Bobe J, Goetz F W
Institut National de la Recherche Agronomique, S.C.R.I.B.E., 35042 Rennes Cedex, France.
Biol Reprod. 2001 Apr;64(4):1048-55. doi: 10.1095/biolreprod64.4.1048.
An up-regulated cDNA fragment was isolated using a differential display polymerase chain reaction between ovulatory and postovulatory brook trout ovarian tissues. Using this fragment as a probe, a full-length cDNA of 1783 base pairs was obtained from an ovarian cDNA library. The cDNA presumably codes for a 383-amino acid protein with strong sequence similarity to an aspartic protease, progastricsin (EC 3.4.23.3), also known as pepsinogen C. On Northern blots of ovarian tissue, the trout progastricsin cDNA hybridized with a 1.8-kilobase transcript that was strongly up-regulated 4-6 days after ovulation. Of all other tissues tested, a transcript was only detected in the stomach. A recombinant trout progastricsin protein was produced and used to raise an antibody. On Western blots of ovarian tissue, the progastricsin antibody recognized a single 39-kDa protein that was present in the ovary only following ovulation. On Western blots of coelomic fluid, the 39-kDa protein was strongly detected 4-10 days after ovulation. The trout progastricsin was immunocytochemically localized to the granulosa cells of postovulatory follicles, suggesting that it is released from this tissue into the coelomic fluid following ovulation. Progastricsin has been found in the stomach, prostate, seminal vesicle, seminal fluid, and pancreas of vertebrates; however, this is the first report of a progastricsin in an animal ovary.
利用排卵和排卵后溪红点鲑卵巢组织之间的差异显示聚合酶链反应,分离出一个上调的cDNA片段。以该片段为探针,从卵巢cDNA文库中获得了一个1783个碱基对的全长cDNA。该cDNA可能编码一种383个氨基酸的蛋白质,其序列与一种天冬氨酸蛋白酶——前胃蛋白酶原(EC 3.4.23.3),也称为胃蛋白酶原C——具有高度相似性。在卵巢组织的Northern印迹中,鳟鱼前胃蛋白酶原cDNA与一个1.8千碱基的转录本杂交,该转录本在排卵后4至6天强烈上调。在所有检测的其他组织中,仅在胃中检测到一个转录本。制备了重组鳟鱼前胃蛋白酶原蛋白并用于制备抗体。在卵巢组织的Western印迹中,前胃蛋白酶原抗体识别出一种单一的39 kDa蛋白,该蛋白仅在排卵后出现在卵巢中。在体腔液的Western印迹中,在排卵后4至10天强烈检测到39 kDa蛋白。鳟鱼前胃蛋白酶原通过免疫细胞化学定位到排卵后卵泡的颗粒细胞中,表明它在排卵后从该组织释放到体腔液中。前胃蛋白酶原已在脊椎动物的胃、前列腺、精囊、精液和胰腺中发现;然而,这是动物卵巢中前胃蛋白酶原的首次报道。