Studies on immunosuppressive substance in seminal plasma and fertility

Research Project

Project/Area Number	62480335
Research Category	Grant-in-Aid for General Scientific Research (B)
Allocation Type	Single-year Grants
Research Field	Urology
Research Institution	Department of Urology, Toyama Medical and Pharmaceutical University
Principal Investigator	KATAYAMA Takashi Professor, Dept. of Urology. Faculty of Med., Toyama Med. Pha. Univ., 医学部, 教授 (10009441)
Co-Investigator(Kenkyū-buntansha)	KAZAMA Taizou Associate, Dept. of Urology, Faculty of Med., Toyama Med. Pha. Univ., 医学部, 助手 (50152624)
Project Period (FY)	1987 – 1988
Project Status	Completed (Fiscal Year 1988)
Budget Amount *help	¥3,700,000 (Direct Cost: ¥3,700,000) Fiscal Year 1988: ¥1,500,000 (Direct Cost: ¥1,500,000) Fiscal Year 1987: ¥2,200,000 (Direct Cost: ¥2,200,000)
Keywords	Human Seminal Plasma / Immunosuppressive Substance / Interlekin 2 / NKC-3 cell / 超遠心 / ゲル濾過 / 入精漿 / NKCー3 cell
Research Abstract	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.