We have compared the relative proportion of protamine 1 (P1) and protamine 2 (P2) bound to DNA in the sperm of a variety of eutherian mammals to obtain insight into how these two proteins interact in sperm chromatin. Gel electrophoresis (combined with microdensitometry) and high performance liquid chromatography (HPLC) were used to determine the content of the two protamines, and the identity of each protein was confirmed by amino-terminal sequencing or amino acid analysis. The sperm of all species examined contained P1, but P2 was found to be present only in certain species. Unlike the fixed ratio of core histones that package DNA into nucleosomes in all somatic cells, the proportion of P2 present in mature sperm was found to be continuously variable from 0 to nearly 80%. These results show that P1 and P2 do not interact with each other or DNA to form a discrete complex or subunit structure that is dependent upon particular P1/P2 stoichiometries. Data obtained from a number of closely and distantly related species also indicate that while the P2 content of sperm chromatin is allowed to vary over a wide range during the course of evolution, the relative proportion of P1 and P2 are tightly regulated within a genus.
We have identified the disulfide cross-links in bull protamine by titrating intact bull sperm with dithiothreitol (DTT) and following the modification of each cysteine residue with tritiated iodoacetate. The derivatization of each cysteine was monitored by a combination of HPLC, peptide mapping, and protein sequencing. Analyses of total free sulfhydryls show that all seven of the bull protamine cysteines are cross-linked as disulfides in mature sperm. The first disulfide is reduced at a DTT:protamine cysteine (DTT:Cys) ratio of 0.3 and the last at a ratio of 2.0. Intra- and intermolecular disulfides were identified by correlating the reduction of specific disulfides with the dissociation of protamine from DNA in partially reduced sperm and sperm treated with N,N'-ethylenedimaleimide, a bifunctional disulfide cross-linking agent. Three intermolecular and two intramolecular disulfides were identified. The results of these experiments demonstrate that the amino- and carboxy-terminal ends of the bull protamine molecule are folded inward toward the center of the molecule and are locked in place, each by a single intramolecular disulfide bridge. Three intermolecular disulfides cross-link neighboring protamine molecules around the DNA helix in such a manner that the protamines cannot be dissociated from DNA without first reducing the interprotamine disulfides.
The nuclei of mouse spermatozoa contain two protamine variants, mouse protamine 1 (mPl) and mouse protamine 2 (mP2). The amino acid sequence predicted from mPl cDNAs demonstrates that mPl is a 50-amino-acid protein with strong homology to other mammalian P1 protamines. Nucleotide sequence analysis of independently isolated, overlapping cDNA clones indicated that mP2 is initially synthesized as a precursor protein which is subsequently processed into the spermatozoan form of mP2. The existence of the mP2 precursor was confirmed by amino acid composition and sequence analysis of the largest of a set of four basic proteins isolated from late-step spermatids whose synthesis is coincident with that of mPl. The sequence of the first 10 amino acids of this protein, mP2 precursor 1, exactly matches that predicted from the nucleotide sequence of cDNA and genomic mP2 clones. The amino acid composition of isolated mP2 precursor 1 very closely matches that predicted from the mP2 cDNA nucleotide sequence. Sequence analysis of the amino terminus of isolated mature mP2 identified the final processing point within the mP2 precursor. These studies demonstrated that mP2 is synthesized as a precursor containing 106 amino acids which is processed into the mature, 63-amino-acid form found in spermatozoa.Protamines are small, basic, arginine-rich proteins that replace histones in the later stages of spermatogenesis in many vertebrates (N. B. Hecht, in G. Stein and J. Stein, ed., Basic Chromosomal Proteins: Structure, Organization and Regulation ofthe Gene, in press). During spermatogenesis in mammals, the histones are not directly replaced by protamines but by a population of basic proteins that are transiently associated with the spermatid nucleus (14,35; Hecht, in press). These testis-specific proteins are removed from the condensing chromatin at later stages of spermiogenesis and are replaced by protamines, the predominant class of proteins found complexed with DNA in the mature spermatozoon. Mammalian protamines contain the amino acid cysteine, which is believed to function in the stabilization and compaction of the sperm nucleus through disulfide bond cross-linking (7).Protamines have been isolated from a large number of vertebrates including fish (3, 28), domestic fowl (38), mice (6, 27, 40; A. R.
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