Danforth D R, Cheng C Y
Department of Obstetrics and Gynecology, Ohio State University, Columbus 43210.
Endocrinology. 1995 Apr;136(4):1658-65. doi: 10.1210/endo.136.4.7895676.
Several lines of evidence suggest that the ovaries of many species produce a nonsteroidal substance, termed gonadotropin surge inhibiting factor (GnSIF), which inhibits the midcycle gonadotropin surge and attenuates the pituitary response to endogenous or exogenous GnRH. We have previously reported the partial purification of GnSIF from porcine follicular fluid (pFF) and its differentiation from inhibin. We present now the purification of GnSIF to homogeneity and determination of the partial NH2-terminal amino acid sequence. The bioassay for GnSIF used rat pituitary cells in short-term culture that were incubated with test fractions for 48 h, washed, and then incubated with 10 nM GnRH plus test fractions for 4 h. GnSIF activity is defined as the suppression of GnRH-stimulated LH secretion. GnSIF was purified from 500 ml of pFF using sequential heparin-Sepharose, anion-exchange, and cation-exchange liquid chromatography followed by gel permeation, hydrophobic interaction, and mono-Q HPLC steps. Using these six purification steps, we have obtained an apparently homogenous preparation that stains as a single band on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. GnSIF has an apparent mol wt of 69K. Limited NH2-terminal sequence analysis reveals that GnSIF has no sequence homology with other reproductive hormones including the inhibins, activins, and follistatins. Over the dose range tested, GnSIF had no effect on basal LH or FSH secretion by pituitary cells in culture and only slightly inhibited GnRH-stimulated FSH secretion at the highest dose tested. In addition, there was no inhibin or follistatin immunoactivity in the GnSIF preparation. As such, GnSIF appears to be a novel protein in pFF that inhibits GnRH-stimulated LH secretion, and which may participate along with other ovarian proteins and steroids in the regulation of pituitary gonadotropin secretion.
多项证据表明，许多物种的卵巢会产生一种非甾体物质，称为促性腺激素高峰抑制因子（GnSIF），它可抑制月经周期中期的促性腺激素高峰，并减弱垂体对内源性或外源性促性腺激素释放激素（GnRH）的反应。我们之前曾报道过从猪卵泡液（pFF）中部分纯化GnSIF及其与抑制素的区分。现在我们展示了将GnSIF纯化至同质状态并确定其部分NH2末端氨基酸序列的过程。用于GnSIF的生物测定采用短期培养的大鼠垂体细胞，将其与测试组分一起孵育48小时，洗涤后，再与10 nM GnRH加测试组分一起孵育4小时。GnSIF活性定义为对GnRH刺激的促黄体生成素（LH）分泌的抑制作用。使用肝素-琼脂糖、阴离子交换和阳离子交换液相色谱，随后进行凝胶渗透、疏水相互作用和单Q高效液相色谱步骤，从500 ml的pFF中纯化GnSIF。通过这六个纯化步骤，我们获得了一种在十二烷基硫酸钠-聚丙烯酰胺凝胶电泳上显示为单一泳带的明显同质制剂。GnSIF的表观分子量为69K。有限的NH2末端序列分析表明，GnSIF与包括抑制素、激活素和卵泡抑素在内的其他生殖激素没有序列同源性。在所测试的剂量范围内，GnSIF对培养的垂体细胞基础LH或促卵泡生成素（FSH）分泌没有影响，仅在测试的最高剂量下略微抑制GnRH刺激的FSH分泌。此外，GnSIF制剂中没有抑制素或卵泡抑素免疫活性。因此，GnSIF似乎是pFF中的一种新型蛋白质，它可抑制GnRH刺激的LH分泌，并可能与其他卵巢蛋白和类固醇一起参与垂体促性腺激素分泌的调节。
