαc on SDS-PAGE.<br /> Giα in medaka oocyte membrane preparations could be ADP-ribosylated with PT after solubilization with CHAPS. This indicates that the solubilization condition used in this study was so mild that Gi retains the heterotrimeric form even after solubilization. As expected, antiserum M1 successfully precipitated solubilized two Giα, Giαa and Giαc. Furthermore, the p subunit of G-protein was also found in the immunoprecipitates. These results indicate that Gi still retains the heterotrimeric form in the immunoprecipitate, because the β and γ subunits are known to tightly associate each other.<br /> Significant amounts of specific 17α, 20β-DP bindings (17α, 20β-DP receptors) were found in the immunoprecipitates, indicating that the 17α, 20β-DP receptor is directly coupled with Gi. This is the first demonstration of direct coupling of the MIH (steroid) receptor and heterotrimeric G-proteins. I have not yet attempted to quantify yield of 17α, 20β-DP binding and Giα in the immunoprecipitates. Several technical problems need to be overcome; these include the difficulty of assaying 17α, 20β-DP binding in solubilized membrane preparations.<br /> In summary, by producing three polyclonal antibodies which recognize Giα, I was able to demonstrate, using immunoprecipitation and ligand binding assays, that the 17α, 20β-DP receptor is coupled directly with Gi, and its a subunit is Giαa and/or Giαc. It should be emphasized that evidence for plasma membrane steroid hormone receptors is not limited to the amphibian and fish oocyte system. For example, it is becoming increasingly apparent that some steroid hormones may effect rapid alterations in brain function independently of classical intracellular receptors. Further studies will focus on the identification of the 17α, 20β-DP receptor using this new coimmunoprecipitation method developed in this study. | |||||
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| 値 | 有 | |||||
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| 内容記述タイプ | Other | |||||
| 内容記述 | application/pdf | |||||
