|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)
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
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.
(1)Bufoの精子核塩基性蛋白質(SBP)は2種のプロタミン(P1,P2)から、Xenopusのそれは4種のコアヒストンおよび6種の精子特異的蛋白質(SP1ー6)から成る。BufoのP1,P2およびXenopusのSP4のcDNAを解析したところによれば、前2者はN末端より28番目が異なる(Glu/Asp)他は39ケのアミノ酸配列が互いに全て同じであり、SP4は78ケのアミノ酸の組成、配列のいずれでも既知のプロタミンと相同性が乏しい。(2)精子形成過程でヒストン群は精母細胞までに合成をやめ、SBPは精子変態が進んで核凝縮の開始とともに出現する。他方、ノザン解析、in situハイブリダイゼ-ションによれば、転写はP1,P2については球形の精細胞(一倍体)期に、SP4については第一精母細胞パキテン(四倍体)期に起こり、両者の間で異なった転写調節機構が存在することが示唆された。(3)成熟卵の抽出液を用いた無細胞系で精子核をインキュベ-トすると、核の脱凝縮とともにSBPが速やかに消失する。SBPを除去する活性は卵母細胞〜胞胚の細胞質に固有の耐熱性因子で、これを精製した結果、分子量、アミノ酸組成、等電点等からヌクレオプラズミンであると結論した。ヌクレオプラズミンは、精子核のSBPに結合してこれを分解することなしに核から除去する。前核は、コアヒストンの他胞胚期までの核に特異的なH1サブタイプを含むことを発見し、これをH1Xと命名した。 Less