Both P1 and P2 protamine genes are expressed in mouse, hamster, and rat

To date, in mammals except for the mouse and human, only one protamine variant has been isolated from sperm. These mammalian protamines share amino acid sequence homology with mouse protamine 1 (mP1), the tyrosine-containing variant. Southern blot analysis of restriction enzyme digests of hamster and rat liver DNA reveals the presence of sequences homologous to mP1, and also to mouse protamine 2 (mP2) cDNAs. Northern blots of hamster and rat total testis RNA probed with mP2 cDNA confirm that the protamine 2 gene in these species is transcribed into two size classes of mRNA of approximately 830 and 700 nucleotides. However, the relative abundance of the rat and hamster protamine 2 mRNAs (rP2 and hP2) in total testis is approximately 50-fold lower and 2- to 5-fold lower, respectively, than the mouse protamine 2 mRNA. Northern blot analysis of hamster and rat testis polysome gradients demonstrates that although the amount of rP2 mRNA and hP2 mRNA is reduced, both are present on polysomes. The decreased expression of rat and hamster protamine 2 mRNA relative to their protamine 1 counterparts contrasts protamine expression in the mouse testis, where approximately equal amounts of mP1 and mP2 protamine mRNAs are present. These results suggest differential expression of the P1 and P2 protamine genes in three closely related mammals.     

Isolation and characterization of trout testis protamine mRNAs lacking poly (A)

Poly(A)+ protamine mRNA was isolated from trout testis cells in a very pure form, and artificial poly(A)- protamine mRNA molecules were derived from it by enzymatic deadenylation with RNAase H from calf thymus after hybridization with oligo(dT). The deadenylated protamine mRNA was found to be active in a wheat germ cell-free system and yielded a labeled product which co-migrated with authentic protamine. These deadenylated mRNA molecules were subsequently used as markers on denaturing polyacrylamide gels to identify and allow the purification of the poly(A)- protamine components known to exist in vivo in the total cellular poly(A)- RNA. RNA species of molecular weights similar to the enzymatically deadenylated subcomponents of protamine mRNA were observed in the natural poly(A)-RNA population of the testis cells. These naturally occurring poly(A)- protamine mRNAs were isolated by preparative gel electrophoresis and further characterized by 3H-poly(U) hybridization assay, by hybridization to complementary DNA made against highly purified poly(A)+ protamine mRNA, and by their ability to direct protamine synthesis in a cell-free system.     

In situ localization of a mammalian protamine gene: parameters affecting specificity of hybridization

Southern hybridization analysis of Indian muntjac genomic DNA with the Eco-Taq bovine genomic protamine probe revealed a simple banding pattern. The pattern of hybridization was identical with that previously observed in the genus Bos. This suggested that the bovine probe specifically hybridized to the Indian muntjac protamine gene. The opportunity was thus provided to assign the chromosomal location of the protamine gene in a comparatively simple system. Accordingly, this probe and the corresponding cDNA probe were used for in situ chromosome hybridization and localization. Various parameters affecting specificity and the resolution of hybridization were examined. Subsequent to optimization, the Indian muntjac gene was shown to be autosomal and distally located in the telomeric region of the p arm of chromosome 1.     

Human male infertility may be due to a decrease of the protamine P2 content in sperm chromatin

Basic chromosomal proteins were extracted from the sperm of fertile and infertile human males. The relative proportions of protamine 1, 2, and 3 were determined by scanning microdensitometry following electrophoresis of total protamine in polyacrilamide gels. The findings were as follows: (1) The proportion of protamine P(2 + 3) in sperm obtained from infertile males was lower than that in fertile males. (2) Protamine P(2 + 3) in infertile human males showed reduced affinity to DNA. The possibility that some cases of human male infertility may be due to mutation within the protamine P2 gene is discussed. © 1993 Wiley-Liss, Inc.     

Isolation and in vitro translation of a mammalian protamine mRNA

mRNA was isolated from sexually mature rat, rabbit, and bovine testes. Poly(A+) and (A-) RNAs were prepared and hybridized to a rainbow-trout protamine probe. The bovine (A+) fraction showed significant hybridization compared to the other species and these related sequences were also found in total bovine DNA. Bovine mRNA programmed the in vitro synthesis of a basic protein that possessed protamine-like properties. The mRNA was fractionated by agarose-gel electro-phoresis and the fractions hybridized to the trout protamine probe. A significant hybridization signal was observed corresponding to a mRNA of approximately 400 nucleotides in length which coded for the protamine-like protein. The data support the view that we have isolated a mammalian (bovine) protamine mRNA.     

Differential distribution of the P1 and P2 protamine gene sequences in eutherian and marsupial mammals and a monotreme

At the protein level, the P1 protamine is the predominant form of mammalian protamine, present in all mammalian spermatozoa analyzed to date. An additional variant, the P2 protamine, has been detected only in spermatozoa of the mouse, hamster and human. Southern blot analysis of a group of restriction enzyme-digested mammalian DNAs has revealed the presence of sequences homologous to the P1 and the P2 mouse protamine genes in diverse species. In agreement with protein studies, nucleotide sequences homologous to the mouse P1 protamine cDNA are widespread, being present in the genomic DNAs of human, rat, dog, ram, horse, bull, hamster, baboon, flying fox (megabat), microbat, boar, North American opossum, and wallaby. Although we detect genomic sequences with strong homology to the mouse protamine 2 cDNA in rat and hamster, we also find weaker but reproducible hybridization to the genomic DNA of human, boar, dog, bull, microbat, wallaby, and platypus. With the exception of the human, the P2 protamine has not been detected in the spermatozoa of these latter species.     

Divergence of protamine gene sequences in fish

Complementary DNA to trout protamine mRNA was hybridized to excess genomic DNA from trout, salmon and yellow perch. Although there was extensive hybridization of the cDNA to trout DNA, no cross-reaction with yellow perch DNA was observed and the hybridization to salmon DNA was noticeably less than in the homologous reaction. To confirm these results, yellow perch protamine mRNA was purified and compared directly to trout protamine mRNA. Yellow perch protamine mRNA was shorter than trout protamine mRNA, when measured by agarose gel electrophoresis in the presence of methyl mercury hydroxide. The two mRNAs did not cross-react in cDNA/RNA hybridizations, although the homologous reactions went to 90% of completion. This lack of sequence homology was confirmed when the oligopyrimidine tracts from the cDNAs were compared. No sequences longer than tetranucleotides were common to both species. Trout protamine cDNA contained oligopyrimidines of composition C₇T₄, C₄T₂, C₃T₂, C₂T₄, C₂T₃, C₁T₅ and C₁T whereas yellow perch protamine cDNA contained C₆T₃ and C₄T₃.     

Cloning of Protamine cDNA of the Medaka (Oryzias latipes) and Its Expression during Spermatogenesis

Protamines or sperm specific basic proteins are highly basic low molecular weight proteins that substitute histones in the chromatin of sperm during spermatogenesis. They condense sperm DNA into a highly compact, stable and inactive complex. In this study, cDNA of protamine of the medaka, Oryzias latipes , was cloned to elucidate the molecular mechanisms involved in spermatogenesis. A medaka testis cDNA library constructed in lambda gt 11 showed 2.78X10⁶ independent recombinants. Several positive clones were obtained by immunoscreening with polyclonal antiserum against medaka protamine. Sequencing showed that one of these positive clones, named MP-1, encoded arginine clusters characteristic of protamine. The putative amino acid sequence of MP-1 revealed a remarkable extent of homology with other fish protamines, such as 71% identity with thynnin Y, a sperm specific basic protein isolated from the bluefin tuna, Thunnus thynnus . Northern hybridization using a MP-1 cDNA probe showed that MP-1 mRNA is present exclusively in the testes and that it gave three detectable bands: a major band of 280 b, and two others of 400 b and 500 b. In situ hybridization of a complementary RNA probe (digoxigenine-UTP-labeled MP-1 RNA) revealed that MP-1 mRNA is localized in some secondary spermatocytes and spermatids, but not in primary spermatocytes or spermatogonia. These results differ from those obtained in studies on the rainbow trout by solution hybridization and in situ hybridization.     

In situ localization of amylase mRNA and protein. An investigation of amylase gene activity in normal human parotid gland

The distribution of human salivary amylase mRNA was studied by in situ hybridization to a [32P]-labeled amylase cDNA probe. Amylase mRNA was localized to the apical portion of acinar cells in frozen sections of human parotid salivary gland. No hybridization was noted in ductal cells, skeletal muscle, or in connective tissue. These results were consistent with immunohistochemical localization of amylase. The technique of in situ hybridization was modified to permit localization of amylase mRNA in variously fixed, paraffin-embedded parotid glands. Although the hybridization signal decreased with all fixatives, the pattern of localization paralleled that obtained with frozen sections. No advantage was noted in fixation with ethanol-acetic acid or Bouin solution over routine fixation with formalin. These results have important implications for researchers interested in studies of gene expression. We have demonstrated that routinely fixed paraffin blocks of human tissue can be used for cellular localization of specific mRNA. In coordination with immunocytochemistry, in situ hybridization offers a powerful tool for studies of mRNA and protein expression in individual cells.     

Protamine messenger RNA: evidence for early synthesis and accumulation during spermatogenesis in rainbow trout

Trout testis cells were separated into various developmental classes by velocity sedimentation in bovine serum albumin gradients and were identified morphologically with particular stages of the process of spermatogenesis. The stage of testis cell differentiation at which protamine mRNA appears in the cell cytoplasm for the first time was determined by hybridization of RNA populations extracted from the separated cells to radioactively labeled protamine cDNA. Primary spermatocytes represent the earliest stage of differentiation at which protamine mRNA can be detected in large quantities in the cell cytoplasm, establishing that the synthesis of this class of mRNA occurs at a much earlier stage than the time of its translation at the spermatid stage. Protamine mRNA sequences were found in both the polysomes and postribosomal supernatant of the spermatid cells which are involved in the synthesis of protamine, while primary and secondary spermatocytes contained the mRNA sequences only in their postribosomal supernatant fractions. These findings strongly suggest that protamine mRNA is synthesized, accumulated, and stored in the cell sap of primary and secondary spermatocytes in the form of “inactive” messenger ribonucleoprotein particles, which are “activated” and translated at the spermatid stage.     

Nucleotide sequence of a protamine component CII gene of Salmo gairdnerii.

We have isolated, using nick-translated cloned protamine cDNA's as probes, several genomic clones containing protamine gene sequences from a Charon 4A library of Eco R1 digested rainbow trout (Salmo gairdnerii) DNA. One clone was chosen for detailed study and the 2.5 kbp Bam HI-Eco R1 restriction fragment containing the gene was subcloned in the plasmid pBR322. A 920 bp Bg1 II - Bam HI restriction fragment contains a sequence coding for protamine component CII as well as regions 5' and 3' to the mRNA coding portion. Present in the region 5' to the mRNA coding sequence are the promoter associated signals "TATA" box and "CAAT" box. The 5' untranslated region of the mRNA whose length and sequence were not established from the cDNA clones (1) was determined by nuclease mapping and starts within a sequence similar to the "capping signal" found in other genes. The protamine gene for CII contains no introns, a situation common to most histone genes, but, unlike the histone genes does not occur close to other protamine genes in a "cluster".     

Temporal translational regulation of the protamine 1 gene during mouse spermatogenesis

Temporal translational control is an important mechanism of gene regulation during mouse spermatogenesis. Studies of the protamine 1 gene, one member of a class of translationally regulated genes, have shown that it is first transcribed post-meiotically in round spermatids, and that the mRNA is stored in an untranslatable form as an inactive ribonucleoprotein particle for up to 1 week before it is translated. The analysis of the expression of fusions between the protamine gene and reporter genes in transgenic mice has demonstrated that sequences mapping in the 3'-untranslated region of the protamine mRNA are sufficient to confer protamine-like translational regulation on the chimeric mRNAs. It is proposed that sequence-specific RNA-binding proteins interact with the protamine 3'-untranslated region and mediate the temporal translational control. Future progress at elucidating the mechanism of translational regulation will come from the identification of translational control factors and their study in vitro and in vivo.     

Ultrastructural localization of mRNA encoding for the EGF receptor in human breast cell cancer line BT20 by in situ hybridization

The EGF receptor (EGF-R), a 170 KD transmembrane glycoprotein, is found at a high level in the BT20 human mammary carcinoma cell line (1 +/- 0.4 x 10(6) sites per cell). In this study, we examined the expression of the EGF-R gene in BT20 cell line by in situ hybridization at the light and electron microscopic level using a human cDNA, corresponding to EGF-R transmembrane and protein kinase domains, labeled with [3H]-, [35S]-, or [32P]-d-ATP. Two treatments were tested to embed cells in Lowicryl resin: the first used fixation and dehydration by progressive lowering of temperature, the second quick freezing and cryosubstitution. The best ultrastructural preservation was obtained with the second procedure without modification of the hybridization signal. EGF-R mRNA was observed principally at the cytoplasmic level, on organelles involved in the protein synthesis process. Labeling was also located on the microvilli which extend into the intercellular space, suggesting that some mRNA would be located in sites where EGF-R is utilized. Some mRNA was observed in the nucleus. This study demonstrates that post-embedding in situ hybridization, after quick freezing and cryosubstitution, is a powerful EM in situ hybridization procedure to study the expression of the EGF-R gene.