: O (streptolysin O, SLO) : 10~12 ICR,, /ml, U/ml SLO 15 min, SLO, (PI) : (P>0.05),,,,, (P<0.05) : SLO,, : O(SLO); ; (PI) ; (IVF)

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1 34 11 Vol.34 No Nov Reproduction & Contraception doi: /j.issn X O ( ) ( ) ( ) : O (streptolysin O ) : 10~12 ICR /ml U/ml 15 min (PI) : (P>0.05) (P<0.05) : : O(); ; (PI) ; (IVF) : R321.1 : A : X(2014) [3] O (streptolysin O ) [4] 30 nm : [1] ; [5] [2] ; : 2013FY : : ; Tel/Fax: ; gu_yihua@hotmail.com; ; Tel: ; chengw911@qq.com [6] [7] 26 (ICSI) [8] ; Rab3A 887

2 [9] Rab3A (IVF) (HTF) [1011] (HSA) (KSOM) Irvine Scientific ; HEPES HTF(mHTF) SAGE; (hcg ) (PMSG) ; (PI) Sigma ; ~10 ICR 10~12 ICR - : SCXK( ) mhtf 5 min 75% Marco (a) (b) (c) 10%HSA mhtf min [12] 2 : 1 ; /ml U/ml 15 min PBS 2 4 HEPES mhtf 4 15 min 37 mhtf PI IVF PI 10 mg/l PI 10 min PBS 2 (Eclipse 50i Nikon ) 3 PI IVF 10 IU PMSG 46~48 h 5 IU hcg HTF 5% CO2 37 ( 10%HSA HTF) ( 10%HSA KSOM) 4 hcg 12 h 10% HSA mhtf min hcg 13 h HTF 20~ %HSA HTF (2~5) 10 6 /ml 10 μl 37 CO2 4~6 h KSOM 24 h (Leica 205MA Leica) SPSS for Windows (Version 15.0; SPSS Inc Chicago IL USA) (x s) (%) χ 2 P<

3 (P>0.05) 1 ( 1A) (5.0% 0.7%); (95% 3.2%) (P<0.01)( 1A) 2.3 IVF IVF 2- (P<0.01) 2 Group Initial motility Experimental 1 (x s)(n=3) Table 1 Motility parameters of the mouse sperms (%) Ratio of sperms a b c Grade a Grade b Grade c (%) Ratio of motile sperms A: morphology of mouse sperms observed by bright-field microscope B: fluorescence of mouse sperms observed at the same field 1 PI (400 ) Figure 1 PI staining of mouse sperms 889

4 1~6 h ( ) min (P<0.01)( 3) 2 IVF 2- Table 2 Ratio of 2-cell embryo formation after the precedure of IVF (n) 2- (%) Group No. of eggs No. of 2-cell embryos (60/73) Experimental (9/80) * *: P<0.01 compared with the control 3 (x s)(n=3) Table 3 Ratio of capacitation of mouse sperms Group 0 h 1.5 h Untreated Experimental * *: P<0.001 compared with the control 3 [7] IVF IVF 1~6 h ( ) [14] Ca 2+ [15] PI ; [13] 2- [7] IVF IVF [1011] 2 [1] Brandner G Mueller N Graessmann A et al. Inhibition by interferon of SV40 tumor antigen formation in cells injected with SV40 crna transcribed in vitro. FEBS Lett (3): [2] Derossi D Chassaing G Prochiantz A. Trojan peptides: the penetrating intracellular delivery. Trends Cell Biol (2):84-7. [3] Plutner H Davidson HW Saraste J et al. Morphological 890

5 analysis of protein transport from the ER to Golgi membranes in digitonin-permeabilized cells: role of the P58 containing compartment. J Cell Biol (5): [4] Bhakdi S Tranum-Jensen J Sziegoleit A. Mechanism of membrane damage by streptolysin-o. Infect Immun (1): [5] Giles RV Spiller DG Grzybowski J et al. Selecting optimal oligonucleotide composition for maximal antisense effect following streptolysin O-mediated delivery into human leukaemia cells. Nucleic Acids Res (7): [6] Walev I Bhakdi SC Hofmann F et al. Delivery of proteins into living cells by reversible membrane permeabilization with streptolysin-o. Proc Natl Acad Sci USA (6): [7] Johnson LR Moss SB Gerton GL. Maintenance of motility in mouse sperm permeabilized with streptolysin O. Biol Reprod (3): [8] Rawe VY Diaz ES Abdelmassih R et al. The role of sperm proteasomes during sperm aster formation and early zygote development: implications for fertilization failure in humans. Hum Reprod (3): [9] Yunes R Michaut M Tomes C et al. Rab3A triggers the acrosome reaction in permeabilized human spermatozoa. Biol Reprod (4): [10] Huang XF Li Y Gu YH et al. The effects of Di-(2- ethylhexyl)-phthalate exposure on fertilization and embryonic development in vitro and testicular genomic mutation in vivo. PLoS One (11):e [11] Gu YH Li Y Huang XF et al. Reproductive effects of two neonicotinoid insecticides on mouse sperm function and early embryonic development in vitro. PLoS One (7): e [12] (3): [13] Claassens OE Wehr JB Harrison KL. Optimizing sensitivity of the human sperm motility assay for embryo toxicity testing. Hum Reprod (7): [14] Tomes CN De Blas GA Michaut MA et al. Alpha -SNAP and NSF are required in a priming step during the human sperm acrosome reaction. Mol Human Reprod (1): [15] Kano F Murata M. The semi-intact cell system and methods for cell resealing: a novel systems biology tool to elucidate protein networks with spatio-temporal information. Adv Syst Biol (1):6-14. ( ) Toxicity of Streptolysin O () on the Mouse Sperm In Vitro Tao SHI 1 Miao LIU 12 Wen-wen GU 12 Peng WANG 1 Yi-hua GU 1 Guo-wu CHENG 13 (1. Shanghai Institute of Planned Parenthood Research Shanghai ) (2. Shanghai Medical College Fudan University Shanghai ) (3. Shanghai JIAI Genetics & IVF Institute Shanghai ) ABSTRACT Objective: To investigate the toxicity of streptolysin O () on the mouse sperm in vitro. Methods: Male mice aged weeks were killed by cervical vertebra dislocation and the cauda epididymis sperms were picked up. The concentration of the sperms was adjusted to /ml and the sperms were separated into two parts. One part was treated with for 15 min as experimental group the other was used as control group. Two groups were treated with the same procedure except that experimental group which was treated by firstly. The sperm motility parameters and the ratio of hyperactivated sperms after capacitation were determined the sperms were then applied for PI staining and in vitro fertilization (IVF) was applied to evaluate the fertilization capability of the -treated sperms. Results: No statistical difference was observed between the two groups in motility parameters. Compared with the control -treated mouse sperms showed a poor status of hyperactivation after capacitation since the sperms were incapable to restore membrane barrier function. Meanwhile with treatment the sperms demonstrated a low capability of fertilization in vitro with a statistically significant difference (P<0.05). Conclusion: has no effect on the motility of the sperm but could adversely affect sperm hyperactivation which could result in dramatically reduced fertility in a procedure of IVF. Key words: streptolysin O (); sperm motility; PI staining; in vitro fertilization (IVF) 891