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Nucleotide sequence of a cDNA encoding mouse transition protein 1
Abstract
We have determined the nucleotide sequence of cDNA clones encoding mouse transition protein 1 (TP1), a basic nuclear protein involved in nuclear condensation during spermiogenesis. The nucleotide sequence predicts that transition protein 1 in rats and mice differs by only one amino acid. The rate of substitution of nucleotides in the coding region of mouse and rat transition protein 1 mRNA is close to the average of many proteins in rats and mice, and the usage of degenerate codons is typical of the mouse. The identification of this cDNA clone, in conjunction with previous work (Kleene et al. (1983) Dev. Biol. 98, 455-464; Hecht et al. (1986) Exp. Cell Res. 164, 183-190), demonstrates that the mRNA for mouse transition protein 1 accumulates during the haploid phase of spermatogenesis.
... TP1 and TP2 are the predominant TPs found in rodent spermatids (24). TP1 is a 6.2-kDa, highly basic chromosomal protein with evenly distributed basic residues (32,34). TP2, by contrast, is a 13-kDa protein with distinct structural domains (39). ...
... Total RNA was extracted from mature mouse testes with the RNAgents Total RNA Isolation System (Promega, Madison Wis.). Plasmids containing Tnp1 and Tnp2 cDNAs were provided by Kenneth Kleene (University of Massachusetts, Boston) (32,33). Prm1 and Prm2 cDNA plasmids (61) were purchased from the American Type Culture Collection. ...
... XP019841056.1) with approximately 20% lysine and 20% arginine spread equally [8]. TNP1 is an extensively produced protein during spermatogenesis regulation, and its sequence is substantially similar among mammals [9]. ...
Transition nuclear proteins (TNPs), the principal proteins identified in the condensing spermatids chromatin, have been found to play a key role in histone displacement and chromatin condensation during mammalian spermatogenesis. One such gene belonging to the TNP family called TNP1 gene is abundantly expressed in the regulation of spermatogenesis, and its sequence is remarkably well conserved among mammals. Genomic analysis, by sequencing and computational approach, was used to identify the novel polymorphisms and to evaluate the molecular regulation of TNP1 gene expression in Sahiwal cattle breeding bulls. DNA samples were sequenced to identify novel single nucleotide polymorphisms (SNPs) in the TNP1 gene. Modern computational tools were used to predict putative transcription factor binding in the TNP1 promoter and CpG islands in the TNP1 promoter region. In the TNP1 gene, four SNPs, three TATA boxes, and one CAAT box were identified. One CAAT box was discovered at 89 bp upstream of start site ATG. The computational analyses indicated that the polymorphisms inside the promoter sequence results in an added HNF-1 transcription factor binding site. In contrast, the other variations may remove the naturally occurring SRF transcription factor binding site. The CpG islands in the TNP1 promoter region were predicted to be absent by the MethPrimer program before and after SNP site mutations. These findings pave the way for more research into the TNP1 gene’s promoter activity and the links between these SNPs and reproductive attributes in the Sahiwal breeding bulls.
... TNPs are first detected in the condensing nucleus of spermatids slightly before than protamines (Heidaran et al., 1989). Whereas TNP2 is a 13 kDa protein with distinct structural domains, containing about 10% arginine, 10% lysine, and 5% cysteine (Meistrich et al., 2003;Zhao et al., 2004); TNP1 is a 6.2 KDa protein with 54 residues of amino acids, of which about 40% are arginine and lysine distributed uniformly and do not contain cysteine (Lanneau and Loir, 1982;Kleene et al., 1988;Alfonso and Kistler, 1993;Meistrich et al., 2003;Zhao et al., 2004). Some studies have suggested different functions for the TNPs like in nuclear shaping, histone removal, transcriptional repression, chromatin condensation and repair of the DNA strand breaks that transiently occur during the displacement of the nucleosomes (Caron et al., 2001;Zhao et al., 2004). ...
The genome of eukaryotes is highly organized within the cell nucleus, this organization per se elicits gene regulation and favors other mechanisms like cell memory throughout histones and their post-translational modifications. In highly specialized cells, like sperm, the genome is mostly organized by protamines, yet a significant portion of it remains organized by histones. This protamine-histone-DNA organization, known as sperm epigenome, is established during spermiogenesis. Specific histones and their post-translational modifications are retained at specific genomic sites and during embryo development these sites recapitulate their histone profile that harbored in the sperm nucleus. It is known that histones are the conduit of epigenetic memory from cell to cell, hence histones in the sperm epigenome may have a role in transmitting epigenetic memory from the sperm to the embryo. However, the exact function and mechanism of histone retention remains elusive. During spermatogenesis, most of the histones that organize the genome are replaced by protamines and their retention at specific regions may be deeply intertwined with the eviction and replacement mechanism. In this review we will cover some relevant aspects of histone replacement that in turn may help us to contextualize histone retention. In the end, we focus on the architectonical protein CTCF that is, so far, the only factor that has been directly linked to the histone retention process.
... Moreover, a dramatic reduction in Prm1 mRNA expression level in the VAE testes, compared to that in control mice, has been noted. Prm1 is expressed exclusively in postmeiotic haploid spermatids (Kleene et al., 1988). The Prm1 transcript was first detected in round spermatids and then translated in elongated spermatids (Esakky et al., 2013). ...
Vitamin A and its derivatives contribute to many physiological processes, including vision, neural differentiation, and reproduction. Vitamin A deficiency causes early cessation of spermatogenesis, characterized by a marked depletion of germ cells. However, there has been no clear understanding about the role of chronic intake of vitamin A excess (VAE) in spermatogenesis. The objective of this study was to investigate whether chronic intake of VAE diet causes arrest of spermatogenesis. To examine the effects of VAE on spermatogenesis, we used ICR male mice fed with control (AIN-93G purified diet: 4 IU/g) diet or VAE (modified AIN-93G diet with VAE: 1,000 IU/g) diet for 7 weeks (from 3 to 10 weeks of age). At 10 weeks of age, the retinol concentration in the testes of VAE mice was significantly higher than that of control mice. Testicular cross sections from control mice contained a normal array of germ cells, while the seminiferous tubules from VAE mice exhibited varying degrees of testicular degeneration. Daily sperm production in VAE testes was dramatically decreased compared to that in control testes. Sperm viability, motility, and morphology were also impaired in VAE mice. Furthermore, we examined the effects of VAE on the expression of genes involved in retinoid signaling and spermatogenesis to determine the underlying molecular mechanisms. Therefore, we are the first to present results describing the long-term dietary intake of VAE impairs spermatogenesis using a mouse model.
... TPs are required for normal chromatin condensation, for reducing the number of DNA breaks and for preventing the formation of secondary defects in spermatozoa and the eventual loss of genomic integrity and sterility. TP1 is a 6.2-kDa, highly basic (about 20% each of arginine and lysine) protein with evenly distributed basic residues [64,65], whereas TP2 is a 13-kDa basic (10% each of arginine and lysine) protein with distinct structural domains. The only similarity between the two is their high basicity, exon-intron genomic patterns, and developmental expression [66]. ...
... Fractions (0.5 ml) were collected and adjusted to 150 mM NaCl, and mRNA was precipitated by the addition of 2.5 volumes of ethanol. The mRNA concentration in each fraction was determined spectrophotometrically, and 1.5 p1g mRNA from each fraction was analyzed by Northern blots probed with radiolabeled rat transition protein 1 (TP) cDNA (kindly provided by Dr. N. Hecht, Tufts University, Boston, MA) [26]. All transcripts larger than 900 bp containing no TP, transcript were pooled and used as a template for cDNA synthesis via oligo(dT) primers (Collaborative Research Inc., Bedford, MA) and SuperScript RNase H-Reverse Transcriptase as specified by the manufacturer (Bethesda Research Laboratories Life Technologies, Inc., Gaithersburg, MD). ...
Subtractive hybridization was used to isolate cDNAs highly expressed in stages IX-XI of the cycle of the seminiferous epithelium in the rat. One of the cloned cDNAs was sequenced and shown to be homologous to a previously described cDNA encoding rat prohibitin. Northern blot analyses showed that 1.9- and 1.2-kb transcripts were present in Sertoli cells whereas 1.5-, 1.2-, and 0.7-kb transcripts were expressed in germ cells. Western blot analyses with anti-peptide antibody to prohibitin revealed only a single 30-kDa protein in testis. Immunocytochemistry demonstrated that prohibitin protein was expressed constitutively in adult Leydig cells and Sertoli cells at all stages. Immunoreactivity of prohibition was very low in preleptotene spermatocytes, very high in leptotene spermatocytes, and very low in zygotene spermatocytes. In pachytene spermatocytes, immunoreactivity was very high in stages VII-XI and was minimal during stages XII and XIV. No protein was detected in spermatogonia and spermatocytes undergoing mitotic and meiotic divisions, respectively. These studies show that the prohibitin gene is expressed differentially in testis. The expression pattern of the prohibitin gene in rat testis appears to correlate with a proposed antiproliferative role of prohibitin.
... The protein synthesized during the terminal stages of spermiogenesis is derived from stored transcripts [24,25]. Examples include the transition proteins and protamines [26][27][28], which are transcribed initially in round spermatids and are stored in the cytoplasm for up to 1 wk before being translated. We found the mRNAs and proteins for both PDE1A and PDE1C genes present in transcriptionally inactive elongating and elongated spermatids (step 9-16 spermatids). ...
Calcium and cyclic nucleotides are second messengers that regulate the development and functional activity of spermatozoa. Calcium/calmodulin-dependent phosphodiesterases (CaM-PDEs) are abundant in testicular cells and in mature spermatozoa and provide one means by which calcium regulates cellular cyclic nucleotide content. We examined the spatial and temporal expression profiles of three knownCaM-PDE genes, PDE1A, PDE1B, and PDE1C, in the testis. In situ hybridization and immunofluorescent staining showed that both PDE1A and PDE1C are highly expressed but at different stages in developing germ cells. However, a very low hybridization signal of PDE1B exists uniformly throughout the seminiferous epithelium and the interstitium. More specifically, PDE1A mRNA is found in round to elongated spermatids, with protein expression in the tails of elongated and maturing spermatids. In contrast, PDE1C mRNA accumulates during early meiotic prophase and throughout meiotic and postmeiotic stages. Immunocytochemistry showed a diffuse, presumably cytosolic distribution of the expressed protein. The distinct spatial and temporal expression patterns of CaM-PDEs suggest important but different physiological roles for these CaM-PDEs in developing and mature spermatozoa.
... TPs are required for normal chromatin condensation, for reducing the number of DNA breaks and for preventing the formation of secondary defects in spermatozoa and the eventual loss of genomic integrity and sterility. TP1 is a 6.2-kDa, highly basic (about 20% each of arginine and lysine) protein with evenly distributed basic residues [64,65], whereas TP2 is a 13-kDa basic (10% each of arginine and lysine) protein with distinct structural domains. The only similarity between the two is their high basicity, exon-intron genomic patterns, and developmental expression [66]. ...
... TPs are required for normal chromatin condensation, for reducing the number of DNA breaks and for preventing the formation of secondary defects in spermatozoa and the eventual loss of genomic integrity and sterility. TP1 is a 6.2-kDa, highly basic (about 20% each of arginine and lysine) protein with evenly distributed basic residues [64,65], whereas TP2 is a 13-kDa basic (10% each of arginine and lysine) protein with distinct structural domains. The only similarity between the two is their high basicity, exon-intron genomic patterns, and developmental expression [66]. ...
The purpose of this chapter is to provide a comprehensive overview of spermatogenesis and the various steps involved in the development of the male gamete, including cellular processes and nuclear transformations that occur during spermatogenesis, to provide a clear understanding of one of the most complex cellular metamorphosis that occurs in the human body. Spermatogenesis is a highly complex temporal event during which a relatively undifferentiated diploid cell called spermatogonium slowly evolves into a highly specialized haploid cell called spermatozoon. The goal of spermatogenesis is to produce a genetically unique male gamete that can fertilize an ovum and produce offspring. It involves a series of intricate, cellular, proliferative, and developmental phases. Spermatogenesis is initiated through the neurological axis by the hypothalamus, which releases gonadotropin-releasing hormone, which in turn signals follicle-stimulating hormone (FSH) and luteinizing hormone (LH) to be transmit-ted to the reproductive tract. LH interacts with the Leydig cells to produce testosterone, and FSH interacts with the Sertoli cells that provide support and nutrition for sperm proliferation and development. Spermatogenesis involves a series of cell phases and divisions by which the diploid spermatogonial cells develop into primary spermatocytes via mitosis. Primary spermatocytes in the basal compartment of Sertoli cells undergo meiosis to produce haploid secondary spermatocytes in the adluminal compartment of Sertoli cells in a process called spermatocyto-genesis. This process gives the cells a unique genetic identity within the A. Agarwal () Professor and Director, Center for Reproductive Medicine, Glickman Urological and Kidney Institute, OB-GYN and Women's Health Institute,
... Between histone removal and protamine deposition in mammalian spermatids, about 90% of the basic chromatin components consists of transition proteins (Fig. 3) [99]. Mouse transition protein 1 (TP1) and 2 (TP2), encoded by Tnp1 and Tnp2, are arginine-and lysine-rich proteins that bind strongly to DNA [100][101][102][103][104][105][106]. The functional activities of each transition protein are still under debate. ...
The function of sperm is to safely transport the haploid paternal genome to the egg containing the maternal genome. The subsequent fertilization leads to transmission of a new unique diploid genome to the next generation. Before the sperm can set out on its adventurous journey, remarkable arrangements need to be made during the post-meiotic stages of spermatogenesis. Haploid spermatids undergo extensive morphological changes, including a striking reorganization and compaction of their chromatin. Thereby, the nucleosomal, histone-based structure is nearly completely substituted by a protamine-based structure. This replacement is likely facilitated by incorporation of histone variants, post-translational histone modifications, chromatin-remodeling complexes, as well as transient DNA strand breaks. The consequences of mutations have revealed that a protamine-based chromatin is essential for fertility in mice but not in Drosophila. Nevertheless, loss of protamines in Drosophila increases the sensitivity to X-rays and thus supports the hypothesis that protamines are necessary to protect the paternal genome. Pharmaceutical approaches have provided the first mechanistic insights and have shown that hyperacetylation of histones just before their displacement is vital for progress in chromatin reorganization but is clearly not the sole inducer. In this review, we highlight the current knowledge on post-meiotic chromatin reorganization and reveal for the first time intriguing parallels in this process in Drosophila and mammals. We conclude with a model that illustrates the possible mechanisms that lead from a histone-based chromatin to a mainly protamine-based structure during spermatid differentiation.
... In Dictyostelium discoideum, different actin genes coding for the same actin are expressed differentially (46). From the results of the Northern blot analysis with the 3'-UT testicular SMGA probe of RNAs from smooth-muscle tissues (Fig. 3 (8,50), and mouse, rat, and human transition proteins (5,19,22,23,26,31; P. C. Yelick, Y. K. Kwon, J. F. Flynn, K. C. Kleene, and N. B. Hecht, Mol. Reprod. ...
... In the spermatids, the stored transcripts are likely to have important functions in the maturation process during spermiogenesis. In the previous study, several genes had been characterized and were reportedly expressed only in haploid spermatids2930313233343536. Following that, a large number of cDNA clones have been isolated from mouse testicular cDNA libraries; by partial cDNA sequencing, plentiful novel cDNA clones previously not described in the literature, have been identified [37]. ...
Prohibitin is essential for intracellular homeostasis and stabilization of mitochondrial respiratory chain complexes. To explore its functions during spermiogenesis of Octopus tankahkeei (O. tankahkeei), we have cloned and sequenced the cDNA of this mammalian PHB homologue (termed ot-PHB) from the testes of O. tankahkeei. The 1165 bp ot-phb cDNA contains a 100 bp 5' UTR, a 882 bp open reading frame and a 183 bp 3' UTR. The putative ot-PHB protein owns a transmembrane domain from 6 to 31 amino acid (aa) and a putative PHB domain from 26 to 178 aa. Protein alignment demonstrated that ot-PHB had 73.3, 73.6, 74.0, 75.1, and 45.4% identity with its homologues in Homo sapiens, Mus muculus, Danio rerio, Xenopus tropicalis and Trypanosoma brucei, respectively. Tissue distribution profile analysis revealed its presence in all the tissues examined. In situ hybridization in spermiogenic cells demonstrated that ot-phb was expressed moderately at the beginning of the spermiogenesis. The abundance of transcripts increased in intermediate spermatids and in drastically remodeling final spermatids. In mature spermatozoa, the residuary transcripts concentrated around the chondriosomal mantle where mitochondria assemble around. In summary, the expression of ot-phb during spermiogenesis implicates a potential function of this protein during mitochondrial ubiquitination. It is the first time to implicate the role of prohibitin in cephalopod spermiogenesis.
... The transition proteins 1 and 2 are small highly basic proteins that are unique to the testis and are believed to contribute to, but perhaps not initiate, the termin¬ ation of RNA synthesis during spermiogenesis (Kistler et ai, 1975, 1976). DNA sequence analyses of transition protein cDNA and genomic clones from the mouse (Kleene & Flynn, 1987; Kleene et ai, 1988) and rat (Cole & Kistler, 1987; Heidaran & Kistler, 1987a; Heidaran et ai, 1988) have established that TP1 and TP2 are distinct. The existence of additional transition proteins currently identified only as protein bands in polyacrylamide gels remains to be established. ...
Excerpt
Department of Biology, Tufts University, Medford, MA 02155, USA
Keywords: spermatogenesis; haploid gene expression; gene regulation; testis
Introduction Spermatogenesis offers an experimental system whereby the gene expression of eukaryotic cells with tetraploid, diploid, and haploid chromosome complements can be compared. Starting from a population of stem cells, the diploid spermatogonia follow one of two lineages. One subpopulation of cells initiates a differentiation process ultimately leading to the spermatozoon while a second, presumably distinct, subpopulation of spermatogonia enters a pathway that maintains and repopulates the stem cells of the testis. The cells destined to become spermatozoa undergo several spermatogonial divisions. The last complete replication of DNA during spermatogenesis, in the preleptotene primary spermatocyte, heralds the start of meiosis. During the lengthy interval of meiotic prophase, homologous chromosomes synapse and genetic recombination occurs, producing the genetic diversity required for survival of a species. Following meiotic recombination, the 4N spermatocytes divide twice
... In Dictyostelium discoideum, different actin genes coding for the same actin are expressed differentially (46). From the results of the Northern blot analysis with the 3'-UT testicular SMGA probe of RNAs from smooth-muscle tissues (Fig. 3 (8,50), and mouse, rat, and human transition proteins (5,19,22,23,26,31; P. C. Yelick, Y. K. Kwon, J. F. Flynn, K. C. Kleene, and N. B. Hecht, Mol. Reprod. ...
Mouse testis contains two size classes of actin mRNAs of 2.1 and 1.5 kilobases (kb). The 2.1-kb actin mRNA codes for cytoplasmic beta- and gamma-actin and is found throughout spermatogenesis, while the 1.5-kb actin mRNA is first detected in postmeiotic cells. Here we identify the testicular postmeiotic actin encoded by the 1.5-kb mRNA as a smooth-muscle gamma-actin (SMGA) and present its cDNA sequence. The amino acid sequence deduced from the postmeiotic actin cDNA sequence was nearly identical to that of a chicken gizzard SMGA, with one amino acid replacement at amino acid 359, where glutamine was substituted for proline. The nucleotide sequence of the untranslated region of the SMGA differed substantially from those of other isotypes of mammalian actins. By using the 3' untranslated region of the testicular SMGA, a highly specific probe was obtained. The 1.5-kb mRNA was detected in RNA from mouse aorta, small intestine, and uterus, but not in RNA isolated from mouse brain, heart, and spleen. Testicular SMGA mRNA was first detected and increased substantially in amount during spermiogenesis in the germ cells, in contrast to the decrease of the cytoplasmic beta- and gamma-actin mRNAs towards the end of spermatogenesis. Testicular SMGA mRNA was present in the polysome fractions, indicating that it was translated. These studies demonstrate the existence of an SMGA in male haploid germ cells. The implications of the existence of an SMGA in male germ cells are discussed.
... It is possible that different NPC proteins may be functioning in the germ cells of later developmental stages, since male germ cell nuclei undergo extensive remodeling involving changes in nuclear morphology during spermiogenesis [1]. This type of gene expression change has been observed for the histones that are replaced by three germ cell-specific DNA-binding proteins, transition protein 1 and protamines 1 and 2, during spermiogenesis [37][38][39]. ...
An mRNA with a substantial similarity to the rat p62 mRNA that encodes a nucleoporin was cloned from the rat testis. A probe derived from a unique sequence in the nucleoporin-related (NPR) cDNA revealed a novel mRNA of 1.3 kb, different from the 2.7-kb transcript attributed to the p62 gene. This 1.3-kb transcript was not detected in Sertoli cells; it was found primarily in the haploid germ cells of the adult testis. The DNA sequencing revealed that the central region of the NPR cDNA sequence was identical to the 3' portion of the p62 cDNA containing heptad repeat sequences. However, the 5' region and the extreme 3' region of the NPR cDNA sequence were different from the p62 cDNA. Interestingly, the extreme 3' untranslated region (UTR) contained a 212-bp inverted repeat of a sequence located in the middle of the NPR cDNA that is identical to the p62 sequence. The inverted repeats of the NPR sequence could potentially hybridize, leading to the formation of circular transcripts. Using antibodies specific for the C-terminal regions of p62, a 26-kDa protein was detected from NPR cDNA hybrid-arrested translational products, and a 28-kDa protein was detected from the testis germ cell extracts but not from Sertoli cell extracts.
... Mouse TP1 contains arginine and histidine but not cysteine (Kleene et al., 1988 when [35S]-cysteine was used ( Figs. 1 and 2). This band was therefore considered to be newly synthesized TP1. ...
Mouse and rat seminiferous tubule fragment cultures were used to examine synthesis and processing of mammalian protamines and transition proteins. The tubule fragments were incubated with [3H]-arginine, [3H]-histidine, [35S]-cysteine, or [32P]-PO4, and radiolabeled proteins were analyzed by acid/urea polyacrylamide gel electrophoresis and fluorography or autoradiography. Newly synthesized protamines were recovered from sonication-resistant nuclei (SRN) and could not be detected in cytoplasmic fractions, indicating that protamines are deposited into nuclei immediately after synthesis. Newly synthesized mouse protamine 1 (mP1) and the precursor to mouse protamine 2 (pre-mP2) migrated more slowly during electrophoresis than their predominant testicular forms, identified by staining with Coomassie blue R-250. Within 1 hour of synthesis, the electrophoretic mobilities of mP1 and pre-mP2 increased to match those of their predominant forms. These changes are consistent with initial charge-neutralizing modifications of the newly synthesized protamines, followed by removal of at least some of the modifying ligands, to unmask protamine basicity. Steady-state phosphorylation rates were high for rat protamine 1 (rP1) and were independent of phosphate content; both rP1 molecules of low and high phosphate content were rapidly phosphorylated. Pre-mP2-3, a major processing intermediate derived by proteolysis of pre-mP2, was also rapidly phosphorylated. Like the protamines, transition protein 2 (TP2) was rapidly phosphorylated and increased in electrophoretic mobility soon after synthesis. In contrast, transition protein 1 (TP1) was not phosphorylated and did not exhibit multiple electrophoretic forms.
... Fractions (0.5 ml) were collected and adjusted to 150 mM NaCl, and mRNA was precipitated by the addition of 2.5 volumes of ethanol. The mRNA concentration in each fraction was determined spectrophotometrically, and 1.5 p1g mRNA from each fraction was analyzed by Northern blots probed with radiolabeled rat transition protein 1 (TP) cDNA (kindly provided by Dr. N. Hecht, Tufts University, Boston, MA) [26]. All transcripts larger than 900 bp containing no TP, transcript were pooled and used as a template for cDNA synthesis via oligo(dT) primers (Collaborative Research Inc., Bedford, MA) and SuperScript RNase H-Reverse Transcriptase as specified by the manufacturer (Bethesda Research Laboratories Life Technologies, Inc., Gaithersburg, MD). ...
Subtractive hybridization was used to isolate cDNAs highly expressed in stages IX-XI of the cycle of the seminiferous epithelium in the rat. One of the cloned cDNAs was sequenced and shown to be homologous to a previously described cDNA encoding rat prohibitin. Northern blot analyses showed that 1.9- and 1.2-kb transcripts were present in Sertoli cells whereas 1.5-, 1.2-, and 0.7-kb transcripts were expressed in germ cells. Western blot analyses with anti-peptide antibody to prohibitin revealed only a single 30-kDa protein in testis. Immunocytochemistry demonstrated that prohibitin protein was expressed constitutively in adult Leydig cells and Sertoli cells at all stages. Immunoreactivity of prohibition was very low in preleptotene spermatocytes, very high in leptotene spermatocytes, and very low in zygotene spermatocytes. In pachytene spermatocytes, immunoreactivity was very high in stages VII-XI and was minimal during stages XII and XIV. No protein was detected in spermatogonia and spermatocytes undergoing mitotic and meiotic divisions, respectively. These studies show that the prohibitin gene is expressed differentially in testis. The expression pattern of the prohibitin gene in rat testis appears to correlate with a proposed antiproliferative role of prohibitin.
The genes encoding three different mammalian testis-specific nuclear chromatin proteins, mouse transition protein 1, mouse protamine 1, and mouse protamine 2, all of which are expressed postmeiotically, are marked by methylation early during spermatogenesis in the mouse. Analysis of DNA from the testes of prepubertal mice and isolated testicular cells revealed that transition protein 1 became progressively less methylated during spermatogenesis, while the two protamines became progressively more methylated; in contrast, the methylation of beta-actin, a gene expressed throughout spermatogenesis, did not change. These findings provide evidence that both de novo methylation and demethylation events are occurring after the completion of DNA replication, during meiotic prophase in the mouse testis.
The synthesis of the protamines, the predominant nuclear proteins of mammalian spermatozoa, is regulated during germ cell development by mRNA storage for about 7 days in the cytoplasm of differentiating spermatids. Two highly conserved sequences, the Y and H elements present in the 3' untranslated regions (UTRs) of all known mammalian protamine mRNAs, form RNA-protein complexes and specifically bind a protein of 18 kDa. Here, we show that translation of fusion mRNAs was markedly repressed in reticulocyte lysates supplemented with a mouse testis extract enriched for the 18-kDa protein when the mRNAs contained the 3' UTR of mouse protamine 2 (mP2) or the Y and H elements of mP2. No significant decrease was seen when the fusion mRNAs contained the 3' UTR of human growth hormone. The 18-kDa protein is developmentally regulated in male germ cells, requires phosphorylation for RNA binding, and is found in the ribonucleoprotein particle fractions of a testicular postmitochondrial supernatant. We propose that a phosphorylated 18-kDa protein plays a primary role in repressing translation of mP2 mRNA by interaction with the highly conserved Y and H elements. At a later stage of male gamete differentiation, the 18-kDa protein no longer binds to the mRNA, likely as a result of dephosphorylation, enabling the protamine mRNA to be translated.
Mouse testis contains two size classes of actin mRNAs of 2.1 and 1.5 kilobases (kb). The 2.1-kb actin mRNA codes for cytoplasmic beta- and gamma-actin and is found throughout spermatogenesis, while the 1.5-kb actin mRNA is first detected in postmeiotic cells. Here we identify the testicular postmeiotic actin encoded by the 1.5-kb mRNA as a smooth-muscle gamma-actin (SMGA) and present its cDNA sequence. The amino acid sequence deduced from the postmeiotic actin cDNA sequence was nearly identical to that of a chicken gizzard SMGA, with one amino acid replacement at amino acid 359, where glutamine was substituted for proline. The nucleotide sequence of the untranslated region of the SMGA differed substantially from those of other isotypes of mammalian actins. By using the 3' untranslated region of the testicular SMGA, a highly specific probe was obtained. The 1.5-kb mRNA was detected in RNA from mouse aorta, small intestine, and uterus, but not in RNA isolated from mouse brain, heart, and spleen. Testicular SMGA mRNA was first detected and increased substantially in amount during spermiogenesis in the germ cells, in contrast to the decrease of the cytoplasmic beta- and gamma-actin mRNAs towards the end of spermatogenesis. Testicular SMGA mRNA was present in the polysome fractions, indicating that it was translated. These studies demonstrate the existence of an SMGA in male haploid germ cells. The implications of the existence of an SMGA in male germ cells are discussed.
The classic studies of Monesi (1,2) in the 1960s revealed an intricate pattern of RNA synthesis during spermatogenesis, characterized by transient transcriptional activity. During the early stages of meiotic prophase, a relatively low rate of [3H]uridine incorporation was detected; however, RNA synthesis increases at the mid-pachytene stage followed by diminution during the meiotic divisions. RNA synthesis once again increases in the round and elongating spermatids (up to step 8), followed by a decline in later stages where nuclear condensation occurs.
Specificity of expression is regarded as a strong indication that genes play a role in determining the phenotype or function of the cells in which they are expressed. A molecular genetic approach to understanding the control of male germ cell differentiation involves, in part, identifying genes that are expressed in a spermatogenic cell type-specific manner. Previous observations from our laboratory and others have shown that some testis-specific genes are expressed in spermatogenic cells in a stage-specific manner. Similarly, more ubiquitously expressed genes can exhibit particularly abundant expression in germ cells, often of uniquely sized transcripts. Both patterns of expression suggest a role for these genes in spermatogenesis or subsequent function of male gametes (rev. in Willison and Ashworth 1987; Hecht 1990; Erickson 1990; Wolgemuth and Watrin 1991).
The term spermatogenesis describes the complex series of events in which a terminally differentiated cell, the spermatozoon, is produced from a stem cell [1-3]. The events leading to the formation of the species-specific-shaped spermatozoon are dependent on the products from a large number of temporally expressed genes, many unique to the testis [4]. Prominent among the organ-specific proteins expressed during spermatogenesis are a group of structural DNA-binding proteins.
Transition nuclear proteins (TPs), the major proteins found in chromatin of condensing spermatids, have been reported to be important for histone displacement and chromatin condensation during mammalian spermatogenesis. In the present study, transition nuclear protein-1 (TNP-1) gene was analyzed using polymerase chain reaction-single strand conformation polymorphism (PCR-SSCP) technique-to detect polymorphism in Murrah bulls. Analysis of TNP-1 gene sequence of Murrah buffalo revealed 3 single nucleotide polymorphisms (SNPs) at 205, 340 and 346 bp positions of intronic region. The effect of this polymorphism was explored on individual motility, mass activity and maturation of spermatozoa. Analysis of variance indicates that two variants C and D of Murrah buffalo had significant effect on spermatozoal maturation. However, their effects on individual motility and mass activity was non significant.
SPERMATOGENESIS is a complex developmental process that occurs in several phases. A large number of genes have been identified that are expressed during spermatogenesis1,2, but the biological significance of many of these is not yet known. We have used gene targeting to selectively eliminate the transcription factor CREM (cyclic AMP-responsive element modulator), which is thought to e important for mammalian spermatogenesis3–5. Male mice deficient for all CREM proteins are sterile, as their developing spermatids fail to differentiate into sperm, and postmeiotic gene expression in the testis declines dramatically. The cessation of sperm development is not accompanied by decreases in the levels of follicle-stimulating hormone or testosterone. Our findings indicate that the CREM gene is essential for spermatogenesis, and mice deficient for this transcription factor could serve as a model system for the study of idiopathic infertility in men.
(Uncorrected OCR) Abstract of thesis entitled Molecular Cloning and Characterization of a Novel Germ Cell-specific Gene, VAD1.3, in Spermatogenesis Submitted by Shum Ka Yee Cathy for the degree of Master of Philosophy at The University of Hong Kong in December 2003 Spermatogenesis is a compex developmental event in which undifferentiated germ cells undergo mitotic and meiotic divisions and dramatic morphological reorganization to generate a cell that is capable of fertilizing an oocyte. To date, the molecular mechanisms of gene regulation III spermatogenesis remain poorly understood. To facilitate the identification of candidate genes implicated in spermatogenesis, mRNA differential display approach was employed for the detection of transcription signals at distinct spermatogenic stages in testes obtained from vitamin A-deficiency (V AD) rat model. Of the 24 differentially expressed cDNA fragments isolated, a novel germ cell- specific gene, VAD 1.3, of 3075 bp appears to be of particular interest. It shared 85% homology to a novel gene encoding Rattus norvegicus vitamin A-deficient testicular protein 11. Subsequent bioinformatics analysis using iPSORT indicated that it contains a signal peptide of 30 amino acids at the N-terminus, suggesting that it may be a membrane protein. Northern blot analysis showed that VADl.3 is testis specific in both rat and mouse, and its mRNA was only detected from 25 days post-partum (dpp) onwards up to adult stages of rat testes. In addition, n situ hybridization and immunohistochemistry further located the VADl.3 transcript and protein, respectively, in the acrosome of the round spermatids residing at the adluminal region of seminiferous tubules at stage VI-VII of the rat spermatogenic cycle. Taken together, V AD 1.3 could be the acrosomal membrane protein and may playa role in the process of spermatogenesis. The study of this novel gene, VADl.3, hopes to provide more information on the complicated process of spermatogenesis. More importantly, the long-term goal is to investigate genetic aspects of human male infertility arising from spermatogenesis dysfunction. This leads to tremendous applicability in the future development of therapeutic approaches for the treatment of male infertility and contraceptive methods.
The nucleotide sequence and exon-intron structure of the bovine transition protein 1 gene was determined. It consists of 2 exons (E1, 139 bp; E2, 29 bp) and a single intron (220 bp). The position of the transcription initiation site was determined 30 nucleotides upstream of ATG. TAAATA- and CAAT-boxes were found 60 and 121 bp upstream of the ATG-translation start point, respectively. It was observed that transition protein 1 is highly conserved in mammals at the nucleotide as well as at the amino acid level.
In mouse spermatozoa, DNA is compacted by two protamines mP1 and mP2. Protamine mP2 (63 residues) is synthesized in spermatid nuclei as a precursor pmP2 (106 residues) which is subsequently processed at the end of spermiogenesis [Yelick, P. C., Balhorn, R., Johnson, P. A., Corzett, M., Mazrimas, J. A., Kleene, K. C. & Hecht, N. B. (1987) Mol. Cell. Biol. 7, 2173–2179].
Six proteins, three of which were described earlier [Chauvière, M., Martinage, A., Debarle, M., Alimi, E., Sautière, P. & Chevaillier, Ph. (1991) C. R. Acad. Sci. 313, 107–112], have molecular and electrophoretic properties similar to those of pmP2. They were isolated from purified testis nuclei and characterized by amino acid composition, N-terminal sequence and peptide mapping. From the amino acid compositions, it appears that all six proteins are rich in arginine, cysteine and histidine and are closely related to pmP2 and mP2. The N-terminal sequence of each protein overlaps a distinct region of the N-terminal part of pmP2. The C-terminal part of protamine mP2 starting at arginine 15 is common to all proteins as assessed by amino acid compositions and peptide maps.
All these structural data demonstrate that the six isolated proteins are products of pmP2 precursor processing. The six intermediate proteins pmP2/5, pmP2/11, pmP2/16, pmP2/20, pmP2/26 and pmP2/32 which contain 102, 96, 91, 87, 81 and 75 residues, respectively, are generated from the pmP2 precursor after N-terminal excision of 4, 10, 15, 19, 25 and 31 residues, respectively. The C-terminal sequence of protamine mP2 is strictly identical to that of its precursor; therefore, no maturation occurs in this part of the molecule. At the present time, the proteolytic pathway involved in the amino-terminal processing leading to the mature form of the protamine mP2 (63 residues) has not been elucidated. However, the different representation of six intermediates in the testis suggests that some stages of processing are faster than others or that some cleavage sites are preferred. The proteins described in this paper could result either from stepwise excision of N-terminal residues or from non-sequential cleavages.
The gene for mouse transition protein 1 (mTP1) was isolated, sequenced, and chromosomally mapped. The nucleotide sequence of 1895 bp of a 6.4-kb mTP1 genomic subclone was determined to include 788 bp of 5' flanking region, 564 bp of coding region including a 396-bp intron and a TAA stop codon, and 543 bp of 3' flanking region. The mTP1 gene contains a B1 repeat sequence within the only intron of the gene. The transcriptional start site of the mTP1 mRNA was determined to be located 31 bases upstream of the ATG translational start codon. Southern blot analysis demonstrated the presence of sequences homologous to the mTP1 cDNA in the genomes of the rat, hamster, bull, boar, dog, horse, ram, human, and two marsupials (the American opossum and Monodelphis), suggesting that the mTP1 gene sequence is widely conserved. The TP1 gene has been mapped by analysis of restriction fragment length variants (RFLV) in an interspecific backcross to a position 0.7 +/- 0.4 cM telomeric of Mylf and 1.2 +/- 0.5 cM centromeric of Vil on mouse chromosome 1.
During elongation and condensation of the spermatid nucleus, histones are replaced by spermatid-specific transition proteins (TNP). TNP1 is well characterized at the cDNA and at the genomic level and was found to be highly conserved during mammalian evolution (similarity between 83 to 98%). We here describe for the first time the nucleotide sequence and organization of the gene for TNP2. The gene was isolated from a bull cosmid library and was found to contain a single intron of 910 bp. The coding sequence consists of 390 bp and has a similarity of about 70% to that of the TNP2 cDNAs of mouse and rat. At the basis of amino-acid sequences, the bull TNP2 is 14 and 15 amino acids longer than that of mouse and rat, respectively, and the similarity is only 45% between bull and mouse and 42% between bull and rat. However, the evolutionary divergence has not occurred at the cost of basic amino acids which are of functional importance in DNA-protein interaction in the condensing spermatid nucleus. The TNP2 gene is closely linked to the protamine genes in the bull genome.
In cuttlefish, as in selachians and mammals, spermiogenesis is characterized by the double nuclear protein transition histones----intermediate protein (protein T)----protamine (protein Sp). The cuttlefish protein T, which consists of two structural variants phosphorylated at different degrees, is the first invertebrate spermatid-specific protein to be fully characterized and sequenced. The primary structures of these two variants were established from sequence analysis and mass spectrometric data of the proteins and their fragments. T1 and T2 are two highly related proteins of 78 and 77 residues, respectively, which differ only by four conservative substitutions, two inversions Ser in equilibrium with Arg, and the deletion of 1 residue of arginine in variant T2. The asymmetrical distribution of the hydrophobic and basic residues determines two well defined domains: an amino-terminal domain (residues 1-21) devoid of arginine and aromatic residues and containing all the aliphatic hydrophobic residues and a highly basic carboxyl-terminal domain (residues 22-77 or 78) that contains 77% of arginine, all the tyrosine residues, and most of the phosphorylated serine residues present in the protein. The complete structural identity of the basic carboxyl-terminal domain of spermatidal proteins T1 and T2 with the protamine variants Sp1 and Sp2 isolated from cuttlefish spermatozoa strongly suggests that T1 and T2 could be precursors of Sp1 and Sp2, respectively.
Two basic proteins, protamines P1 and P2, are present in chromatin of mouse spermatozoa. Protamine P1, the less abundant protein in mouse, has a homolog in most mammals, and its synthesis follows a conventional route. In contrast, protamine P2 has been found only in certain other mammals, including humans, and it is synthesized as a precursor nearly twice as long as the mature protein. Processing of this precursor is not yet understood, although it necessarily takes place in elongating spermatids and is likely to play a role in the chromatin condensation occurring in these haploid cells.
We have fractionated basic proteins from mouse testis chromatin and have identified six proteins on electrophoretic gels which, like protamines, are insoluble in SDS. All six were also soluble at the same trichloroacetic acid concentration as protamine P2 and were present in chromatin of elongating spermatids. Radioactive labelling patterns acquired by these SDS‐insoluble proteins during translation in vitro of testis RNA indicate that the largest represents the precursor of protamine P2, and suggest that the others represent intermediates generated by proteolytic cleavage of the precursor. Results from pulse ³ H labelling in vivo were also consistent with the conclusion that a precursor/product relationship exists between these proteins and protamine P2. Conclusions concerning the kinetics of processing have, in addition, been drawn from this data. Hypotheses concerning possible functional roles played by the precursor are presented.
The methylation patterns of genes expressed in the mouse male germ line have been examined. Int-1, Hox-2.1, and Prm-1, all of which contain 5' CpG islands, were found to be completely unmethylated at many sites in these domains, both in somatic tissues and in sperm DNA. Many other testis-specific genes have a similar structure and are probably also constitutively unmethylated. Pgk-2, a non-CpG-island gene, is similar to somatic tissue-specific genes in that it is highly methylated in nonexpressing cell types but undermethylated in pachytene spermatocytes and round spermatids, where it is actively transcribed. At later stages of spermatogenesis, however, the gene becomes remethylated and thus acquires the full modification pattern in sperm DNA. In all these cases, the sperm DNA that emerges from the testis does not contain any germ-line-specific unmethylated sites and thus carries the methylation pattern typical of that in somatic tissues.
The ram transition protein 1 (TP1) is present in spermatid cell nuclei in the nonphosphorylated, monophosphorylated and diphosphorylated forms. Its primary structure was determined by automated Edman degradation of S-carboxamidomethylated protein and of peptides generated by cleavage with thermolysin and endoproteinase Lys-C.
The ram TP1 is a small basic protein of 54 residues and structurally very close to other mammalian TP1. The mass spectrometric data obtained from the protein and its fragments reveal that ram TP1 is indeed a mixture (approximately 5:1) of two structural variants (Mr 6346 and 6300). These variants differ only by the nature of the residue at position 27 (Cys in the major variant and Gly in the minor variant). The study of phosphorylation sites has shown that four different serine residues could be phosphorylated in the monophosphorylated TP1, at positions 8, 35, 36 or 39.
From previous physical studies, it has been postulated that the Tyr32 surrounded by two highly conserved basic clusters was responsible for the destabilization of chromatin by intercalation of its phenol ring between the bases of double-stranded DNA.
The presence of three phosphorylatable serine residues in the very conserved sequence 29–42 is another argument for the involvement of this region in the interaction with DNA.
Transition protein 1 (TNP1) is a highly basic nuclear protein of 54 amino acids that is found in haploid spermatogenic cells during the period of transition of histones to protamines. Using the cDNA clone for human TNP1, we have isolated the gene encoding human TNP1 from human genomic libraries. The gene contains an intron of 200 bp; 1104 bp of the 5'- and 276 bp of the 3'-noncoding region have been sequenced. Comparison with the rat TNP1 gene yielded a similarity of 77% over the region between the transcription start point and the polyadenylation signal. The gene contains typical CAAT and TATAA boxes at conventional distances from the transcriptional start site. Using a series of human-rodent somatic cell hybrids containing variant complements of human chromosomes, the TNP1 gene was found to cosegregate with human chromosome 2. By in situ hybridization, the gene was assigned to the q35 and q36 bands of the long arm of chromosome 2. This chromosomal region encodes several genes, including TNP1, that are located on murine chromosome 1.
The boar late spermatid nuclei retaining transition proteins (TPs) could be obtained from the testis by the use of antipain to inhibit TP-degrading proteinases of the nuclei. The enzymes detected in acid extract including the basic proteins were inactivated by reduction and carboxymethylation of the proteins. The reduced and carboxymethylated basic proteins were fractionated by differential precipitation between 3% trichloroacetic acid (TCA) and 3-20% TCA. From the 3% TCA-precipitate, boar TP2 and TP4 were isolated by high-performance liquid chromatography (HPLC) on Nucleosil 300 7C18. The two TPs were characterized by acid urea- and SDS-polyacrylamide gel electrophoreses and amino acid analysis. Boar TP2 closely resembled rat and mouse TP2s, and ram protein 3 in its high content of serine and basic amino acids, the presence of cysteine and molecular weight. Boar TP4 was similar to ram protein P1 in its high content of basic amino acids, the presence of cysteine and molecular weight. But the TP2 and TP4 differed in electrophoretic mobility on acid urea-gel and solubility in 3% TCA from those of the other species. The HPLC used here also enabled us to efficiently separate boar TP1, TP2, TP3 and TP4, and to estimate that the amount of the TP2, TP3 and TP4 was about 1/8, 1/4 and 1/4 that of the TP1, respectively.
The genes encoding three different mammalian testis-specific nuclear chromatin proteins, mouse transition protein 1, mouse
protamine 1, and mouse protamine 2, all of which are expressed postmeiotically, are marked by methylation early during spermatogenesis
in the mouse. Analysis of DNA from the testes of prepubertal mice and isolated testicular cells revealed that transition protein
1 became progressively less methylated during spermatogenesis, while the two protamines became progressively more methylated;
in contrast, the methylation of beta-actin, a gene expressed throughout spermatogenesis, did not change. These findings provide
evidence that both de novo methylation and demethylation events are occurring after the completion of DNA replication, during
meiotic prophase in the mouse testis.
I have compared the quantity and the length of the poly(A) tracts of five haploid-expressed mRNAs in the polysomal and nonpolysomal fractions of round and elongating spermatids in mice: transition proteins 1 and 2, protamines 1 and 2, and an unidentified mRNA of about 1050 bases. Postmitochondrial supernatants of highly enriched populations of round and elongating spermatids (early and late haploid spermatogenic cells) were sedimented on sucrose gradients, and the size and amount of each mRNA in gradient fractions were analyzed in Northern blots. In round spermatids, all five mRNAs are restricted to the postpolysomal fractions, but in elongating spermatids about 30-40% of each mRNA is associated with the polysomes. The distribution of these mRNAs in sucrose gradients suggests that all five mRNAs are stored in a translationally repressed state in round and early elongating spermatids, and that they become translationally active in middle and late elongating spermatids. The translationally repressed forms of all five mRNAs are long and homogenous in size, whereas the polysomal forms are shorter and more heterogenous due to shortening of their poly(A) tracts. The relationship between translational activity and poly(A) size exemplified by these five mRNAs may be typical of mRNAs which are translationally repressed in round spermatids and translationally active in elongating spermatids.
Spermatid transition protein 1 (TP1) is a 54 amino acid (aa), highly basic chromosomal protein found in mammals during the brief period when histones are being replaced by protamines in the haploid phase of spermatogenesis. Using a cDNA clone as probe, we have isolated the gene (Stp-1) coding for rat TP1 from a population of recombinant bacteriophage lambda. The nucleotide (nt) sequence was established from a point 126 nt upstream from the mRNA cap site to a point about 30 nt downstream from the predicted site of polyadenylation. The gene contains a single intron separating the codon for aa 45 of the mature protein. Comparison of the nucleotide sequences for Stp-1 and the mouse gene coding for protamine P1 suggests a possible evolutionary relationship. Southern blot hybridization to genomic DNA isolated from a panel of mouse-hamster somatic cell hybrids unambiguously mapped Stp-1 to mouse chromosome 1.
Transition protein 1 (TP1) is a small basic nuclear protein that functions in chromatin condensation during spermatogenesis in mammals. Here, recently identified cDNA clones encoding mouse transition protein 1(mTP1) were used to characterize the expression of the mTP1 mRNA during spermatogenesis. Southern blot analysis demonstrates that there is a single copy of the gene for transition protein 1 in the mouse genome. Northern blot analysis demonstrates that mTP1 mRNA is a polyadenylated mRNA approximately 600 bases long, which is first detected at the round spermatid stage of spermatogenesis. mTP1 mRNA is not detectable in poly(A)+ RNAs isolated from mouse brain, kidney, liver, or thigh muscle. mTP1 mRNA is translationally regulated in that it is first detected in round spermatids, but no protein product is detectable until approximately 3 days later in elongating spermatids. In total cellular RNA isolated from stages in which mTP1 is synthesized, the mTP1 mRNA is present as a heterogeneous class of mRNAs that vary in size from about 480 to 600 bases. The shortened, heterogeneous mTP1 mRNAs are found in the polysome region of sucrose gradients, while the longer, more homogeneous mTP1 mRNAs are present in the postmonosomal fractions.
The expression of mRNAs for a transition protein (TP1) and two variants of protamines (P1 and P2) during rat and mouse spermiogenesis was investigated using cDNA hybridization techniques. Slot-blot analyses from 1-mm segments of seminiferous tubules and in situ hybridization from testis sections showed that the levels of mRNA for TP1 increased in step-7 round spermatids at substage VIIb of the seminiferous epithelial cycle, earlier than that of P1 and P2 at substage VIIc. The mRNA levels of all transcripts remained high during steps 8-13 in both species. In the rat, the mRNA of TP1 disappeared during step 14 between substages XIVa and XIVb. The P1 mRNA levels decreased during steps 15-16 (stages I-III) and the P2 mRNA during step 15 (stage I). In the mouse, TP1 mRNA disappeared during step 13 (stage I). The P1 mRNA level decreased before P2 in step 14 (stage II), whereas P2 was detected up to step 15 (stage V). Northern-blot analyses with all three cDNA probes revealed two sizes of mRNA and their stage-specific expression. The shorter transcripts appeared later than the longer ones, at the steps of spermiogenesis where translation is known to begin. The results suggest that transcription of TP1, P1, and P2 mRNAs starts at specifically defined times during spermiogenesis and that the temporal translational regulation of these mRNAs is different.
During spermatogenesis, the nucleoproteins undergo several dramatic changes as the germinal cells differentiate to produce the mature sperm. With nuclear elongation and condensation, the histones are replaced by basic spermatidal transition proteins, which are themselves subsequently replaced by protamines. We have isolated cDNA clones for one of the transition proteins, namely for TP1, of bull and boar. It turned out that TP1 is a small, but very basic protein with 54 amino acids (21% arginine, 19% lysine) and is highly conserved during mammalian evolution at the nucleotide as well as at the amino-acid level. Gene expression is restricted to the mammalian testis, and the message first appears in round spermatids. Thus production of TP1 is an example of haploid gene expression in mammals. The size of the mRNA for TP1 was found to be identical in 11 different mammalian species at around 600 bp. Hybridization experiments were done with cDNAs from boar and bull, respectively. The positive results in all mammalian species give further evidence for the conservation of the TP1 gene during mammalian evolution and its functional importance in spermatid differentiation.
Several recent articles have reported localization of specific mRNAs in the rat testis to stage IX and X seminiferous tubules using in‐situ hybridization. In all cases the expression was located basally in the tubules and appeared as discrete round clusters of grains close to the lamina propria. The localization was interpreted as being in Sertoli cells or leptotene spermatocytes. In this study we demonstrate that this pattern is most probably due to artefactual binding of probes to the residual body (RB).
In the present study testicular tissue, perfusion‐fixed with Bouin's and embedded in paraffin, was used, as this resulted in excellent morphological preservation such that RBs within tubules at stages VIII—X were clearly distinguishable. RNA content of the FU3s was demonstrated at stages VIII—X using methyl green pyronin staining, and could be eliminated by pretreatment with RNAse or trichloroacetic acid. Localization of mRNAs for 11 seminiferous tubule proteins was assessed using ³⁵ S‐labelled and digoxigenin‐labelled riboprobes (activin receptor‐11, α‐inhibin, transferrin, androgen‐binding protein (ABP), cyclic protein‐2 (CP‐2), CREM, sulphated glycoproteins 1 and 2 (SGP‐1 and SGP‐2), transition protein 2 (TP‐2) and cystatin‐C), and digoxigenin‐labelled oligonucleotide probes (transition protein‐1 (TP‐l), TP‐2 and protamine‐1). All of these probes showed localization to the correct cell type(s) within the seminiferous epithelium. In addition, six antisense riboprobes (activin receptor‐II, CREM, SGP‐2, CP‐2, cystatin C and α‐inhibin) showed hybridization to basally located residual bodies in tubules at stages IX—X on one or more occasions, whereas residual bodies around the edge of the lumen (stage VIII) or in transit through the seminiferous epithelium showed no hybridization; sense probes showed no localization to residual bodies.
A common feature of the probes which localized to the basal RBs was that they had been prepared using cDNA cloned into Bluescript SK— vector such that the antisense strand was generated from the T7 polymerase promotor. A cRNA prepared using T7 polymerase and Bluescript vector alone and a GC‐rich 27mer oligonucleotide corresponding to the region of the multiple cloning site of Bluescript adjacent to the T7 site both localized uniquely to basal RB.
It is concluded that the hybridization seen within RBs is probably a subtle artefact unique to RBs undergoing dissolution following fusion with Sertoli cell lysosomes, and may reflect nonspecific hybridization to GC‐rich fragments of RNA.
To understand the spermatid-specific regulation of the protamine-1 (Prm-1) gene, we examined the nuclear proteins that bind to regions of the Prm-1 promoter known to regulate transcription. We focused on the Prm-1 promoter region between bp -224 and -37 because this region directs spermatid-specific expression of a heterologous reporter gene in transgenic mice and because regulatory function has been demonstrated for several subregions of this fragment. Testis nuclear proteins that bind to this promoter region were identified by means of gel mobility shift assays, and the tissue distribution of these proteins was examined through nuclear extracts derived from several mouse tissues. Nuclear extracts prepared from prepubertal and mutant mouse testes were used to demonstrate the developmental appearance of these DNA-binding proteins during spermatogenesis. These studies indicate that a testis-specific protein that appears after Day 12 interacts with a sequence (between bp -37 and -77) shown to be essential for Prm-1 transcription in vivo. In addition, a number of proteins that are not restricted to the testis interact with other functionally important regions of the Prm-1 promoter.
Protamines are small arginine-rich proteins that package DNA in spermatozoa. The mouse protamine 1 (Prm-1) gene is transcribed exclusively in post-meiotic spermatids. To identify elements in the Prm-1 promoter required for spermatid-specific transcription, we generated transgenic mice by microinjection of transgenes containing Prm-1 5' flanking sequences with 5' truncations or internal deletions of conserved sequences linked to a marked Prm-1 gene. We also tested Prm-1 promoter regions with a heterologous human growth hormone reporter gene. We conclude that a 113-bp region can direct spermatid-specific transcription and we have defined sequences within this region that are essential for proper function. These results will facilitate the isolation and characterization of transcription factors essential for post-meiotic gene expression.
The synthesis of the protamines, the predominant nuclear proteins of mammalian spermatozoa, is regulated during germ cell
development by mRNA storage for about 7 days in the cytoplasm of differentiating spermatids. Two highly conserved sequences,
the Y and H elements present in the 3' untranslated regions (UTRs) of all known mammalian protamine mRNAs, form RNA-protein
complexes and specifically bind a protein of 18 kDa. Here, we show that translation of fusion mRNAs was markedly repressed
in reticulocyte lysates supplemented with a mouse testis extract enriched for the 18-kDa protein when the mRNAs contained
the 3' UTR of mouse protamine 2 (mP2) or the Y and H elements of mP2. No significant decrease was seen when the fusion mRNAs
contained the 3' UTR of human growth hormone. The 18-kDa protein is developmentally regulated in male germ cells, requires
phosphorylation for RNA binding, and is found in the ribonucleoprotein particle fractions of a testicular postmitochondrial
supernatant. We propose that a phosphorylated 18-kDa protein plays a primary role in repressing translation of mP2 mRNA by
interaction with the highly conserved Y and H elements. At a later stage of male gamete differentiation, the 18-kDa protein
no longer binds to the mRNA, likely as a result of dephosphorylation, enabling the protamine mRNA to be translated.
A unique acid-soluble basic protein was detected in the testes of sexually mature rats and a number of other mammalian species. This protein was not found in comparable acid extracts prepared from many other rat organs or from the testes of juvenile rats. It makes its appearance only after spermatids are evident in normally developing testes and is absent from the gonads of male rats rendered artificially cryptorchid for periods of 10 days or longer. This protein was purified extensively and was found to be devoid of phenylalanine, tryptophan, glutamic acid, glutamine, isoleucine, and cyst(e)ine.
The specific basic protein from rat testes could not be extracted from epididymal sperm by a variety of procedures. In contrast, certain techniques reproducibly extracted from both epididymal spermatozoa and whole testis a highly basic protein fraction that is strikingly rich both in arginine and cyst(e)ine.
The amino acid sequence of the COOH-terminal cyanogen bromide fragment (residues 12 to 54) of the testis-specific basic protein of the rat has been determined. This analysis completes the primary structure of the whole protein by over-lapping the sequence of the 23 residues from the NH-2 terminus previously published (Kistler, W. S., Noyes, C., and Heinrikson, R.L. (1974) Biochem. Biophys. Res. Commun. 57, 341-347). The complete sequence of this small, highly basic protein is: (see article for formular).
A whole-mount electron microscope technique has allowed direct visualization of the transcription process in mouse spermatids. Thes observations have been supported by light and electron microscope autoradiographic techniques that employ [3H]uridine and [3H]arginine in attempts to clarify mechanisms of RNA synthesis and their relationship to nuclear histone changes throughout spermiogenesis. Early spermatid genomes are dispersed almost completely, whereas in later spermiogenic steps the posterior or flagellar nuclear region is readily dispersed and the anterior or subacrosomal nuclear region remains compact. Display of genome segments permits identification of regions where transcription complexes, presumably heterogeneous nuclear RNA species, are seen related to chromatin. These complexes appear as ribonucleoprotein chains, some of them of considerable length, decreasing progressively in number in late spermiogenic steps. This decrease coincides with diminishing rates of [3H]uridine incorporation. Two distinct patterns of chromatin have been identified: a beaded chromatin type associated with transcription complexes encounterd in early spermatids; and a smooth chromatin type not involved in transcriptive activity observed in advanced spermiogenic genomes. Protein particles staining densely with phosphotungstic acid become apparent in nuclei of spermatids after [3H]arginine incorporation becomes significant. There is no structural or autoradiographic evidence for the presence of nucleoli during spermiogenesis. From these data and from previous experimental findings, we conclude that: (a) spermatogonia, spermatocytes and Sertoli cells are transcriptionally expressed into heterogeneous nuclear RNA and preribosomal RNA species whereas transcription in spermatids is predominantly heterogeneous nuclear RNA; and (b) the modification of the chromatin patterns in late spermiogenic steps indicates a stabilized genome that restricts transcriptive functions.
An algorithm was developed which facilitates the search for similarities between newly determined amino acid sequences and sequences already available in databases. Because of the algorithm's efficiency on many microcomputers, sensitive protein database searches may now become a routine procedure for molecular biologists. The method efficiently identifies regions of similar sequence and then scores the aligned identical and differing residues in those regions by means of an amino acid replacability matrix. This matrix increases sensitivity by giving high scores to those amino acid replacements which occur frequently in evolution. The algorithm has been implemented in a computer program designed to search protein databases very rapidly. For example, comparison of a 200-amino-acid sequence to the 500,000 residues in the National Biomedical Research Foundation library would take less than 2 minutes on a minicomputer, and less than 10 minutes on a microcomputer (IBM PC).
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.
The spermatid transition proteins comprise a set of basic chromosomal proteins that appear during the period when spermatids are undergoing nuclear elongation and condensation, about midway between the end of meiosis and the release of spermatozoa from the seminiferous tubule. The transition proteins replace the histones but are themselves subsequently replaced by protamines, and they are not found in sperm nuclei. We have used a cDNA clone for the smallest transition protein (TP1, 54 amino acids) to show that its message first appears postmeiotically in late round spermatids. Thus production of TP1 is an example of haploid gene expression. The message remains translationally inactive for some 3-4 days before translation occurs in early elongating spermatids. While translationally repressed, TP1 message is nonpolysomal and has a discrete size of about 590 bases, including a 140 residue poly(A) tail. In contrast, polysome-associated message is of heterogeneous size due to variability of poly(A) lengths.
A cDNA clone encoding a small cysteine and serine-rich basic protein has been isolated from a mouse testis cDNA library. This cDNA clone encodes the mouse homologue of a protein involved in the initial phases of condensation of chromatin during spermiogenesis in rats, TP2, based on similarities in the sequence of the carboxyl terminus, composition, molecular weight, and electrophoretic mobility. Mouse TP2 can be divided into a highly basic domain comprising about one-third of the polypeptide chain at the carboxyl terminus and a much less basic domain comprising the remaining two-thirds at the amino terminus. The 5' end of the mouse TP2 mRNA contains two in-phase initiation codons both of which may be used generating two polypeptides which differ in length at the amino terminus. Southern blots demonstrate that there is a single copy of the TP2 gene in the mouse genome and Northern blots demonstrate that the polyadenylated TP2 mRNA is present at high and essentially equal levels in early and late haploid cells, and that it is virtually absent from meiotic cells.
The nucleic acid binding properties of the testis protein, TP, were studied with the help of physical techniques, namely, fluorescence quenching, UV difference absorption spectroscopy, and thermal melting. Results of quenching of tyrosine fluorescence of TP upon its binding to double-stranded and denatured rat liver nucleosome core DNA and poly(rA) suggest that the tyrosine residues of TP interact/intercalate with the bases of these nucleic acids. From the fluorescence quenching data, obtained at 50 mM NaCl concentration, the apparent association constants for binding of TP to native and denatured DNA and poly(rA) were calculated to be 4.4 X 10(3) M-1, 2.86 X 10(4) M-1, and 8.5 X 10(4) M-1, respectively. UV difference absorption spectra upon TP binding to poly(rA) and rat liver core DNA showed a TP-induced hyperchromicity at 260 nm which is suggestive of local melting of poly(rA) and DNA. The results from thermal melting studies of binding of TP to calf thymus DNA at 1 mM NaCl as well as 50 mM NaCl showed that although at 1 mM NaCl TP brings about a slight stabilization of the DNA against thermal melting, a destabilization of the DNA was observed at 50 mM NaCl. From these results it is concluded that TP, having a higher affinity for single-stranded nucleic acids, destabilizes double-stranded DNA, thus behaving like a DNA-melting protein.
DNA in mammalian, and most vertebrate sperm, is packaged by protamines into a highly condensed, biochemically inert form of chromatin. A model is proposed for the structure of this DNA-protamine complex which describes the site and mode of protamine binding to DNA and postulates, for the first time, specific inter- and intraprotamine interactions essential for the organization of this highly specialized chromatin. In this model, the central polyarginine segment of protamine binds in the minor groove of DNA, crosslinking and neutralizing the phosphodiester backbone of DNA while the COOH- and NH2-terminal ends of protamine participate in the formation of inter- and intraprotamine hydrogen, hydrophobic, and disulfide bonds. Each protamine segment is of sufficient length to fill one turn of DNA, and adjacent protamines are locked in place around DNA by multiple disulfide bridges. Such an arrangement generates a neutral, insoluble chromatin complex, uses all protamine sulfhydryl groups for cross linking, conserves volume, and effectively renders the chromatin invulnerable to most external influences.
The present studies were designed to identify mouse spermatid proteins into which intratesticularly injected [³H] arginine and [³H] lysine were initially incorporated and to determine the fates of those proteins during subsequent spermatid differentiation. At intervals between 2 h and 7 days after injections, elongated spermatid nuclei were isolated from the testes by virtue of their resistance to sonication, and mature sperm nuclei were isolated from the epididymides. Basic proteins were extracted from isolated spermatid and sperm nuclei and subjected to electrophoresis on acid-urea polyacrylamide gels. Two hours after injection, [³H] arginine was seen in a number of spermatid basic proteins, including both the "testis-specific" protein (TP) and the protamines. As expected from previous studies, only one class of these labeled proteins, protamine, was retained through the completion of spermiogenesis and sperm maturation 7 days later. In striking contrast, [³H] lysine was initially incorporated only into the spermatid TP protein, was retained for only 3 days, and was then lost. Our previous autoradiographic study (Mayer and Zirkin, 1979) demonstrated that intratesticularly injected [³H] lysine was initially incorporated into elongating spermatid nuclei at the initiation of chromatin condensation (late step 12 and step 13), was retained for 3 days through the completion of chromatin condensation (step 14), and was then lost. The present results, taken together with the results of our previous autoradiographic study, demonstrate striking temporal relationships between the first appearance of newly synthesized TP protein and the initiation of chromatin condensation in spermatid nuclei of late step 12, and between the loss of TP protein and the completion of chromatin condensation in spermatid nuclei of step 14.
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.
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 R10 restriction fragment containing the gene was
subcloned in the piasmid pBR322. A 920 bp Bgl II-Bam HI restriction fragment contains a sequence coding for protamine component
C 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”.
In order to investigate the sequence of changes in nuclear basic proteins throughout ram spermiogenesis, we have used different techniques to obtain populations of spermatid nuclei in specific stages of maturation. Sedimentation of testis cells at 1 gravity followed by treatment with Triton X-100 resulted in one population of round spermatid nuclei (steps 1–a), one of non-round spermatid nuclei (steps 8b-15), and one of elongated spermatid nuclei (steps 12–15). Populations of non-round spermatid nuclei were obtained by treatment with EDTA (steps 9–15), by sonication (steps 12–15) and digestion by DNase (steps 13–15). Nuclear proteins, extracted either directly with dilute acid or following a reducing treatment with 2-mercaptoethanol were characterized by polyacrylamide gel electrophoresis.The most striking alterations in protein composition occur during the elongation phase (steps 8–12). The five histones are displaced from chromatin at the same rate. When they are freed of histones (step 12), the nuclei start to accumulate the sperm-specific protein (BNSP) which is then partly extractable by dilute acid without a thiol that is required for its extraction from more mature nuclei. This stepwise replacement process is accompanied by a reduction of the basic protein amount bound to DNA. As soon as histones begin to disappear, eight spermatidal protein fractions are present in the nuclei until the BNSP synthesis reaches its maximum rate. Except for one, they all contain cysteine and are partially intermolecularly cross-linked in the chromatin. After in vivo and in vitro labelling experiments, they are synthesized in elongating spermatids (steps 8–11). None are degradation products of histones.Correlations of the times of onset of EDTA, sonication and DNase resistances with changes in the basic nuclear proteins point out that stabilization and condensation of spermatid chromatin is promoted through a progressive increase in disulfide bridges.
Dramatic transitions occur in the nuclear proteins during spermiogenesis in rats. In order to determine more precisely when these transitions occur, we have employed centrifugal elutriation, velocity sedimentation at unit gravity, centrifugation in metrizamide gradients, and sonication to obtain relatively homogeneous populations of testis cells and nuclei. The results indicate that histones are present in step 1–8 spermatid nuclei but are not detectable after step 12. Nuclear proteins designated TP and TP2 are not detectable in step 1–8 spermatids but are present and actively synthesized in step 13–15 spermatids. These two proteins are turned over within 5 days after synthesis. A spermatidal basic nuclear protein, designated TP3, and the sperm basic nuclear protein, S1, are present in step 16–19 spermatids. Biochemical characterization of TP2 and TP3 are presented.
The basic chromosomal proteins (SCP) of human, mouse, rabbit and guinea pig sperm nuclei were characterized by polyacrylamide gel electrophoresis and amino acid analysis. Spermatozoa were decapitated with 1% SDS and the nuclei recovered by density gradient centrifugation. Examination by Nomarski and electron microscopy revealed the nuclei to be intact and 99% pure. The basic proteins were extracted from nuclei, aminoethylated and purified by ion exchange chromatography and gel filtration chromatography.The SCP of human, rabbit and guinea pig gave single protein bands with similar mobilities when subjected to polyacrylamide gel electrophoresis. In contrast, aminoethylated mouse SCP consisted of two proteins, SCP·AE1 and SCP·AE2, which had different electrophoretic mobilities. The SCP of these mammalian species were characteristically rich in arginine (47–54.4%) and cysteine (7.7–12.2%). Major differences existed in the amino acid compositions of these proteins. Mouse and human SCP were rich in histidine (12.2 and 7.7%, respectively) and guinea pig was high in tyrosine (11.7%) and phenylalanine (3.5%). Valine was detected only in rabbit SCP and proline in human and guinea pig. Aspartic acid, methionine and tryptophan were not detected in all four species. Studies on the incorporation of [3H]arginine into mouse SCP demonstrated that these basic proteins are synthesized during the terminal stages of spermatogenesis and are subsequently conserved.
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).
Since previous studies have suggested that the mammalian protamine mRNAs are translated poorly in cell-free systems, we directly measured the efficiency of translation of mouse protamine 1 mRNA. We found that mouse testis poly(A)+ mRNA stimulates the synthesis in the wheat germ and reticulocyte cell-free systems of three prominant translation products which can be resolved by electrophoresis through acid urea polyacrylamide gels containing 8 M urea. These translation products have been identified as testis-specific protein, protamine 1, and the precursor to protamine 2 by several criteria, including labeling with amino acids, [35S]cysteine, and [3H]leucine, which are known to be specific to some of these proteins from the nucleotide sequences of recombinant DNAs. Surprisingly, the mobility of the testis-specific protein translation product is slightly reduced and the mobility of both protamine translation products is drastically reduced unless the extracts of cell-free translations are coelectrophoresed with the appropriate carrier. The fraction of [35S]cysteine- labeled protamine 1 translation product was compared with the fraction of testis poly(A)+ mRNA as protamine 1 mRNA which we measured in dot blots with the use of an SP6 RNA polymerase transcript for protamine 1. The results demonstrate that protamine 1 mRNA is translated only slightly less efficiently than the average testis poly(A)+ mRNA.
We have isolated a cDNA clone for rat transition protein 1 (TP1), a major chromosomal protein of mammalian spermatids. The clone was identified initially by hybrid selection of TP1 mRNA. The sequence of the 251-nucleotide cDNA includes the entire coding region for the protein, thereby confirming the identity of the clone as well as predicting two changes in the published amino acid sequence.
The nucleotide sequence of a 404-base cDNA encoding the cysteine-rich, tyrosine-containing mouse protamine has been determined. This insert, isolated from a mouse testis cDNA library, encodes a polypeptide of 50 amino acids of which 28 are arginine, 9 are cysteine, and 3 are tyrosine. The insert contains the complete 3' noncoding region of 151 bases and most of the 5' noncoding region. The predicted amino acid sequence of mouse protamine 1 is about 80% homologous to boar protamine and 67% homologous to bull protamine and contains the central, highly basic domain of four arginine clusters found in the trout protamines. The identification of a cDNA clone for a mouse protamine will facilitate studies of the evolution, regulation, and protein-DNA interaction of this nuclear protein unique to haploid spermatogenic cells.
The sequences of four histone H3 genes coding for the replication variant proteins H3.1 and H3.2 have been determined. Three of these genes, two coding for H3.1 proteins and one for an H3.2 protein, are located on chromosome 13 and expressed at low levels. The fourth gene, encoding an H3.2 protein, is located on chromosome 3 and expressed at a high level. The coding regions of the three genes on chromosome 13 are more similar to each other than to the H3 gene on chromosome 3, and equally divergent from it, suggesting that either gene duplication or gene conversion has occurred since the genes were dispersed onto two chromosomes. A 14-base sequence including the CCAAT sequence and located 5' to the genes on chromosome 13 has been conserved. The histone H3 gene on chromosome 3 has multiple potential binding sites for the Sp1 transcription factor. The coding regions show greater than 95% conservation among the four genes. This is due to the strict pattern of codon usage and the presence of two long (greater than 60 base) regions of completely conserved nucleic acid sequence. These conserved regions in the coding sequence may have an important functional role at the mRNA or DNA level.
Protamines are abundant basic proteins involved in the condensation of sperm chromatin. In the mouse, protamine genes are transcribed postmeiotically in round spermatids. We have cloned and sequenced the mouse protamine 1 gene. Ten lines of transgenic mice harboring marked protamine 1 sequences were generated by microinjection of fertilized eggs. Transcription of the transgene is restricted to round spermatids and in several cases exceeds that of the endogenous gene. The cis-acting sequences required for tissue-specific protamine expression reside on a 2.4-kilobase restriction fragment. Prospects for using transgenic mice to address fundamental questions of male germ-cell development are discussed.
The nucleic acid sequences coding for 23 H3 histone genes from a variety of species have been analyzed using a computer assisted alignment and analysis program. Although these histones are highly conserved within and between highly divergent species, they represent various classes of histones whose patterns of expression are distinctively regulated. Surprisingly, in dendrograms derived from these comparisons, H3 sequences cluster according to their modes of regulation rather than phylogenetically. These clusters are generated from highly distinctive patterns of codon usage within the functional gene classes. We suggest that one factor involved in specifying the differing codon usage patterns between functional classes is a difference in requirements for rapid translation of mRNA. In addition, the data presented here, together with structural and sequence information, suggest a heterodox evolutionary model in which genes related to the intron-bearing, basally expressed H3.3 vertebrate genes are the ancestors of the intronless H3.1 class of genes of higher eukaryotes. The H3.1 class must have arisen, therefore, following duplication of a primitive H3.3 gene, but prior to the plant-animal divergence. Implications of the data presented are discussed with regard to functional and evolutionary relationships.
A statistical analysis of extensive DNA sequence data from primates, rodents, and artiodactyls clearly indicates that no global molecular clock exists in mammals. Rates of nucleotide substitution in rodents are estimated to be four to eight times higher than those in higher primates and two to four times higher than those in artiodactyls. There is strong evidence for lower substitution rates in apes and humans than in monkeys, supporting the hominoid slowdown hypothesis. There is also evidence for lower rates in humans than in apes, suggesting a further rate slowdown in the human lineage after the separation of humans from apes. By contrast, substitution rates are nearly equal in mouse and rat. These results suggest that differences in generation time or, more precisely, in the number of germline DNA replications per year are the primary cause of rate differences in mammals. Further, these differences are more in line with the neutral mutation hypothesis than if the rates are the same for short- and long-living mammals.
The primary sequences were compared among several proteins: gene product 5 protein (GP5) from phage M13; PIKE from phage Ike; gene product 32 protein (GP32) from phage T4; RecA, SSB and SSF from Escherichia coli. These proteins bind strongly and cooperatively to single-stranded DNA with no sequence specificity. GP5 is the smallest in this group and its three-dimensional structure is well-characterized. Using the entire sequence of GP5 as a template we searched for the regions in other single-stranded DNA binding proteins yielding the best alignment of aromatic and basic residues. The identified domains show alignment of five aromatic and four charged residues in these proteins. The domains in PIKE, GP32 and RecA exhibit statistically significant sequence homology with GP5. These observations strongly favor the hypothesis that the protein-single-stranded DNA complex in this class of proteins is stabilized by the stacking interaction of the aromatic residues with the bases of the DNA, and by the electrostatic interaction of the basic residues with the phosphate groups of the DNA. We also find that the DNA binding domains of these proteins have similar secondary structural preferences, mainly beta structures. The triple-stranded beta-sheet may be a common motif in the DNA binding domains of these proteins.
A clone containing a 445-bp cDNA insert was isolated from a cDNA library synthesized from dog-fish testes mRNA. The nucleotide sequence was determined and corresponded to a 50-amino-acid protein. The known five-amino-acid N-terminal sequence corresponded exactly to our deduced amino acid sequence. After in vitro transcription of this cDNA using SP6 RNA polymerase, the translated polypeptide comigrated with the Z1 scylliorhinine marker. Analysis of the cDNA 3' flanking region of our Scylliorhinus protamine Z1 revealed an inverted repeat sequence, an ACAA motif and a CAGGAAAGA box known as regulatory signals for transcription termination in histone genes. In addition, sequences homologous to the simian virus (SV 40) and polyoma virus core enhancer elements were identified in the 5' and 3' flanking regions.
A 500-base cDNA encoding the bovine protamine was used for hybridization experiments with total RNA prepared from testes of prepubertal and sexually mature bulls and from pachytene spermatocytes and spermatids isolated from mature testes. The mRNA for protamine was first detected in the 7-month old testis containing 10-15% of round spermatids but was absent in testes of younger animals containing only diploid spermatogenic cells. Hybridization of the protamine cDNA with the RNA of isolated spermatids of the mature testis resulted in a prominent hybridization signal, while the faint signal obtained with the RNA of pachytene spermatocytes was found to be due to contamination of the cell preparation by spermatids. As demonstrated by in situ hybridization on testis-sections the transcripts are confined to the central cell layers of the tubuli seminiferi corresponding to the spatial arrangement of postmeiotic spermatogenic cells. The results indicate that the protamine gene in the bull is postmeiotically expressed and the mRNA is synthesized as a 680 nucleotide long molecule.
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.
A cDNA library was constructed from a protamine-enriched fraction of dogfish (Scylliorhinus caniculus) mRNA. The nucleotide sequence of a 440-bp insert was determined, and its produced protein sequence confirmed its identification as a cysteine-rich protamine Z2 [Martinage, A., Gusse, M., Belaiche, D., Sautiere, P. and Chevaillier, P. (1985) Biochim. Biophys. Acta 831, 172-178]. The frequency of utilization of the different triplets coding for arginine, which represents 30-70% of the total amino acid residues for trout, mouse and dogfish protamines, is discussed. An alternative repetitive sequence of CGC-AGG was found in the N terminus of the protein. Analysis of the 3' flanking region after the mRNA-terminating TAA codon identified an inverted repeat sequence and an ACCA sequence, which may be possible vestiges of a histone-like termination signal.
A bovine P1 protamine cDNA from a bull testis cDNA library was isolated utilizing a series of oligonucleotide probes. Sequence analysis showed that the cloned cDNA insert extended 317 bp to the poly(A) tail. The 51-residue 6750-dalton protamine primary translated protein is encoded within a 156-bp segment. The protamine sequence predicted from the cDNA sequence differs from that previously reported for the amino acid sequence of bovine protamine P1 by the insertion of the tripeptide Cys-Arg-Arg from residues 39-41 in the carboxy-terminal region of the mature protein. Consistent with previous hybridization analysis, nucleotide sequence comparisons showed that trout protamine cDNA was more closely related to that of bovine than to that of mouse. However, bovine P1 protamine cDNA shared greater sequence homology with mouse P1. A common nucleotide sequence of 30 bp is conserved among all three of these species. Primer extension analysis revealed that, as with trout protamine mRNAs, the majority of the untranslated portion of the mRNA lies 3' to the coding segment. Comparisons of their mRNA secondary structures by computer modeling indicate that the mRNAs fold back onto themselves, producing similar, extensively hydrogen-bonded, convoluted forms. These models support the view that translational regulation of protamine mRNA may be partially dependent on secondary structure. Southern analysis suggests that the bovine protamine P1 gene is not sex-linked and is present as one (or relatively few) copy within the bovine genome.
Ram spermatidal proteins P1, 3 and T were isolated from non-round spermatid nuclei and characterized by amino acid analysis. Spermatidal proteins are small arginine- and cysteine-rich basic proteins. Proteins P1 and T are unusually rich in serine and the histidine content of P1 is particularly high. The NaCl molarities required to dissociate these proteins from the spermatid nuclei were determined. These proteins are present only during the reorganization of the spermatid chromatin.
A small region (220 bases) of SV40 sequence information--141 bases before the polyadenylation site and 79 beyond--are sufficient for cleavage of an messenger RNA precursor (that is, the formation of a mature 3' terminus), the addition of polyadenylic acid, and the transport of messenger RNA from the nucleus to the cytoplasm. These 220 bases include a highly conserved sequence--AAUAAA (A, adenine; U, uracil). Four point mutations in this sequence--AACAAA, AAUUAA, AAUACA, and AAUGAA (C, cytosine; G, guanine)--prevent cleavage.
We have studied the exact sequence requirement for the formation of 3' termini of the sea urchin H2A mRNA in frog oocyte injection experiments. Point mutations destroying the symmetry of the inverted DNA repeat in the mRNA trailer coding sequences prevent the generation of genuine 3' termini. Mutants containing complementary base changes are pseudorevertants and allow the production of H2A mRNA with faithful 3' termini at wild-type levels. Our transcription analyses show that it is primarily the sequence of the transcribed strand that decides whether or not true 3' mRNA termini are produced. This is evidence that an RNA stem-loop structure, rather than a DNA cruciform, is essential for this process. Spacer sequences are absolutely required, because in their absence only H2A mRNA with spacer transcript extensions are found. Once the canonical CAAGAAAGA and flanking sequences are linked to the H2A gene, H2A messenger is synthesized at a suboptimal rate, which can be increased to wild-type levels by the addition of 80 bp of the spacer immediately adjacent to the H2A gene.
The distribution of the mRNA for one of the two mouse protamines, the cysteine-rich, tyrosine-containing protamine (MP1), was examined in the polysomal and nonpolysomal compartments of total testis and purified populations of round and elongating spermatids using Northern blots. In postmitochondrial supernatants prepared from total testis, about 10-15% of MP1-mRNA sediments with the small polysomes. The nonpolysomal molecules of MP1-mRNA are homogeneous in size, about 580 bases, while the polysomal molecules are heterogeneous with a mode of about 450 bases. Digestion with RNase H and thermal chromatography on poly(U) Sepharose reveals that the difference in size of polysomal and nonpolysomal MP1-mRNA is due to a shortening of the poly(A) from about 160 to 30 bases. In round spermatids, essentially all of MP1-mRNA is 580 bases long and is in the nonpolysomal fraction. Elongating spermatids contain roughly equal proportions of the homogeneous, 580 base form in the nonpolysomal compartment, and the heterogeneous 450 base form solely in the polysomal compartment. These results indicate that mRNA for one of the mouse protamines is stored as an untranslated RNP in round spermatids, and that it is partially deadenylated when it is translated in elongating spermatids.
We have isolated several cDNA clones encoding cytoplasmic poly(A)+ RNAs which are enriched in postmeiotic (haploid) spermatogenic cells in the mouse. Seventeen of 750 clones from a testis cDNA library hybridized more strongly to 32P-labeled cDNA copied from cytoplasmic poly(A) RNA of round spermatids than pachytene spermatocytes. Northern gel blots demonstrated that these 17 plasmids hybridized to RNA(s) approximately 0.5 kb (1 clone), 0.7 kb (13 clones), 0.8 kb (1 clone), and 0.9 kb (2 clones). Four plasmids hybridizing to RNAs 0.7 and 0.9 kb were further characterized by Northern blots. The levels of hybridization were about 10-fold greater with RNA from round spermatids, elongating spermatids and residual bodies than from pachytene spermatocytes from adult testis. These plasmids did not hybridize with cytoplasmic poly(A)+ RNA from sexually immature testis, adult liver, or brain, larger precursors in adult testis nuclear RNA, total RNA from cultured Sertoli cells, poly(A)- RNA from adult testis or the mouse mitochondrial genome. These results demonstrate that certain poly(A)+ RNAs are abundant in haploid cells but barely or not detectable in meiotic cells suggesting the accumulation of these RNAs in round spermatids requires transcription in haploid cells.
5′-Noncoding sequences have been tabulated for 211 messenger R'JAs from higher eukaryotic cells. The 5′-proximal AUG triplet
serves as the initiator codon in 95% of the mRNAs examined. The most conspicuous conserved feature is the presence of a purine
(most often A) three nucleotides upstream from the AUG initiator codon; only 6 of the mRNAs in the survey have a pyrimidine
in that position. There is a predominance of C in positions −1, −2, −4 and −5, just upstream from the initiator codon. The
sequence (G) thus emerges as a consensus sequence for eukaryotic initiation sites. The extent to which the ribosome binding site in
a given mRNA matches the −1 to −5 consensus sequence varies: more than half of the mRNAs in the tabulation have 3 or 4 nucleotides
in common with the CCACC consensus, but only ten mRNAs conform perfectly.
Spermatogenesis in the dogfish is characterized by the synchronous development of germinal cells inside follicles. This particularity has permitted studies on precise stages of cell differentiation, especially on the evolution of chromatin structure. A microelectrophoretic method has been devised for the determination of the basic nuclear protein content of accurately identified homogeneous stages of spermatid differentiation. No significant difference was observed during the first stages of spermiogenesis, i.e., in round spermatids, where a typical histone complement was present. At the beginning of nuclear elongation, two new basic protein fractions appeared and coexisted for some time with typical histones; they replaced somatic histones progressively. Later, during elongation, four proteins of high electrophoretic mobility appeared and gradually replaced the intermediary basic proteins. In elongated spermatids, DNA was found tightly packed by these four proteins: three are arginine- and cysteine-rich (Z1, Z2 and S4), the fourth is arginine-rich (Z3). At first, these fractions are all soluble in 0.25 M HCl but during sperm maturation only one (Z3) remains acid-soluble, the others being extractable only after reducing and alkylating treatments. This modification of solubility of Z1, Z2 and S4 corresponded to the oxidation of cysteine residues to form SS crosslinks in chromatin of mature sperm cells. Thus spermiogenesis of the dogfish shows two basic nuclear protein transitions which both occur during nuclear elongation.
Using standardized methods for protein extraction and analysis, the testes of rams, bulls, goats, boars, stallions, rats, cats, hedgehogs, European mink and ferrets were examined for basic spermatid nucleoproteins by electrophoresis. The results suggest that differences exist in the total number of these proteins as well as in the number and amount of the cross-linked cystein-containing proteins. These differences appear to be more family-specific than species-specific.
Developing trout testis has an unusually high steady-state level of histone H4 hyperacetylation and therefore provides an excellent system in which to investigate the role(s) of this modification. We have studied the pattern of H4 hyperacetylation as a function of trout testis cell differentiation by using (a) whole testes at progressive stages of development, and (b) individual testis cell populations separated in a stage-dependent manner. The transcriptional activity associated with the differentiating cell types has also been investigated. The results provide strong evidence that the proliferative and transcriptionally active spermatogonia and spermatocytes possess nearly undetectable steady-state levels of H4 hyperacetylation. The content of hyperacetylated H4 rises dramatically (approaching 50% of the total H4) during spermiogenesis—the process comprises the transition of spermatids to mature sperm and is characterized by declining transcriptional activity. We conclude that the majority of the hyperacetylation of H4 in this case is not associated with the proliferative, transcriptionally active stages of testis development, but rather is associated with an event occurring late in development, during spermiogenesis. The most obvious process associated with this stage is the complete replacement of the histones with the sperm-specific proteins, the protamines. It is suggested that the unusually high level of histone H4 hyperacetylation observed in trout testis is acting, in some manner, to assist in this replacement process.
A variety of biochemical and histochemical techniques have been used to compare the composition of chromatin in sperm nuclei isolated from the epididymides of five mouse strains. The DNA content was determined by phosphorus analysis, deoxyribose analysis, absorption spectroscopy at 260 nm, and cytomorphometry following gallocyanine chrome alum staining. All four methods indicate that the mouse sperm nucleus contains approx. 3.3 pg DNA and that the DNA content does not vary significantly among the strains tested. Three different techniques, quantitative amino acid analysis, absorption spectroscopy at 230 nm, and sperm head density analysis in cesium chloride, were used to determine the protein content. Sperm nuclei from each strain of mouse were found to have a protein to DNA ratio of 0.9 and a chromatin protein content of 3 pg/nucleus. Comparisons of the basic proteins by disc gel electrophoresis demonstrate that the sperm nuclei contain only protamine and lack significant levels of somatic histones or transition proteins. The sperm from each strain contained both mouse protamine variants and the relative distribution of the two proteins did not appear to differ among strains. Using this information, we have been able to draw certain conclusions regarding DNA-protamine interactions and the mode of DNA packaging in the sperm nucleus. The most important of these is that the DNA in the mouse sperm nucleus cannot be packaged in nucleosomes. The protamines in sperm chromatin do not function as structural proteins, providing a subunit core around which the DNA is wrapped, but appear to completely neutralize the phosphodiester backbone of the DNA molecule, thereby minimizing the repulsion between neighboring segments of DNA and allowing it to be condensed into a biochemically inactive particle of genetic information.
The present studies were designed to identify mouse spermatid proteins into which intratesticularly injected [3H]arginine [3H]lysine were initially incorporated and to determine the fates of those proteins during subsequent spermatid differentiation. At intervals between 2 h and 7 days after injections, elongated spermatid nuclei were isolated from the testes by virtue of their resistance to sonication, and mature sperm nuclei were isolated from the epididymides. Basic proteins were extracted from isolated spermatid and sperm nuclei and subjected to electrophoresis on acid-urea polyacrylamide gels. Two hours after injection, [3H]arginine was seen in a number of spermatid basic proteins, including both the 'testis-specific'protein (TP) and the protamines. As expected from previous studies, only one case of these labeled proteins, protamine, was retained through the completion of spermiogenesis and sperm maturation 7 days later. In striking contrast, [3H]lysine was initially incorporated only into the spermatid TP protein, was retained for only 3 days, and was then lost. Our previous autoradiographic study (Mayer and Zirkin, 1979) demonstrated that intratesticularly injected [3H]lysine was initially incorporated into elongating spermatid nuclei at the initiation of chromatin condensation (late step 12 and step 13), was retained for 3 days through the completion of chromatin condensation (step 14), and was then lost. The present results, taken together with the results of our previous autoradiographic study, demonstrate striking temporal relationships between the first appearance of newly synthesized TP protein and the initiation of chromatin condensation in spermatid nuclei of late step 12, and between the loss of TP protein and the completion of chromatin condensation in spermatid nuclei of step 14.