| Budget Amount *help |
¥6,900,000 (Direct Cost: ¥6,900,000)
Fiscal Year 1991: ¥1,800,000 (Direct Cost: ¥1,800,000)
Fiscal Year 1990: ¥1,800,000 (Direct Cost: ¥1,800,000)
Fiscal Year 1989: ¥3,300,000 (Direct Cost: ¥3,300,000)
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| Research Abstract |
The sperm nuclear bsic proteins (SBPs) in two anuran amphibians, Xenopus laevis and Bufo japonicus, were characterized, and their synthesis during spermatogenesis and removal during fertilization were studied. The SBPs in Bufo consist exclusively of two protamines (Pl, P2), and those in Xenopus consist of 6 proteins (SPl-6) in addition to four nucleosomal core histones. Amino acid sequence analyses based on cloned cDNAs indicated that the Bufo P1 and P2 contain 39 amino acid residues, with a high sequence homology with fish protamines, and differ with each other only in the 28th amino acid residue (Pl, Asp ; P2, Glu). The Xenopus SP4 comprises 78 amino acid residues whose sequence has no indications of homology with protamines found in many other classes of vertebrates. During spermatogenesis, the histonesare synthesized before the end of primary spermatocyte stage, while the SBPs are first detectable in nuclei at the beginning of chromatin granulation and increase sharply at the last
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step of spermiogenesis. Both Northern analyses and in situ hybridization studies employing cDNA clones encoding Bufo P2 and Xenopus SP4 as probes revealed that in Bufo the transcripts of PI genes are present in the round spermatids (haploid stage), while in Xenopus the mRNAs for SP4 are present in the pachytene stage spermatocytes (tetraploid stage), and round spermatids. Thus the results suggested the occurrence of different regulatory mechanisms for transcription of SBP genes between Xenopus and Buf, as well as translational regulation of SBP gene products during spermiogenesis. When lysolecithinpermeabilized sperm were induced to form pronucleus by incubation with the egg extract, protamines were lost from nuclei within 1 min, accompanied by nuclear decondensation. The activities that induce both selective removal of SBPs and decondensation in sperm nuclei were found in extracts from growing and mature oocytes and pregastrula embryos, but not in postneurula embryos and adult tissues. The protamine-removing activity (PRA) was purified from egg extracts as the unique, heat-stable proteins with mobilities of l4OkD and 36kD on native- and SDS-PAGE, respectively, with the isoelectric points in the range 4.2 - 4.5, and possessing amino acid composition quite similar to that reported for Xenopus nucleoplasmin. We propose that in fertilized eggs the protamines are removed from sperm nuclei by nucleoplasmin by binding to but not by enzymatic degradation of the protamine. Less
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