On the expression of protamine genes in the testis of man and other mammals.

Protamines are low molecular weight, highly basic nuclear proteins involved in the condensation of sperm chromatin. cDNA clones for human protamine 1 and 2 (PRM1 and PRM2) were used for Northern blot experiments with RNA from different human tissues. Protamine transcripts, 0.6 kb and 0.9 kb in lengths for PRM1 and PRM2, respectively, were detected only in testicular RNA. The hybridization signals though did not produce sharp bands but enclosed a minor fraction of significant smaller transcripts. When polysomal RNA fractions were used in the hybridization, these shorter transcripts, 0.45 kb in length for PRM1 and 0.7 kb for PRM2, were specifically enriched. As demonstrated by in situ hybridization on human testis sections, the transcripts of both protamine genes are restricted to the central cell layer of the tubuli seminiferi corresponding to the spatial arrangement of postmeiotic cells. This result indicates that the protamine genes in the human are postmeiotically and haploid expressed. When cDNA clones of both protamines of the boar (BPrm-1 and BPrm-2) were used for hybridization experiments with the testicular RNA of those mammalian species which lack protamine 2 in their spermatozoa, the presence of transcripts for both protamines was detected. It can be assumed that mammals in general are endowed with at least two protamine genes which are both transcribed but are translationally regulated in a species-specific manner.

The lack of protamine 2 (P2) in boar and bull spermatozoa is due to mutations within the P2 gene.

The nuclei of spermatozoa in all mammals examined so far contain P1 protamine. A second protamine variant, protamine P2, has to date been isolated only from human and murine spermatozoa where it represents the major fraction of basic nuclear protein. In order to elucidate the reason for this unusual distribution of the protamine variants among mammals we have investigated the expression of protamine P2 in boar and bull. It can be shown that also in these species protamine 2 is transcribed and translated on low levels. Various mutational events though have altered the primary structure of the protein: In boar, a deletion of 8 aminoacids has removed a sequence motif from the amino-terminus of the molecule, which highly probable is of functional relevance. The bovine sequence, as a consequence of numerous point mutations has accumulated neutral and hydrophobic aminoacids which reduce the affinity of the protamine 2 to DNA.

Mouse preproacrosin: cDNA sequence, primary structure and postmeiotic expression in spermatogenesis.

The primary structure of mouse preproacrosin was deduced by nucleotide sequencing of cDNA clones isolated from a mouse testis cDNA library. The largest cDNA, with 1373 bp, consists of a 11-bp 5'untranslated sequence, a 1254-bp open reading frame terminated by a TGA triplet and a 105-bp 3' untranslated end, including one potential polyadenylation signal. The NH2-terminus of the polypeptide contains a hydrophobic 15-amino acid signal peptide. This cleavable signal sequence is followed by 403 amino acids, representing the acrosin light and the heavy chain of 23 and 380 amino acid residues, respectively. The proteolytic active site segments His, Asp and Ser are part of the heavy chain, as well as a proline-rich COOH-terminus, which is not present in any other serine proteinase studied so far. Furthermore the postmeiotic expression of the preproacrosin gene during mouse spermatogenesis was studied.

Molecular cloning of human preproacrosin cDNA.

Complementary DNA-clones for human preproacrosin have been isolated from a human testis cDNA library in lambda gt11. The nucleotide sequence of the 1402 bp cDNA insert includes a 20 bp 5' noncoding region, an open reading frame of 1263 bp corresponding to 421 amino acids (45.9 kdalton), and a 105 bp 3' untranslated region. The deduced amino acid sequence is compared with that recently evaluated from a cDNA clone for boar preproacrosin. The sequence identity is 70%; the leader sequence, the catalytic triad (His, Asp, Ser; which is characteristic for serine proteinases) and the positions of the cysteine residues crosslinking the light and the heavy chain of the active enzyme, acrosin, are conserved in both species. At the C-terminal end, a proline-rich sequence is present in both species; this may represent the species-specificity of acrosin.

Molecular cloning of preproacrosin and analysis of its expression pattern in spermatogenesis.

Complementary DNA clones for the boar preproacrosin have been isolated from a randomly primed testis cDNA library in lambda gt10 and from an oligo(dT)-primed testis cDNA in lambda gt11. The nucleotide sequence of the 1418-bp cDNA insert includes a 46-bp 5'-untranslated region, an open reading frame of 1248 bp corresponding to 416 amino acids (45.59 kDa) and a 121-bp 3'-untranslated region. The deduced amino acid sequence includes the active-site residues histidine, asparagine and serine of the catalytic triad of the serine proteinase super-family and is colinear with that determined by amino acid sequencing of the boar acrosin light chain and of a small region of the NH2-terminal sequence of the heavy chain. The preproacrosin cDNA contains at the 3' end a 381-bp sequence which codes for an amino acid sequence not yet found in any other serine proteinase. This amino acid sequence is rich in proline (42 out of 127 amino acids) and is suggested to be involved in the recognition and binding of the spermatozoa to the zona pellucida of the ovum. The mRNA for preproacrosin is synthesized as an approximately 1.6-kb-long molecule only in the postmeiotic stages of boar and bull spermatogenesis.