Winger Q A, de los Rios P, Han V K, Armstrong D T, Hill D J, Watson A J
Department of Physiology, The University of Western Ontario, London, Canada.
Biol Reprod. 1997 Jun;56(6):1415-23. doi: 10.1095/biolreprod56.6.1415.
Bovine oviductal monolayer and vesicle primary cultures express insulin-like growth factor (IGF)-I and -II mRNAs and polypeptides. Early bovine embryos also express IGF-I, IGF-II, IGF-I receptor, IGF-II receptor, and insulin receptor mRNAs. This study reports the expression of IGF binding protein (IGFBP) mRNAs and polypeptides in bovine oviduct primary cultures and IGFBP mRNAs in preattachment embryos. Release of immunoreactive IGF-I and IGF-II by oviduct cultures and bovine blastocysts was also determined. IGFBP-2, -3, -4, and -5 transcripts were observed in oviduct primary cultures throughout an 8-day interval. IGFBP-1 and -6 mRNAs were consistently not detected in the oviduct. Messenger RNAs encoding IGFBPs -2, -3, and -4 were detected throughout bovine preattachment development, while transcripts encoding IGFBP-5 were detected only in blastocysts. IGFBP-1 and -6 transcripts were not detected in early embryos. Ligand blot analysis with 125I-labeled IGF-II revealed the presence of four prominent polypeptide bands of approximate molecular masses 24, 31, and 36 kDa, and a broad band extending from 46 to 53 kDa, in conditioned media samples prepared from oviduct primary cultures. Western immunoblot analysis confirmed the identity of the 24-kDa, 31-kDa, and 36-kDa species as IGFBP-4, -5, and -2, respectively. Levels of the release of IGF-II from oviductal vesicle cultures were significantly greater than levels observed for monolayer cultures (p < 0.005). No significant difference in the levels of IGF-I release between monolayer and vesicle cultures was observed. Pools of 10 blastocysts released on average 36.2 +/- 3.9 pg of IGF-II per embryo, while the release of embryonic IGF-I was below the levels of detection for our assay. The results suggest that maternally derived IGF may be regulated by IGFBPs to support bovine preattachment development.
牛输卵管单层细胞和囊泡原代培养物表达胰岛素样生长因子(IGF)-I和-II的mRNA及多肽。早期牛胚胎也表达IGF-I、IGF-II、IGF-I受体、IGF-II受体和胰岛素受体的mRNA。本研究报道了牛输卵管原代培养物中IGF结合蛋白(IGFBP)的mRNA和多肽表达以及着床前胚胎中的IGFBP mRNA表达。还测定了输卵管培养物和牛囊胚释放的免疫反应性IGF-I和IGF-II。在8天的时间间隔内,在输卵管原代培养物中均观察到IGFBP-2、-3、-4和-5的转录本。在输卵管中始终未检测到IGFBP-1和-6的mRNA。在整个牛着床前发育过程中均检测到编码IGFBP-2、-3和-4的mRNA,而编码IGFBP-5的转录本仅在囊胚中检测到。在早期胚胎中未检测到IGFBP-1和-6的转录本。用125I标记的IGF-II进行的配体印迹分析显示,从输卵管原代培养物制备的条件培养基样品中存在四条明显的多肽带,其分子量约为24、31和36 kDa,以及一条从46至53 kDa延伸的宽带。Western免疫印迹分析证实,24-kDa、31-kDa和36-kDa的条带分别为IGFBP-4、-5和-2。输卵管囊泡培养物释放IGF-II的水平显著高于单层培养物(p < 0.005)。单层培养物和囊泡培养物之间IGF-I释放水平未观察到显著差异。10个囊胚的集合平均每个胚胎释放36.2±3.9 pg的IGF-II,而胚胎IGF-I的释放低于我们检测方法的检测水平。结果表明,母体来源的IGF可能受IGFBP调节以支持牛着床前发育。