McNutt T, Rogowski L, Vasilatos-Younken R, Killian G
Department of Dairy and Animal Science, Pennsylvania State University, University Park.
Mol Reprod Dev. 1992 Nov;33(3):313-23. doi: 10.1002/mrd.1080330313.
125I-labeled oviductal fluid (ODF) proteins and antiserum to ODF were used to determine whether ODF proteins associate with the sperm membrane during in vitro capacitation. Luteal and nonluteal pools of ODF were obtained from oviduct catheters during the estrous cycle. Washed sperm (50 x 10(6) sperm/ml) were incubated up to 4 h in a protein-free modified Tyrode's medium (MTM), or MTM supplemented with 40% ODF, or 0.5 ng 125I-labeled ODF proteins. Solubilized sperm membrane proteins and incubation media containing ODF proteins were separated by gel electrophoresis. Membranes isolated from bovine sperm, previously incubated with ODF, adsorbed five 125I-proteins: A doublet at 85-95 kDa, and others at 24, 34, 53, and 66 kDa. The amount of 66 kDA 125I-protein associated with the sperm decreased during the incubation, whereas the amount of 85 to 95-kDa protein did not. Western blot analyses also detected the presence of ODF proteins (53, 66, 85-95, and 116 kDa) in solubilized membranes from sperm incubated in ODF. The 85 to 95-kDa protein in ODF decreased in apparent molecular weight by 5 kDa when associated with the sperm membrane. At 53 kDa, ODF proteins which associated with sperm were transformed from two to three separate proteins. These studies indicate that the surface of sperm is modified by adsorption of ODF proteins to the membrane during in vitro capacitation.
使用125I标记的输卵管液(ODF)蛋白和抗ODF血清来确定在体外获能过程中ODF蛋白是否与精子膜结合。在发情周期中,从输卵管导管获取黄体期和非黄体期的ODF样本。将洗涤后的精子(50×10⁶精子/ml)在无蛋白的改良Tyrode's培养基(MTM)、添加40% ODF的MTM或0.5 ng 125I标记的ODF蛋白中孵育长达4小时。通过凝胶电泳分离溶解的精子膜蛋白和含有ODF蛋白的孵育培养基。从先前与ODF孵育的牛精子中分离出的膜吸附了五种125I蛋白:85 - 95 kDa的双峰,以及24、34、53和66 kDa的其他蛋白。在孵育过程中,与精子结合的66 kDa 125I蛋白量减少,而85至95 kDa蛋白量未减少。蛋白质印迹分析还检测到在ODF中孵育的精子溶解膜中存在ODF蛋白(53、66、85 - 95和116 kDa)。当与精子膜结合时,ODF中85至95 kDa的蛋白表观分子量降低了5 kDa。在53 kDa时,与精子结合的ODF蛋白从两种转变为三种不同的蛋白。这些研究表明,在体外获能过程中,精子表面通过ODF蛋白吸附到膜上而发生修饰。