Buhi W C, O'Brien B, Alvarez I M, Erdos G, Dubois D
Department of Obstetrics and Gynecology, University of Florida, Gainesville 32610-0294.
Biol Reprod. 1993 Jun;48(6):1274-83. doi: 10.1095/biolreprod48.6.1274.
The objectives of the present study were to develop an antibody probe to the porcine estrogen-dependent oviductal glycoproteins and to determine, by use of immunogold electron microscopy, whether these glycoproteins become associated with oviductal and uterine oocytes and early embryos. Polyclonal antibody, prepared using the M(r) 75,000-85,000 glycoprotein, separated from other proteins by two-dimensional SDS-PAGE, specifically recognized all three estrogen-dependent glycoproteins (acidic 75,000-85,000 M(r); acidic 100,000 M(r); basic 100,000 M(r)). In ampullary tissue collected from ovariectomized and estrogen-treated gilts and from gilts at Day 1 of estrus, gold particles were clustered over putative secretory granules restricted to the apical region of secretory epithelial cells. While follicular oocytes did not react with immunoreactive colloidal gold, oviductal and uterine unfertilized oocytes were found to be densely and uniformly labeled by colloidal gold throughout the zona pellucida, associated with flocculent material in the perivitelline space, and associated with microvilli and vitelline membrane. Similarly, in oviductal (1-4-cell) and unhatched uterine (4-cell/blastocyst) embryos, colloidal gold particles were distributed throughout the zona pellucida, heavily associated with flocculent material in the perivitelline space, and associated with the plasma membrane of the blastomeres. Immunoreactive colloidal gold remained detectable within Day 7 hatched uterine embryos, but not with embryos from later days. These results further support the proposal that porcine estrogen-dependent oviductal glycoproteins are released into the oviductal lumen, become associated with oviductal and uterine oocytes and early embryos, and are retained by oocytes and early embryos in the uterus.
本研究的目的是开发一种针对猪雌激素依赖性输卵管糖蛋白的抗体探针，并通过免疫金电子显微镜确定这些糖蛋白是否与输卵管和子宫中的卵母细胞及早期胚胎相关联。使用通过二维SDS-PAGE从其他蛋白质中分离出的分子量为75,000 - 85,000的糖蛋白制备的多克隆抗体，特异性识别所有三种雌激素依赖性糖蛋白（酸性分子量75,000 - 85,000；酸性分子量100,000；碱性分子量100,000）。在从切除卵巢并经雌激素处理的后备母猪以及发情第1天的后备母猪收集的壶腹组织中，金颗粒聚集在局限于分泌上皮细胞顶端区域的假定分泌颗粒上。虽然卵泡卵母细胞不与免疫反应性胶体金发生反应，但发现输卵管和子宫未受精卵母细胞在整个透明带中被胶体金密集且均匀地标记，与卵周隙中的絮状物质相关联，并与微绒毛和卵黄膜相关联。同样，在输卵管（1 - 4细胞）和未孵化的子宫（4细胞/胚泡）胚胎中，胶体金颗粒分布在整个透明带中，大量与卵周隙中的絮状物质相关联，并与卵裂球的质膜相关联。在第7天孵化的子宫胚胎中仍可检测到免疫反应性胶体金，但在之后几天的胚胎中则检测不到。这些结果进一步支持了以下观点：猪雌激素依赖性输卵管糖蛋白被释放到输卵管腔中，与输卵管和子宫中的卵母细胞及早期胚胎相关联，并被子宫中的卵母细胞和早期胚胎保留。
