Immunobiochemical evidence for the loss of sperm specific histones during male pronucleus formation in monospermic zygotes of sea urchins

Abstract

To obtain information on the remodeling of sperm chromatin during male pronuclei formation, we have followed the sperm specific histones (SpH) that form the nucleosomal core by Western immunoblot analysis with polyclonal antibodies directed against the core SpH. The results obtained indicate that the complete set of SpH is absent from zygote chromatin at the beginning of the first S phase. The disappearance of SpH is not coincidental for the five histone classes: SpH4 and SpH3 are lost 5-15 min post insemination (p.i.), SpH2B and SpH2A disappear 20-40 min p.i., and SpH1 is progressively diminished up to 30 min p.i. This order of sperm chromatin remodeling is not affected by the inhibition of protein synthesis by emetine, indicating that the factor(s) responsible for SpH disappearance are present in unfertilized eggs. The lost SpH's are not replaced by newly synthesized CS variants, since the basic proteins synthesized de novo during male pronuclei formation are not incorporated into chromatin remaining in the cytoplasm. These newly synthesized proteins are different from the CS variants as judged by their electrophoretic migration.

Identification of a cysteine protease responsible for degradation of sperm histones during male pronucleus remodeling in sea urchins

“…Such selective loss was previously suggested to be due to degradation of sperm histones (Imschenetzky et al, 1991a]. This mechanism would require a protease recognition of the SpH, leaving the maternally derived CS histone variants intact.…”

“…The procedures followed to produce antibodies against sperm histones and for the electrophoretic transfer, blotting and immunodetection were described previously [Imschenetzky et al, 1991a[Imschenetzky et al, , 1996a. One extra lane containing whole SpH or alternatively the native CS histone variants was included in each SDS-PAGE as positive and negative controls, respectively.…”

“…Unfertilized eggs, sperms, and zygotes were obtained as described previously [Imschenetzky et al, 1984[Imschenetzky et al, , 1986[Imschenetzky et al, , 1991a.…”

“…In addition, we have previously reported that the complete set of sperm histones were no longer present in zygotes at 30 min postinsemination (p.i.) [Imschenetzky et al, 1991a]. It was suggested that this disappearance could be due to selective proteolysis activated concomitantly with other fertilization-related events.…”

“…It was suggested that this disappearance could be due to selective proteolysis activated concomitantly with other fertilization-related events. It was also demonstrated that the lost sperm-specific histones (SpH) are replaced by maternal cleavage-stage (CS) histone variants that are forming the chromatin of unfertilized eggs and the initial two cleavage divisions [Green and Poccia, 1985;Imschenetzky et al, 1991a]. These changes in chromatin composition result in differences in chromatin organization that may be followed by micrococcal nuclease digestion studies.…”