Fowler Paul A, Sorsa-Leslie Tarja, Cash Phillip, Dunbar Bryan, Melvin William, Wilson Yvonne, Mason Helen D, Harris William
Department of Obstetrics and Gynaecology, University of Aberdeen, Aberdeen, AB25 2ZD, UK.
Mol Hum Reprod. 2002 Sep;8(9):823-32. doi: 10.1093/molehr/8.9.823.
We aimed to confirm the ovarian site of gonadotrophin surge-attenuating factor (GnSAF) production and produce granulosa/luteal cell-conditioned medium (G/LCM) containing GnSAF for purification studies. Blue dye affinity chromatography followed by pseudochromatofocusing of G/LCM yielded bioactive fractions at pH 5.74 and 5.77. The former had a major 60-66 kDa band with an internal amino acid sequence of EPQVYVHAP following tryptic digestion. A rat polyclonal antiserum (rPAb) raised against this band completely blocked in-vitro GnSAF bioactivity in human follicular fluid, serum and G/LCM. GnSAF bioactivity was localized to a 64 kDa band of serum-free G/LCM and following 2D gel electrophoresis, one of the spots recognized by Western blotting with the GnSAF rPAb had an N-terminal amino acid sequence of NH-XVPQGNAXXN. Neither amino acid sequence had significant homology with proteins in the human genome database. When ovarian tissues from spontaneously cycling women were cultured under serum-free conditions, neither theca- nor stroma-conditioned media contained GnSAF bioactivity. However, granulosa cell-conditioned medium significantly reduced GnRH-induced LH secretion, an effect that was reversed by incubation with the GnSAF rPAb. In conclusion, we have confirmed that human granulosa cells produce GnSAF within the ovary and have two candidate amino acid sequences for GnSAF. We have also demonstrated that serum-free granulosa cell culture constitutes the method of choice for the characterization of GnSAF since recovery of bioactivity is superior in the presence of fewer serum proteins.
我们旨在确定促性腺激素激增衰减因子（GnSAF）的产生卵巢部位，并制备含有GnSAF的颗粒细胞/黄体细胞条件培养基（G/LCM）用于纯化研究。对G/LCM进行蓝色染料亲和层析，然后进行假色聚焦，在pH 5.74和5.77时得到生物活性组分。前者有一条主要的60 - 66 kDa条带，经胰蛋白酶消化后内部氨基酸序列为EPQVYVHAP。针对该条带产生的大鼠多克隆抗血清（rPAb）完全阻断了人卵泡液、血清和G/LCM中的体外GnSAF生物活性。GnSAF生物活性定位于无血清G/LCM的一条64 kDa条带，二维凝胶电泳后，用GnSAF rPAb进行Western印迹识别的一个斑点的N端氨基酸序列为NH - XVPQGNAXXN。这两个氨基酸序列与人类基因组数据库中的蛋白质均无显著同源性。当对自然周期女性的卵巢组织进行无血清培养时，卵泡膜细胞条件培养基和基质细胞条件培养基均不含有GnSAF生物活性。然而，颗粒细胞条件培养基显著降低了GnRH诱导的LH分泌，与GnSAF rPAb孵育可逆转该效应。总之，我们已经证实人颗粒细胞在卵巢内产生GnSAF，并且有两个GnSAF的候选氨基酸序列。我们还证明，无血清颗粒细胞培养是GnSAF特性鉴定的首选方法，因为在血清蛋白较少的情况下生物活性回收率更高。
