IDENTIFICATION OF A CYSTEINE PROTEASE RESPONSIBLE FOR DEGRADATION OF SPERM HISTONES DURING MALE PRONUCLEUS REMODELING IN SEA-URCHINS

We have identified a 60-kDa cysteine protease that is associated with chromatin in sea urchin zygotes. This enzyme was found to be present a s a proenzyme in unfertilized eggs and was activated shortly after fer tilization. Ar a pH of 7.8-8.0, found after fertilization, the enzyme degraded the five sperm-specific histones (SpH), while the native clea vage-stage (CS) histone variants remained unaffected. Based on its req uirements for reducing agents, its inhibition by sulfhydryl blocking c ompounds and its sensitivity to the cysteine-type protease inhibitors (2S,3S)-trans-epoxysuccinyl-L-leucyl-amido- 3-methylbutane-ethyl-ester (E-64 d), cystatin and leupeptin, this protease can be defined as a c ysteine protease. Consistently, this protease was not affected by the serine-type protease inhibitors phenylmethylsulfonyl fluoride (PMSF) a nd pepstatin. The substrate selectivity and pH modulation of the prote ase activity strongly suggest its role in the removal of sperm-specifi c histones, which determines sperm chromatin remodeling after fertiliz ation. This suggestion was further substantiated by the inhibition of sperm histones degradation in vivo by E-64 d. Based on these three lin es of evidence, we postulate that this cysteine protease is responsibl e for the degradation of sperm-specific histones which occurs during m ale pronucleus formation. (C) 1997 Wiley-Liss, Inc.