GENE EXPRESSION AND FUNCTION OF CYTOKINE DURING MATURATION AND AFTER FERTILIZATION IN PORCINE OVA

猪卵子成熟过程中和受精后细胞因子的基因表达和功能

• 批准号: 06806032
• 负责人: YOSHIDA Mitsutoshi
• 金额: $ 1.34万
• 依托单位: SHIZUOKA UNIVERSITY
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for General Scientific Research (C)
• 财政年份: 1994
• 资助国家: 日本
• 起止时间: 1994 至 1995
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-06806032/
• 关键词: CYTOKINE; CYTOKINE RECEPTOR; RT-PCR; OOCYTE MATURATION; FERTILIZATION; DEVELOPMENT; GENE EXPRESSION; PORCINE; 成長因子; 成長因子レセプター; 成熟; チロシンキナーゼ; 卵; レセプター; 体外成熟

This study was carried out examine, 1) by the reverse transcription-polymerase chain reaction (RT-PCR), whether cytokine mRNAs [epidermal growth factor (EGF), basic fibroblast growth factor (bFGF), insulin-like growth factor-I (IGF-I), platelet derived growth factor-A (PDGF-A) and transforming growth factor-a (TGF-a)] and cytokine-receptor (R) mRNAs (EGF-R,bFGF-R,IGF-I-R,PDGF-Ra) express in porcine ova during maturation and after fertilization in vitro, 2) the effect of inhibitors for receptor-tyrosine kinase (genistein and herbimycin A) on cumulus mass expansion (CME) and nuclear maturation of porcine cumulus-oocytes complexes (COCs) in vitro, and 3) the effect of delayd insemination in vitro on the developmental capacity to blastocyst in porcine embryos derived from in vitro maturation and fertilization. The results are as follows ; EGF,bFGF,IGF-I and PDGF-A mRNAs were detectable at all stages of maturation and fertilization, but were not detected at 8-to 16-cell stages. TGF-a mRNA was only dctectable after 2-cell stage of development. IGF-I-R and PDGF-Ra mRNAs were detectable at all stages of maturation, fertilization and development. EGF-R and bFGF-R mRNAs were detectable at all stages of maturation and development, but were not detected at early stage of fertilization. The addition of genistein or herbimycin A to maturation medium significantly decreased the rates of CME and oocytes with metaphase II in dose-dependent fashion. The delayd insemination in vitro was effective for the increase of rate of embryos developed to the blastocyst stage. These results indicate that porcine ova show the temporal and stage-specific expression patterns of cytokine and its receptor mRNAs during in vitro maturation and after fertilization, and that the signal transduction pathway for induction of CME and the progression of oocytes to metaphase II involve cytokine-sensitive process.

本研究采用逆转录-聚合酶链反应（RT-PCR）技术，检测细胞因子[表皮生长因子（EGF）、碱性成纤维细胞生长因子（bFGF）、胰岛素样生长因子-I（IGF-I）、血小板源性生长因子-A（PDGF-A）、转化生长因子-α（TGF-α）]和酪氨酸受体（R）mRNAs在体外培养条件下，（2）受体酪氨酸激酶抑制剂对猪卵母细胞体外成熟过程中和体外受精后表达的影响染料木黄酮和除莠霉素A对猪卵丘-卵母细胞复合体（COCs）体外卵丘质量扩增（CME）和核成熟的影响3）延迟授精对猪体外成熟受精胚胎囊胚发育能力的影响。结果如下：EGF、bFGF、IGF-I和PDGF-A mRNA在成熟和受精的所有阶段都可检测到，但在8- 16-细胞阶段未检测到。TGF-α mRNA在2细胞期后才能检测到。IGF-I-R和PDGF-Ra mRNA在成熟、受精和发育的所有阶段均可检测到。EGF-R和bFGF-R mRNA在成熟和发育的各个阶段都可检测到，但在受精早期检测不到。在成熟培养液中添加染料木黄酮或除莠霉素A可显著降低CME和中期II卵母细胞的比率，并呈剂量依赖性。延迟授精对提高胚胎发育到囊胚期的比率有明显效果。这些结果表明，猪卵母细胞在体外成熟过程中和受精后，细胞因子及其受体mRNA的表达具有时间和阶段特异性，CME诱导和卵母细胞向中期II发育的信号转导途径涉及对精氨酸敏感的过程。

项目成果

Kawarasaki T,Kosaka T,Sone M,Yoshida M,Bamba K.: "Detectiun of Y-bearing porcine spermurogoubyiu situ hybridization using digoxigenin-labelled,porcine wale-specific DNA probe produced by polymerase chain reaction." Mole cular Reproduction and Developwent.

Kawarasaki T、Kosaka T、Sone M、Yoshida M、Bamba K.：“使用聚合酶链式反应产生的地高辛标记的猪威尔士特异性 DNA 探针检测 Y 型猪精子原位杂交。”

Kawarasaki,T. et al: "Detection of Y-bearing porcine spermatozoa by in situ hybridization using digce xigenin-labelled,porcine male-specific DNA probe." Molecular Reproduction and Development. 40. 455-459 (1995)

川原崎，T.

Kawarasaki T,Kosaka T,Sone M,Yoshida M,Bamba K.: "Detection of Y-bearing porcine spermatozoa by in situ hybridization using digoxigenin-labelled, porcine male-specific DNA probe produced by polymerase chain reaction." Mol.Reperod.Dev.40. 455-459 (1995)

Kawarasaki T、Kosaka T、Sone M、Yoshida M、Bamba K.：“使用聚合酶链式反应产生的地高辛标记的猪雄性特异性 DNA 探针，通过原位杂交检测携带 Y 的猪精子。”

Kawarasaki,T.et al: "Rapid and simultaneous detection of chrocesome Y-and I-bearing porcine spermatozoa by fluorescence in situ bybridization." Molecular Reproduction and Development. (in press).

Kawarasaki,T.et al：“通过荧光原位杂交快速同时检测携带染色体 Y 和 I 的猪精子。”

Yoshida Y,Yosida M,BamBa K.: "Effect of genistein on cumulus mass lxpan sion and huclearmaturation of Pigaud cow cumulus-occpes complexes in vitro." Molecular Reproduction and Development. (accepted for publication). (1996)

Yoshida Y，Yosida M，BamBa K.：“金雀异黄酮对体外 Pigaud 牛卵丘-occpes 复合物的积云质量膨胀和核成熟的影响。”