Protamine 3'-untranslated sequences regulate temporal translational control and subcellular localization of growth hormone in spermatids of transgenic mice.

Although the mouse protamine 1 gene (mP1) is first transcribed in round spermatids, its mRNA is not translated until about 1 week later in elongating spermatids. To determine what mP1 sequences are important for its transcriptional and translational regulation, we have constructed fusions between mP1 and the human growth hormone (hGH) structural gene and analyzed their expression in transgenic mice. We show that mP1 sequences 5' to the start of transcription are sufficient to confer spermatid-specific expression on the hGH gene. We also show that 156 nucleotides of mP1 3'-untranslated sequence is sufficient to confer mP1-like translational regulation on the hGH mRNA. Interestingly, the subcellular localization of hGH was dependent on the time during spermiogenesis that it was made. Synthesis of hGH in early round spermatids resulted in localization in the acrosome, whereas synthesis in late elongating spermatids resulted in intracellular, but not acrosomal, localization.

Localization of protamine 1 mRNA in different stages of the cycle of the rat seminiferous epithelium.

A mouse protamine 1 cDNA probe was used to study P1 protamine gene expression during the cycle of the seminiferous epithelium in the rat. In situ hybridization experiments showed that transcription of the P1 protamine mRNA starts in the middle of step 7 of spermiogenesis during substage VIIc. The mRNA levels stay high in steps 7-14 spermatids but decrease during steps 15-16 and are virtually undetectable in steps 17-19 spermatids. Northern blot analyses of RNAs isolated from microdissected pools of seminiferous tubules show high P1 protamine mRNA concentrations during stages VIIc-XIV-III of the cycle and lower levels during stages IV-VIIb. Owing to a post-transcriptional shortening of the poly(A) tail by 130 bases, a decrease in the size of protamine 1 mRNA from approximately 580 to 450 nucleotides was observed in stages XIII-XIV suggesting an initiation of protamine 1 synthesis in step 13-14 spermatids. In stages II-VI (steps 16-18 spermatids), only the smaller size protamine 1 mRNA was detectable. The expression of protamine 1 mRNAs has been localized in the very last phase of the haploid gene activity. Although the in situ hybridization suggests a disappearance of protamine 1 mRNA after step 16 of spermiogenesis, Northern blot analysis shows that low levels of mRNA are present during the period of final condensation of the chromatin, reflecting the association of protamine with DNA.

Translational regulation of the novel haploid-specific transcripts for the c-abl proto-oncogene and a member of the 70 kDa heat-shock protein gene family in the male germ line.

Expression of the c-abl proto-oncogene in the mouse testis is characterized by the production of a unique 4.7-kb transcript present in germ cells that have entered the haploid phase of spermatogenesis. A similar developmental stage specificity of expression is observed for a member of the 70-kDa heat-shock protein (hsp 70) gene family. A unique-sized hsp 70 transcript (T-hsp 70) is produced in haploid spermatids and is stable throughout spermatogenesis. In the present study, we examined the regulation of expression of these genes by examining their association with polyribosomes. The germ cell-specific c-abl and T-hsp 70 mRNA variants were both associated with the polysomal fractions of mouse testis cells, suggesting that they are functional mRNAs. However, both c-abl and T-hsp 70 mRNAs were also found in the ribonucleoprotein particle fractions. The distribution of these mRNAs in both the polysomal and nonpolysomal fractions is comparable to that seen for the mRNA of protamine-1, a gene whose expression in the testis is known to be regulated at the level of translation. In contrast, transcripts from the beta-tubulin gene were seen predominantly in the polyribosomal fractions. These findings suggest that translation of the novel c-abl and T-hsp 70 transcripts is confined to subpopulations of testicular cells.

Mouse protamine 2 is synthesized as a precursor whereas mouse protamine 1 is not.

The nuclei of mouse spermatozoa contain two protamine variants, mouse protamine 1 (mP1) and mouse protamine 2 (mP2). The amino acid sequence predicted from mP1 cDNAs demonstrates that mP1 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 mP1. 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.

DNA methylation pattern and restriction endonuclease accessibility in chromatin of a germ-line specific gene, the rainbow trout protamine gene.

The chromatin structure of a germ-line specific gene, the TPG-3 gene, one of the rainbow trout protamine genes was analyzed in various tissues. The protamine genes are expressed in early stage testis but not in late stage testis, liver or erythrocyte. Five potential CpG methylation sites in the coding and flanking regions of the TPG-3 protamine gene were monitored in early and late stage testis, nucleoprotamine, liver and erythrocyte. In all cases the patterns of methylation were identical with only one CpG site at position -740 being methylated. Thus, the methylation pattern of this protamine gene remained the same independently of the expression of the gene. Two Msp I sites at positions -293 and/or -275 and +155 were accessible to the enzyme in the TPG-3 chromatin of early stage testis. Since the Msp I site at position -293 and/or -275 was also present in the TPG-3 chromatin of liver, only the site at position +155 within the transcribed region correlated with the expression of the protamine gene.

Nucleotide sequence of a bovine protamine cDNA.

The nucleotide sequence of a 441-base cDNA encoding the bovine protamine has been determined. This insert, isolated from a bovine spermatid-specific cDNA library, encodes a polypeptide of 50 amino acids of which 26 are arginine, 7 are cysteine, and 2 are tyrosine. The insert contains the complete 3'-noncoding region of 150 bases and most of the 5'-noncoding region. The predicted amino-acid sequence of bovine protamine is about 96% homologous to ram protamine, 76% to boar protamine, 64% to mouse protamine 1 and 52% to human protamine 1 and contains the central, highly basic domain of four arginine clusters found in the trout protamines. Our results show that bovine protamine is 50 amino-acid residues in length and not 47 residues as previously published (Coelingh, J.P. et al. (1972) Biochim. Biophys. Acta 285, 1-14).

Evidence for haploid expression of mouse testicular genes.

Hybridization of RNA blots of total testicular RNA from prepuberal and sexually mature CD1 mice with several mouse testicular cDNA probes reveals that the mRNA encoding the two mouse protamines, an actin sequence of 1.5 kb, and a post-meiotically expressed 620 nucleotide mRNA are first detected in the testes of mice 22 days of age. These experiments and other studies analysing RNA preparations from isolated populations of testicular cell types with cDNA probes [1, 2] demonstrate that haploid gene expression occurs in the mammalian testis.

The increase by spermidine of fidelity of protamine synthesis in a wheat-germ cell-free system.

The influence of spermidine on the fidelity of natural mRNA-directed protein synthesis has been investigated. With protamine mRNA as a template for protamine synthesis, misincorporation of lysine, histidine, threonine and cysteine for arginine was measured in the presence and absence of spermidine. It was found that misincorporation of these four amino acids in the presence of spermidine was less than or nearly equal to that occurring in the absence of spermidine; however, incorporation of arginine was stimulated greatly by spermidine. These results clearly show that spermidine induced an increase of fidelity in protamine synthesis. The increase of fidelity in the presence of spermidine occurred mainly at the level of binding of aminoacyl-tRNA to ribosomes. The frequency of misreading the 5' base of the codon (misincorporation of cysteine) was greater than that of the middle base of the codon (misincorporation of histidine), but spermidine reduction of misreading was more marked at the middle base of the codon. Misincorporation of lysine (misreading of G to A residue at the middle base of the codon) was greater than that of threonine (misreading of G to C residue), but spermidine reduction of misreading was more marked in the misincorporation of threonine. It was deduced from these results that spermidine inhibited low-frequency misreading more effectively than high-frequency misreading.

DNA packaging in mouse spermatids. Synthesis of protamine variants and four transition proteins.

A comparison of the protein compositions of mouse late-step spermatids and cauda epididymal sperm has revealed that the relative distribution of the two amino acid sequence variants of mouse protamine differ markedly in spermatids and sperm. Sonication-resistant spermatids contain the two variants in a ratio of 1:1, while the ratio of these two proteins in cauda epididymal sperm is approx. 2:1. Labeling studies in vivo have shown that this difference is due, in part, to an asynchrony in the time of synthesis of the two protamine variants. Both proteins are synthesized in late-step spermatids, but synthesis of the tyrosine variant in sperm chromatin begins approximately one day before synthesis of the more predominant histidine variant. Analyses of the time of synthesis of protamine and the four transition proteins in late-step spermatids allowed us to estimate the spermatid stage in which these proteins are deposited on DNA and relate these events to the onset of sonication resistance in maturing spermatids. These results indicate that: (1) synthesis and deposition of protamine begins coincident with the onset of sonication resistance in early step 12 spermatids; (2) protamine deposition is complete by mid-step 15; and (3) synthesis of the transition proteins occurs coincident with protamine synthesis.

Red cell ghost-mediated microinjection of RNA into HeLa cells. II. Cellular translation of protamine mRNA; post-translational modifications and nuclear binding of newly-synthesized protamine.

Red cell ghosts loaded with protamine messenger RNA (pmRNA) were fused to HeLa cells using polyethylene glycol, as a means of introducing the mRNA into heterologous cells. The recipient cells were capable of translating the RNA into the three protamine polypeptides, which may be resolved as three peaks (CI, CII, and CIII) by cation exchange chromatography. The synthesis of components CII and CIII was easily observed with possible traces of CI as well. The HeLa cells also phosphorylated CII after synthesis. However, this phosphorylation did not occur with CIII. In addition, CII but not CIII localized in the nucleus of the HeLa cells after synthesis. Thus, a correlation of post-translational modification with nuclear entry was observed. Localization in the nucleus, however, was not accompanied by the same tight binding of protamine to chromatin as is seen in the homologous trout testis spermatid cells. In the spermatid cells, protamine elutes from chromatin at a salt concentration of 1.2 M NaCl. In contrast, in the HeLa cells, the newly synthesized CII which had entered the nucleus, could be eluted with 0.6 M NaCl. Thus, the tight binding of protamine to chromatin in trout testis may require a series of concomitant developmental events, such as core histone hyper-acetylation (Christensen, M E & Dixon, G-H. In press) [17], which would be lacking in the HeLa cells.

Purification and characterization of cytoplasmic protamine messenger ribonucleoprotein particles from rainbow trout testis cells.

Poly(A)-containing protamine messenger ribonucleoprotein particles [poly(A+) pmRNP particles] have been isolated from the polysomal and free cytoplasmic subcellular fractions of trout testis cells by a two-step isolation procedure. Ethylenediaminetetraacetic acid (EDTA) treated particles from both cytoplasmic fractions were first fractionated by sucrose gradient centrifugation and the putative pmRNP particles localized by utilizing 3H-labeled protamine complementary DNA (pcDNA) probes. In addition, particles present in these fractions were characterized by their translational activity in the heterologous, rabbit reticulocyte cell-free system and the protein components of crude mRNP complexes analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoesis. The final purification step involved affinity chromatography of pooled gradient fractions on oligo(dT)-cellulose from which intact pmRNP could be eluted with distilled water at 40 degrees C. Highly purified particles from both polysomal and free cytoplasmic fractions prepared by this procedure had buoyant densities of 1.35-1.37 g/cm3 in CsCl or a protein content of approximately 82%. Particles isolated from EDTA-dissociated polysomes were actively translated in vitro, while their free cytoplasmic counterparts were not. High salt washed pmRNP particles or the RNA extracted from pmRNP preparations, however, directed the synthesis of trout protamines in this system. A model of the activation of stored pmRNP particles in vitro and in vivo is presented.

The release of high mobility group protein H6 and protamine gene sequences upon selective DNase I degradation of trout testis chromatin.

Limited digestion of trout testis nuclei with DNase I selectively degrades the protamine genes. Concomitant with the degradation of transcribed DNA sequences a series of chromosomal proteins are released; among these, the major species corresponds to the high mobility group protein H6. The amounts of H6 released from chromatin by limited DNase I action and that in the residual nuclear pellet have been determined. A very high proportion of H6 is associated with DNase I sensitive chromatin regions.

Heterogeneity of biologically active deadenylated protamine mRNA components isolated from rainbow trout testes.

Poly(A)+ protamine mRNA's were isolated from rainbow trout testes and deadenylated by treatment with calf thymus RNase H. Four subcomponents of deadenylated PmRNA (PmRNA1-4) were purified by electrophoresis on a 6% polyacrylamide gel in 8 M urea. Translation of each PmRNA subcomponent in the wheat germ S-30 cell-free system showed that all subcomponents are biologically active but each codes for two or more protamine polypeptides suggesting molecular heterogeneity. However, the deadenylated mRNA's can be categorized into two groups based on the spectrum of protamines whose synthesis they stimulate.

Molecular cloning of three major sequence species from Rainbow trout protamine mRNA.

Double stranded cDNA molecules complementary to purified Rainbow trout protamine mRNA have been cloned in the bacterial plasmid pBR322. In order to circumvent the problems associated with a heterogeneous cDNA probe when identifying recombinants, a comparative hybridisation technique was used which can resolve between closely related cloned sequences. Using this technique, selected recombinants were shown to carry sequences corresponding to separate major fractions of protamine mRNA. Partial nucleotide sequences of the inserts in two clones confirms this conclusion.

Translation of partially purified poly(A)+ protamine messenger RNA components in wheat germ and rabbit reticulocyte cell-free systems. Evidence for translational control mechanisms.

The coding properties of individual poly(A)+ protamine mRNA subcomponents have been explored by analysis of their translation products in two different cell-free protein synthesis systems, the rabbit reticulocyte lysate and the wheat germ S-30, both of which can translate total protamine mRNA. The products synthesized in the reticulocyte lysate in the presence of total poly(A)+ PmRNA consisted mainly of protamine components CII and CIII with component CI only a minor product. However, in the wheat germ S-30, the same mRNA preparation supported the synthesis of all three protamine components, in approximately equal amounts. In addition a new polypeptide, a putative fourth protamine component, labelled CO, was also synthesized. The translation products of subcomponents of poly(A)+ PmRNA separated as individual bands on polyacrylamide gels were similarly analyzed and it was shown that each of the isolated poly(A)+ PmRNA species could stimulate the incorporation of [3H]arginine into protamines in both translational systems. Although each mRNA band stimulated the synthesis of one particular protamine polypeptide predominantly in a given cell-free system, the same RNA preparation was found to direct preferentially the synthesis of a different protamine component in the second cell-free system. The products synthesized in the rabbit reticulocyte lysate in the presence of the individual mRNA species still showed component CI present as a minor product.