Studies on immunosuppressive substance in seminal plasma and fertility

用重组白介素2(rIL-2)和IL-2依赖细胞株(NKC-3)研究精浆免疫抑制物质(IS)的作用机制和性质。SP耐煮15分钟，冷冻干燥。然而，用链霉蛋白酶、木瓜酶、高碘酸对SP的抑制作用几乎是减弱的。这些结果表明，糖蛋白是SP生物学功能所必需的。在含IL-2的NKC-3细胞系中加入SP后，可明显抑制~3H-胸腺嘧啶核苷的摄取。在不依赖IL-2的细胞系如NS-1或YAC-1中加入SP，这种抑制作用不能被发现。在培养开始时加入SP，即可观察到SP的抑制作用。而在采收前6小时加入SP，则未观察到这种现象。用Sephacry1Sephacry1 S-300凝胶过滤，发现对IL-2反应的抑制作用最强的是分子量740,000以上，分布广泛。证明SP的IS是一种超离心性大分子，经105,000G超速离心2小时后沉淀。将SP和rIL-2混合后，在105,000G下超速离心2小时。IL-2活性完全保留在培养上清液中。NKC-3细胞在SP存在下培养2小时，通过洗涤去除SP来恢复IL-2反应。SP可抑制NKC-3细胞与I-rIL-2的结合。用抗IL-2受体的单抗(3C7、7D4)进行流式细胞术分析表明，加入SP后，NKC-3细胞中抗IL-2受体的单抗的结合强度降低。这些结果表明，SP的IIs不直接与IL-2结合，也不中和IL-2本身，而是阻止IL-2与IL-2受体结合。

The mechanism and the character of immunosuppressive substance (IS) of seminal plasma (SP) were investigated using recombinant interleukin 2 (rIL-2) and IL-2 dependent cell line (NKC-3). Is of SP resisted to boiling for 15 minutes and lyophilization. However, prior treatment with pronase, papain, periodic acid of SP virtually reduced the inhibitory effect. These results suggested that glycoprotein was essential for biological function of SP. Remarkable inhibition of ^3H-thymidine uptake was observed, when SP was added to the NKC-3 cell line culturing in IL-2 containing medium. Such inhibitory effect could not be found by addition of SP to IL-2 independent cell line such as NS-1 or YAC-1. The inhibitory effect of SP was observed when SP was added at the initiation of culture. But it was not observed when SP was added 6 hours before harvest. It was revealed that the most intensive inhibition of IL-2 response existed over M.W. 740,000 with broad distribution by gel filtration using Sephacry1 S-300.It was demonstrated that IS of SP was an ultracentrifugable macromolecule, because it was sedimented by ultracentrifugation at 105,000G for 2 hours. SP and rIL-2 were mixed and then ultracentrifuged at 105,000G for 2 hours. IL-2 activity was completely retained in the supernatant. NKC-3 cells culturing in the presence of SP for 2 hours restored IL-2 response by washing to remove SP. The binding of ^<125>I-rIL-2 to NKC-3 cells was inhibited by SP. Flow cytometry using monoclonal antibodies (3C7, 7D4) against IL-2 receptors indicated that the biding of the monoclonal antibodies to IL-2 receptors decreased by addition of SP to NKC-3 cells. These results indicated that IIs of SP did not bind directly with nor neutralize IL-2 itself but prevented IL-2 from binding to IL-2 receptors.