Transition Protein Research Articles

Stage-specific expression of genes associated with rat spermatogenesis: characterization by laser-capture microdissection and real-time polymerase chain reaction.

Spermatogenesis in the rat consists of 14 unique morphologic cellular associations between Sertoli cells and developing germ cells within the seminiferous epithelium. The complexity of the cellular associations leads to difficulty in the isolation of individual cells at a defined stage of development for the study of their unique patterns of gene or protein expression. Thus, laser-capture microdissection is an ideal technique to permit such analysis. This study used laser-capture microdissection and real-time reverse transcription-polymerase chain reaction (RT-PCR) to quantitate the stage-specific expression of a series of genes of functional significance in hormonal regulation and cell-cell interactions in spermatogenesis, including cathepsin-L, CREM-tau, transition protein-1, androgen receptor, beta1-integrin, N-cadherin, and hypoxanthine phosphoribosyltransferase (HPRT). Frozen sections (10 micro m) were obtained from normal adult rat testes. Laser-capture microdissection (LCM) was used to capture all cells in cross-sections of seminiferous tubules that were grouped into stages I-V, VII-VIII, and IX-XIII. Transition protein-1 expression was lowest during stages I-V and increased 5.9-fold during stages VII-VIII and IX-XIII (P < 0.01). Cathepsin-L expression was highest during stages I-V and VII-VIII, falling 4.9-fold during stages IX-XIII (P < 0.05). Similarly, CREM-tau expression was highest during stages I-V and VII-VIII, falling 1.6-fold during stages IX-XIII (P < 0.05). A novel CREM-tau isoform lacking the phosphorylation domain was also characterized but was not stage-specific. beta1-Integrin, N-cadherin, and androgen receptor expression did not change between the spermatogenic stages examined. HPRT housekeeper expression was lowest during stages I-V but increased 1.5-fold during stages VII-VIII and IX-XIII (P < 0.05). This study is the first to apply LCM and real-time RT-PCR analysis to quantitate stage-specific changes in the expression of multiple genes in the seminiferous epithelium.

In vitro production of haploid germ cells from fresh or frozen-thawed testicular cells of neonatal bulls.

Improved methods for culturing spermatogenic cells will facilitate the study of spermatogenesis, treatment of male factor infertility, and genetic modification of the male germ line. The objective of this study was to develop a procedure for achieving male germ cell progression through meiosis in vitro. Testes from 3-day-old bulls were decapsulated and seminiferous tubules were dissociated enzymatically to recover Sertoli and germ cells. Dissociated cells were reaggregated by phytohemagglutinin and encapsulated by calcium alginate, then cultured for up to 14 wk in modified Dulbecco modified Eagle medium/F12 (32 degrees C, 5% CO(2) in air). At 2, 5, and 10 wk, cultured cells were examined and evaluated by reverse transcription-polymerase chain reaction (RT-PCR) and Northern blot analysis for protamine-2 (PRM-2) and transition protein-1 (TP-1) mRNA, expressed specifically in round spermatids. Ploidy was characterized by flow cytometric analysis of DNA content of cultured cells. Only Sertoli cells and gonocytes were observed in seminiferous tubules of 3-day-old testes. By 10 wk of culture, small spherical cells (7-10 microm) were apparent at the margin of cell associations in culture. Following RT-PCR and Northern blot analysis, specific bands corresponding to PRM-2 and TP-1 were detected only in adult testis RNA or after 10 wk of culture. Based on flow cytometry, a haploid population of cells appeared in vitro that was not in 3-day-old bull testis. The novel culture system developed in this study is the first to promote differentiation of gonocytes to presumptive spermatids in vitro based on the expression of spermatid-specific genes.

Stimulation of DNA repair by the spermatidal TP1 protein.

The chromatin remodeling process that takes place during spermiogenesis in mammals is characterized by a transient increase in DNA single-strand breaks (SSB). The mammalian transition proteins (TPs) are expressed at a high level at mid-spermiogenesis steps coincident with chromatin remodeling and could be involved in the repair of these lesions since SSB are no longer detected in terminally differentiated spermatids. We report that TP1 can stimulate the repair of SSB in vitro and demonstrate that in vivo repair of UV-induced DNA lesions is enhanced in mammalian cells stably expressing TP1. These results suggest that, aside from its role in DNA compaction, this major transition protein may contribute to the yet unidentified enzymatic activity responsible for the repair of SSB at mid-spermiogenesis steps. These results also suggest that the TP1 proteins have the potential to participate in the repair process following genotoxic insults and therefore may play an active role in the maintenance of the integrity of the male haploid genome during spermiogenesis.

Spermiogenesis and exchange of basic nuclear proteins are impaired in male germ cells lacking Camk4.

Ca2+/calmodulin-dependent protein kinase IV (Camk4; also known as CaMKIV), a multifunctional serine/threonine protein kinase with limited tissue distribution, has been implicated in transcriptional regulation in lymphocytes, neurons and male germ cells. In the mouse testis, however, Camk4 is expressed in spermatids and associated with chromatin and nuclear matrix. Elongating spermatids are not transcriptionally active, raising the possibility that Camk4 has a novel function in male germ cells. To investigate the role of Camk4 in spermatogenesis, we have generated mice with a targeted deletion of the gene Camk4. Male Camk4-/- mice are infertile with impairment of spermiogenesis in late elongating spermatids. The sequential deposition of sperm basic nuclear proteins on chromatin is disrupted, with a specific loss of protamine-2 and prolonged retention of transition protein-2 (Tnp2) in step-15 spermatids. Protamine-2 is phosphorylated by Camk4 in vitro, implicating a connection between Camk4 signalling and the exchange of basic nuclear proteins in mammalian male germ cells. Defects in protamine-2 have been identified in sperm of infertile men, suggesting that our results may have clinical implications for the understanding of human male infertility.

Analysis and significance of messenger RNA in human ejaculated spermatozoa.

Human ejaculate spermatozoa contain multiple mRNA species carried over from earlier stages in spermatogenesis. To date, gene-specific RT-PCR or in situ hybridization has detected transcripts for beta-actin, heat shock proteins (HSP) 70 and 90, protamines (PRM) 1 and 2, transition protein (TNp) 2, HLA II, beta-integrins, and, most recently, phosphodiesterase subtypes. We have further evidence for a complex population of transcripts based on screening a human testis cDNA library with a heterologous spermatozoal probe. High levels of transcribed repetitive sequences are present in human spermatozoa, including medium reiteration repeats (MERs) and short and long nuclear interspersed repeats (SINES and LINES). Both SINES and LINES belong to the retroposon class of repeat elements, which are thought to proliferate via an intermediate RNA that is converted to DNA by an RNA-dependent DNA polymerase (reverse transcriptase). We have circumstantial evidence for the presence of an RT in ejaculate sperm based on the detection of transcripts for ORF2 of LINE 1 encoding such an enzyme. Our data suggests the following: 1. Ejaculate spermatozoa may be a very useful tool in the identification of genes linked to an infertile phenotype. 2. Spermatozoa (or spermatids) may express a reverse transcriptase, the role of which is unknown. 3. RNA isolated from spermatozoa or washed semen samples may facilitate the detection of mutations and deletions in testis-expressed AZF-linked genes.

Temporal expression of the transgenic human protamine gene cluster

Objective: To ascertain the fidelity of expression of the genes from the transgenic human sperm-specific nuclear packaging protamine-1→protamine-2→transition protein-2 (PRM1→PRM2→TNP2) locus. Design: Controlled human transgene study. Setting: Basic science laboratory. Animal(s): Age-matched transgenic and nontransgenic mice. Intervention(s): Transgenic mice containing the human protamine locus were mated. One testis from each offspring was frozen at −80°C and the other was preserved in formalin. Main Outcome Measure(s): The temporal expression of the human and mouse protamines was evaluated by Northern blot analysis. Orientation of the transgenic locus was determined by Southern blot analysis. Tissue morphology was assessed histologically. Result(s): Conservation of transgenic morphology was confirmed. Head-to-tail integration of the PRM1→PRM2→TNP2 locus was shown. Temporal expression of the mouse and human protamine genes was maintained in the transgenic state. Conclusion(s): These results show that the head-to-tail concatomer of the PRM1→PRM2→TNP2 locus contains all the necessary elements for appropriate temporal expression while maintaining testicular structure and function.

Co‐localization of HP1 and TP1 transcripts in human spermatids by double electron microscopy in situ hybridization

Nuclear changes in the basic nucleoprotein complement occur during spermiogenesis in man. Somatic type histones are displaced by transition proteins which are replaced themselves by protamines, the major nuclear proteins present in late spermatids and sperm nuclei. Sense and antisense 35S-labelled riboprobes, coding respectively for human transition protein 1 (TP1) and protamine 1 (HP1), were synthesized with modified specific oligonucleotides and were used for light microscopy in situ hybridization. A double EM in situ hybridization was performed using a digoxigenin-labelled probe for TP1 and a biotin-labelled probe for HP1, and hybrids were revealed, respectively, with specific antibodies coupled to colloidal gold particles of different sizes (10 nm and 15 nm). For both types of transcripts, histological study revealed a specific distribution of the silver grains in the adluminal region of the seminiferous tubules where spermatids are localized. Quantitative ultrastructural analysis of the nuclear and cytoplasmic labelling densities for the mRNAs coding for TP1 and HP1 showed that the transcripts were found in both the nucleus and cytoplasm of round spermatids and persisted until the elongation phase. Transcripts accumulated in the spermatid cytoplasm without any particular cellular compartmentalization. At the end of the spermatid elongation phase, the disappearance of TP1 and HP1 transcripts may be related to the arrest of transcriptional activity, while the deposition of transition proteins and protamines occurs successively within spermatid nuclei.

The amino acid sequence of the ram spermatidal protein 3--a transition protein TP3 or TP4?

As in other mammals, several nuclear basic proteins replace histones during the differentiation of germinal cells into spermatozoa in the ram. These proteins called transition proteins (TP) are later replaced by protamines. The amino acid sequence of the ram spermatidal protein 3 has been established by Edman degradation of the protein and of its fragments generated from digestion with endoproteinase Lys-C and pepsin and from the coding sequence of the gene and of the cDNA. The ram protein 3 is a basic protein of 109 residues (calculated Mr 13,200) with arginine and lysine residues uniformly distributed along the polypeptide chain. Of the 13 serine and threonine residues, 9 are located in structural motifs where they could be phosphorylated and, thus, modulate the binding of the protein to DNA. The tyrosine residues at position 33 and position 93, located in a basic environment, and the tryptophan residue at position 29 could be involved in the interactions of the protein with DNA through the stacking of their aromatic ring between the nucleotide bases. The ram protein 3 differs completely from the two well-defined transition protein families TP1 and TP2, which are also synthesised transiently during mammal spermiogenesis. In contrast with the rat TP3, ram protein 3 does not correspond to a precursor of a protamine. However, it shares structural similarities with both transition proteins TP3 and TP4 of the boar. The ram protein 3 and the boar transition proteins TP3 and TP4 probably belong to the same transition-protein group and would play similar functions in the chromatin remodelling during spermiogenesis. As protamine and transition-protein TP1 and TP2 genes from mammals, the coding sequence of the gene of ram protein 3 is interrupted by one intron but its organisation is different.

Genesis of a novel human sequence from the protamine PRM1 gene

The members of the male haploid expressed protamine 1 (PRM1)→protamine 2 (PRM2)→transition protein 2 (TNP2) locus exist as a single, coordinately expressed genic domain. Previous analysis has revealed that the genes within the human PRM1→PRM2→TNP2 domain are inter-related, as they share significant sequence similarity at both the nucleotide and amino acid levels. Analysis described here supports the view that a fourth candidate coding region, gene4/Prm3, was derived from PRM1 during the genesis of the PRM1→PRM2→TNP2 domain. In some species, gene4 has diverged to a great extent, which can limit its expression.

Expression of mRNA and protein of nucleoproteins during human spermiogenesis.

The most important event determining the nuclear status of sperm cells is the replacement of histones by protamines, which are the basic nuclear proteins of mature spermatozoa. A first step in this exchange is the displacement of histones by transition proteins (TP). Our study demonstrates, for the first time, the sequential expression of the testis-specific histone (H1t) and the transition proteins (TP1 and TP2) during normal human spermatogenesis. H1t mRNA could only be detected in the cytoplasm of mid and late pachytene spermatocytes. Concomitant with the onset of H1t transcription, the H1t protein appeared in the nuclei of pachytene spermatocytes and remain as a nuclear protein constituent up to step 5 spermatids. While transition protein 1 gene TNP-1 mRNA was present in spermatids from step 2 to early step 4, the TP1 protein occured, with temporal delay, in the nuclei of step 3 and step 4 spermatids. The TP2 protein was observed in the nuclei of spermatids from step 1 to step 5. The transition protein 2 gene TNP-2 mRNA was only detected by reverse transcription-polymerase chain reaction, but not on paraffin sections. These data demonstrate a strong temporal association between H1t gene transcription and synthesis of the H1t protein. Since the TP1 protein appeared with temporal delay we can assume that the corresponding TNP-1 mRNA is translationally delayed.

Extended analysis of the region encompassing the PRM1→PRM2→TNP2 domain: Genomic organization, evolution and gene identification

The human male haploid expressed protamine 1 (PRM1)-->protamine 2 (PRM2)-->transition protein 2 (TNP2) locus comprises a coordinately regulated multigenic domain. This region of 16p13.13 has been used as a model to address how the organization of genes and genic domains within the human genome may influence tissue specific gene expression. Toward this goal, we have completed an extensive computational and biological analysis of the region encompassing the PRM1-->PRM2-->TNP2 domain. These analyses have revealed the likely genesis of this domain. Interestingly, the SOCS-1 gene and an hnRNPC-class pseudogene lies just 3' of this domain. Regions of nuclear matrix attachment also mark these newly identified genes.

Comparative gene mapping: cytogenetic localization of PROC, EN1, ALPI, TNP1, and IL1B in cattle and sheep reveals a conserved rearrangement relative to the human genome

The cytogenetic locations of the genes for protein C (PROC), transition protein 1 (TNP1), intestinal alkaline phosphatase (ALPI), engrailed (EN1), and human protointerleukin β (IL1B) have been compared between cattle (Bos taurus, BTA) and sheep (Ovis aries, OAR). Bovine YAC and cosmid clones were used as FISH probes to determine the order (centromere to telomere) of four of these genes on OAR 2q, as well as the location of IL1B on OAR 3p. In cattle, IL1B and EN1 were assigned to BTA 11 and BTA 2, respectively. Alignment of the ovine, bovine, and human physical maps based on these data shows that segments of conserved synteny and chromosomal rearrangements detected between cattle and human are also found in sheep, where the order in cattle is conserved.

Assessment of the degree of contamination of rat germ cell preparations using specific cDNA probes.

Recent reports showing a decrease in sperm count in men have brought new concerns about male infertility. Animal models have been widely used to provide some relevant information about the human male gamete, and extrapolations are made to men and to the clinical context. The present-study assesses one of the methods used for separation of germ cells of the adult rat testis, namely centrifugal elutriation followed by density gradients (Percoll). This method was chosen since it presents the best results for cell purity in separating germ cells from the rat testis. A comparison between continuous and discontinuous Percoll gradients was performed in order to identify the best type of gradient to separate the cells. Maximal cell purity was obtained for spermatocytes (81 +/- 8.2%, mean +/- SEM) and spermatids (84 +/- 2.6%) using centrifugal elutriation followed by continuous Percoll gradients. A significant difference in purity was observed between elongating spermatids harvested from continuous Percoll gradients and from discontinuous gradients. Molecular analysis was used to assess cell contamination by employing specific probes, namely transition protein 2 (TP2), mitochondrial cytochrome C oxidase II (COX II), and sulfated glycoprotein 1 (SGP1). Molecular analysis of the samples demonstrated that morphological criteria are efficient in characterizing the main composition of the cell suspension, but are not reliable for identifying minimal contamination from other cells. Reliable cell purity data should be established using molecular analysis.

Specific binding of a 47-kilodalton protein to the 3' untranslated region of rat transition protein 2 messenger ribonucleic acid.

The synthesis of transition protein (TNP) 2, one of the predominant nuclear proteins of mammalian spermatids, was shown to be posttranscriptionally regulated, by storing the untranslated mRNA for about 3-5 days in the cytoplasm of differentiating spermatids. It has been proposed that binding of a cytoplasmic protein to a conserved motif of 8 nucleotides (nt) in the 3' untranslated region (3'UTR) of TNP2 mRNA is involved in this translational control mechanism. In this report, we show that deletion or variation of the conserved 8-nt motif (GCCAT-CAC) in rat TNP2-3'UTR abolishes the capacity of the in vitro-transcribed RNA to reconstitute specific RNA-protein complexes in RNA bandshift assays. Using Northwestern analysis, we identified specific binding to the TNP2-3'UTR of four proteins of 45, 47, 49, and 60 kDa, all of which are stage-specifically regulated in male germ cell differentiation. Deletion of the 8-nt motif in the 3'UTR specifically prevented binding of the 47-kDa protein, the interaction of which is thought to be mediated by the RNA secondary structure. Analysis of the RNA secondary structure revealed that the 8-nt motif is an essential element of a specific stem-loop structure that is predicted for rat wild-type TNP2-3'UTR. Therefore we assume that the 47-kDa protein plays an important role in specific RNA-protein complex formation of rat TNP2-3'UTR that may be a central event in the translational control of rat TNP2 mRNA.

Sequential Expression of Nucleoproteins during Rat Spermiogenesis

Transition proteins and protamines are highly basic sperm-specific nuclear proteins that serve to compact the DNA during late spermiogenesis. To understand their sequential role in this function, transition protein 1 (TP1), transition protein 2 (TP2), and protamine 1 (P1) were assayed by polyacrylamide gel electrophoresis in pools of microdissected, staged seminiferous tubule segments in the rat. The results were compared with immunocytochemical analyses of squash preparations from accurately identified stages of the epithelial cycle. TP2 was the first to appear as a faint band at stages IX–XI, followed by high levels at stages XII–XIV of the cycle. TP1 showed a low expression at stage XII of the cycle and peaked at stages XIII–I, whereas protamine 1 first appeared at stage I of the cycle and remained high throughout the rest of spermiogenesis. Immunocytochemical analyses and Western blots largely confirmed these results: TP2 in steps 9–14, TP1 in steps 12–15, and P1 from late step 11 to step 19 of spermiogenesis. We propose that TP2 is the first nucleoprotein that replaces histones from the spermatid nucleus, and its appearance is associated with the onset of nuclear elongation. TP1 shows up along with the compaction of the chromatin. The two transition proteins seem to have distinct roles during transformation of the nuclei and compaction of spermatid DNA.

Chromatin reorganization in rat spermatids during the disappearance of testis-specific histone, H1t, and the appearance of transition proteins TP1 and TP2.

Transition proteins replace testis-specific histones and are finally replaced by protamines in the nucleus of germ cells during spermiogenesis. In this study, immunoperoxidase and immunogold localization were used to determine both qualitatively and quantitatively the intracellular distribution of testis-specific histone (H1t), transition protein 1(TP1), and transition protein 2 (TP2) during rat spermatogenesis. H1t labeling was concentrated over heterochromatin in the nucleus of late-pachytene spermatocytes and spermatids up to mid-steps 10. In step 9 spermatids, H1t was confined to the caudal end of the nucleus where heterochromatin was still present, while in early step 10 spermatids, only a few of the nuclei remained caudally labeled. In late step 10 spermatids, a fibrillar chromatin network was distributed throughout the nucleus coincident with the loss of H1t. A statistically significant rise in TP1 and TP2 labeling density over control values was first encountered in the nucleus of step 11 spermatids coincident with the initiation of condensation of the fibrillar chromatin. The TP1 and TP2 labeling density progressively increased in nucleus of step 11-13 spermatids with the apical to caudal condensation of the fibrillar chromatin, In step 13 spermatids, the chromatin was homogeneously condensed throughout the nucleus. In the case of TP1, the nuclear labeling density gradually declined after step 13 and disappeared by step 17. In the case of TP2, the nuclear labeling density disappeared by step 16. This study shows that, coincident with the loss of H1t, the chromatin of the spermatid is reorganized into a fibrillar network, whereas, coincident with the appearance and progressive increase of TP1 and TP2, the fibrillar chromatin condenses in an apical to caudal direction in the nucleus of the spermatid. Thus the remodeling of chromatin structure during spermiogenesis appears to be a two-step process that is sequentially influenced by the loss of spermatid-specific histones and the appearance of transition proteins.

Colocalization of mRNA and protein using in situ hybridization and immunohistochemistry in testicular tissue.

Using testes fixed by perfusion with Bouin's fluid and embedded in paraffin wax, this study has established methods for combining in situ hybridization and immunocytochemistry on the same section to colocalize mRNA and protein for transition protein-1 (TP-1) and sulfated glycoprotein-1 (SGP-1), respectively. It was found that SGP-1 could be detected in tissue sections subsequent to the detection of TP-1 mRNA in situ. The finding that 1) the tissue pretreatments required to permeabilize the section and to allow access to the probe, and 2) the hybridization conditions themselves, had no adverse effect on the detection of antigen, eases the performance of this technique. On this basis, important information could be obtained on the transcriptional and translational activity of spermatogenic cells, if related probes and antibodies are utilized.

DNA condensation by the rat spermatidal protein TP2 shows GC-rich sequence preference and is zinc dependent.

Transition protein-2 (TP2), isolated from rat testes, was recently shown to be a zinc metalloprotein. We have now carried out a detailed analysis of the DNA condensing properties of TP2 with various polynucleotides using circular dichroism spectroscopy. The condensation of the alternating copolymers by TP2 (incubated with 10 microM ZnSO4), namely, poly(dG-dC).poly(dG-dC) and poly(dA-dT).poly(dA-dT), was severalfold higher than condensation of either of the homoduplexes poly(dG).poly-(dC) and poly(dA).poly(dT) or rat oligonucleosomal DNA. Between the two alternating copolymers, poly(dG-dC).poly(dG-dC) was condensed 3.2-fold more effectively than poly(dA-dT).poly(dA-dT). Preincubation of TP2 with 5 mM EDTA significantly reduced its DNA-condensing property. Interestingly, condensation of the alternating copolymer poly(dI-dC).poly(dI-dC) by TP2 was much less as compared to that of poly(dG-dC).poly(dG-dC). The V8 protease-derived N-terminal fragment (88 aa) condensed poly(dA-dT).poly(dA-dT) to a very small extent but did not have any effect on poly(dG-dC).poly-(dG-dC). The C-terminal fragment (28 aa) was able to condense poly(dA-dT).poly(dA-dT) more effectively than poly(dG-dC).poly(dG-dC). These results suggest that TP2 in its zinc-coordinated form condenses GC-rich polynucleotides much more effectively than other types of polynucleotides. Neither the N-terminal two-thirds of TP2 which is the zinc-binding domain nor the C-terminal basic domain are as effective as intact TP2 in bringing about condensation of DNA.

Identification of a functional cyclic adenosine 3',5'-monophosphate response element in the 5'-flanking region of the gene for transition protein 1 (TP1), a basic chromosomal protein of mammalian spermatids.

Transition protein 1 (TP1) is a small basic chromosomal protein that appears in mammalian spermatids during the period of chromatin condensation. The gene for TP1 from several species contains an apparent cAMP response element (CRE) in the immediate 5'-flanking region. The recent identification of high expression of the novel CRE-activating protein (CREM tau) in advanced testicular germ cells provided a stimulus to ask whether or not the TP1 CRE is functional. To this end we show both by gel retardation and by footprint assays that TP1 CRE forms specific bound complexes with proteins in whole testis nuclear extracts and that these complexes involve CREM as evidenced by recognition by a specific antibody. In addition, the TP1 CRE forms specific bound complexes with bacterially expressed CREM tau. Finally, the TPI CRE conveys protein kinase A-dependent induction to a linked chloramphenicol acetyl transferase gene when transfected into JEG-3 cells. Accordingly, TP1 is a good candidate for a testis-specific gene subject to CREM tau regulation.

Genetics: Disturbances of nuclear condensation in human spermatozoa: search for mutations in the genes for protamine 1, protamine 2 and transition protein 1

During spermiogenesis, the successive replacement of the somatic histones by basic proteins, the transition proteins and protamines, allows normal sperm nuclear condensation. It was suggested that disturbances in nuclear condensation may result in male infertility. Here we report the first molecular analysis of the structure of three genes which code for germ cell-specific nuclear proteins, namely protamine 1 (PRM1), protamine 2 (PRM2) and transition protein 1 (TNP1) in infertile men with disturbed sperm chromatin condensation. In 36 infertile men whose spermatozoa showed a positive reaction with aniline blue, which is an indication for the presence of histones in the nuclei, the complete nucleotide sequences of the coding regions and 5' and 3' untranslated regions of the three genes were evaluated. In addition, 10 infertile patients with oligoasthenoteratozoospermia were studied in the same way, as well as nine infertile patients whose spermatozoa showed a reduction of the protamine 2 content. We did not detect any mutation in the three genes in any of the patients. We assume that the disturbances in the sperm chromatin condensation of our patients, and those described in the literature, are not primarily due to mutations in the genes for PRM1, PRM2 and TNP1.