Fielder P J, Thordarson G, Talamantes F, Rosenfeld R G
Department of Pediatrics, Stanford University Medical School, California 94305.
Endocrinology. 1990 Nov;127(5):2270-80. doi: 10.1210/endo-127-5-2270.
Characterization of insulin-like growth factor binding proteins (IGFBPs) and their pituitary regulation were investigated in intact and hypophysectomized (HX) pregnant and nonpregnant mice. Serum samples were obtained from pregnant mice killed on days 5, 8, 10, 12, 14, and 18 of gestation and fetal mice killed on day 18 of gestation. Some animals were HX or sham operated (SH) on days 9, 11, and 14 of gestation; HX and SH control animals were killed 3 days post surgery. Identification and relative quantities of IGFBPs were determined by Western ligand blotting (WLB) of whole serum, or immunoprecipitated and/or deglycosylated serum, using [125I]IGF-II as the ligand. Serum IGF-I and -II concentrations were determined in both HX and SH day 14 pregnant, nonpregnant, and day 18 fetal mouse sera. WLB analysis of nonpregnant mouse serum demonstrated IGFBPs with apparent Mr of approximately 45-40, 31-27, and 24 K. The 45-40 K IGFBPs appeared to be the mouse equivalent of IGFBP-3, and one of the 31-27 K IGFBPs appeared to be IGFBP-2. The other IGFBPs present are not yet fully identified. During pregnancy, the amount of IGFBP-3 present in serum (as measured by WLB) gradually decreased to 42%, 17%, and 10% of virgin levels on days 5, 8, and 10 of gestation. The amount of IGFBP-3 present in pregnant serum after day 10 of gestation was not measurable by scanning laser densitometry. Additionally, an IGFBP of approximately 27 K appeared during gestation; after treatment with Endoglycosidase F, this IGFBP decreased to 24 K. The major IGFBP in fetal serum appeared to be IGFBP-2, with lesser amounts of IGFBP-3 and the 27 and 24 K IGFBPs. Hypophysectomy significantly decreased IGFBP-3, IGF-I, and IGF-II in nonpregnant mouse serum, increased the amount of IGFBP2 found in pregnant mouse serum, and had no effect on IGF-I or -II concentration in day 14 pregnant mouse serum. Incubation of nonpregnant serum with late (day 17) pregnant serum for 5 h at 37 C, decreased the amount of IGFBP-3 (as measured by WLB) in the mixed sample to 93% of the amount present in the nonmixed sample. Iodinated recombinant IGFBP-3 ([125I]human (h)IGFBP-3) was incubated with different mouse serum samples, and proteolytic fragments were visualized after separation by sodium dodecyl sulfate polyacrylamide gel electrophoresis and autoradiography.(ABSTRACT TRUNCATED AT 400 WORDS)
在完整及垂体切除（HX）的怀孕和未怀孕小鼠中，研究了胰岛素样生长因子结合蛋白（IGFBPs）的特征及其垂体调节。从妊娠第5、8、10、12、14和18天处死的怀孕小鼠以及妊娠第18天处死的胎鼠获取血清样本。部分动物在妊娠第9、11和14天接受垂体切除或假手术（SH）；垂体切除和假手术对照动物在术后3天处死。使用[125I]IGF-II作为配体，通过对全血清、免疫沉淀和/或去糖基化血清进行Western配体印迹（WLB）来确定IGFBPs的鉴定及其相对含量。测定了垂体切除和假手术组第14天怀孕、未怀孕小鼠以及第18天胎鼠血清中的IGF-I和-II浓度。对未怀孕小鼠血清进行WLB分析显示，存在表观分子量约为45 - 40、31 - 27和24 K的IGFBPs。45 - 40 K的IGFBPs似乎相当于小鼠的IGFBP-3，31 - 27 K的IGFBPs之一似乎是IGFBP-2。其他存在的IGFBPs尚未完全鉴定。在怀孕期间，血清中存在的IGFBP-3量（通过WLB测量）在妊娠第5、8和10天逐渐降至未孕水平的42%、17%和10%。妊娠第10天后怀孕血清中存在的IGFBP-3量通过扫描激光密度测定法无法测量。此外，在妊娠期间出现了一种约27 K的IGFBP；用内切糖苷酶F处理后，这种IGFBP降至24 K。胎鼠血清中的主要IGFBP似乎是IGFBP-2，IGFBP-3以及27 K和24 K的IGFBPs含量较少。垂体切除显著降低了未怀孕小鼠血清中的IGFBP-3、IGF-I和IGF-II，增加了怀孕小鼠血清中发现的IGFBP2量，并且对第14天怀孕小鼠血清中的IGF-I或-II浓度没有影响。将未怀孕血清与妊娠晚期（第17天）怀孕血清在37℃孵育5小时，混合样本中IGFBP-3量（通过WLB测量）降至未混合样本中该量的93%。将碘化重组IGFBP-3（[125I]人（h）IGFBP-3）与不同的小鼠血清样本孵育，通过十二烷基硫酸钠聚丙烯酰胺凝胶电泳和放射自显影分离后观察蛋白水解片段。（摘要截于400字）
