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Single-Nucleotide Polymorphisms and Mutation Analyses of the TNP1 and TNP2 Genes of Fertile and Infertile Human Male Populations

HIROMI NISHIMURA

YOSHITAKE NISHIMUNE

HIROMITSU TANAKA

From the ^* Department of Urology, Osaka University Graduate School of Medicine and the Department of Science for Laboratory Animal Experimentation, Research Institute for Microbial Diseases, Osaka University, Suita City, Osaka, Japan.

Correspondence to: Dr Hiromitsu Tanaka, Department of Science for Laboratory Animal Experimentation, Research Institute for Microbial Diseases, Osaka University, 3-1 Yamadaoka, Suita City, Osaka 565-0871, Japan (e-mail: tanaka{at}biken.osaka-u.ac.jp).

Received for publication April 12, 2005; accepted for publication June 17, 2005.

	Abstract

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Previously, we examined the relationship between protamine gene variations and human male infertility. In this study, we show specific variability in the transition nuclear protein genes (TNPs) of sterile male patients. Transition nuclear proteins (TPs) are major nuclear proteins that replace nuclear histones, leading to eventual substitution by protamines during human spermiogenesis. Analysis of the human TNP1 and TNP2 gene sequences in 282 sterile male patients and 270 (TNP1) and 266 (TNP2) proven-fertile male volunteers revealed 5 amino acid substitution-causing single nucleotide polymorphisms (SNPs) in the open-reading frame of the TNP2 gene. On the other hand, a deletion of 15 nucleotides, which encompassed the recognition site for the cAMP response element (CRE) transcription factor, was found in the 5'-promoter region of the TNP1 gene in infertile men. This deletion reduces TNP1 expression and may cause human male infertility.

     Key words: Protamine, transition nuclear protein, sperm, male infertility, genome, promoter, SNPs

During spermiogenesis, round spermatids undergo complex morphologic, physiologic, and biochemical modifications that result in the formation of mature spermatozoa. These specific events are supported by spermiogenesis-specific gene products (Tanaka and Baba, 2005). The sperm nucleus undergoes marked rearrangement, which involves the removal of histones and their replacement by various nuclear proteins. Finally, the DNA of human sperm is highly condensed in the sperm head by highly positively charged protamines (PRMs; Tanaka and Baba, 2005). The replacement of histones and the deposition of protamines is supported by different nuclear proteins, including the transition nuclear proteins (TPs), for major remodeling of the chromatin (Meistrich et al, 2003). Almost all of these nuclear basic proteins, including PRMs, are derived from histone H1 and undergo complex processes of modification in mammals (Lewis et al, 2004). The PRM1, PRM2, and TNP2 genes of these nuclear proteins, which are expressed during spermiogenesis, are clustered on 16p13.13 (Martins et al, 2004). PRMs are highly charged, arginine rich, and bind to DNA in a nonspecific manner. However, the mechanism of condensation of sperm chromatin has not been resolved. On the other hand, the technique of gene targeting to produce knockout animals allows the study of gene function in vivo. Disruption of PRM1 or PRM2 in mice has shown that the PRM1 and PRM2 proteins are essential for fertility and that haploinsufficiency is caused by a mutation in 1 protamine allele (Cho et al, 2001). Moreover, when PRM2 is disrupted, the resultant sperm nuclei are infertile, even via intracytoplasmic sperm injection (ICSI) (Cho et al, 2003). These results indicate that PRM2 is essential for the process of nuclear compaction during spermiogenesis. It has been reported that mouse null mutants for either TNP1 or TNP2 are subfertile (Yu et al, 2000; Zhao et al, 2001), while mice that lack both these TNPs are infertile (Zhao et al, 2004). These results indicate that these basic proteins are important players in nuclear formation during spermiogenesis. In previous studies of single-nucleotide polymorphisms (SNPs) in the open-reading frames (ORFs) of the PRM1 and PRM2 genes in the protamine gene cluster on 16p13.13, we found that a mutation leading to a single nucleotide replacement induced a nonsense mutation in the PRM2 gene in a group of infertile patients (Tanaka et al, 2003). This mutation would be expected to cause male infertility, even in the hemizygous condition, because haploinsufficiency of either PRM1 or PRM2 is known to cause infertility in male mice.

In the present study, we assessed the prevalence of TNP2 gene SNPs in the protamine gene cluster at 16p13.13. We also examined the prevalence of alterations in the gene for another transition nuclear protein, TP1. DNA samples were analyzed from 548 men: 282 infertile patients who were undergoing fertility evaluation and 270 (TNP1) and 266 (TNP2) proven-fertile volunteers. We discovered a deletion mutation of the recognition site for the cyclic adenosine monophosphate (cAMP) response element (CRE) transcription factor (Sassone-Corsi, 1998) in the promoter region of the TNP1 transcription unit. The cAMP-responsive element modulator (CREM) plays an important role in regulating spermiogenesis by binding to the recognition site for the CRE transcription factor (Nantel et al, 1996). Mice that are CREM deficient have reduced testis weights and a complete lack of mature spermatozoa in the seminal fluid. The promoter regions of many genes expressed during spermiogenesis include the recognition site for the CRE transcription factor, which regulates gene expression in spermiogenesis (Sassone-Corsi, 1998). Deletion of the recognition site for the CRE transcription factor in TNP1 dramatically decreases the expression of TNP1 mRNA (Kistler et al, 1994). TNP1 plays an important role in nuclear formation during spermiogenesis in mice (Yu et al, 2000). This deletion may be associated with human male infertility, although mouse null mutants for TNP1 and TNP2 are subfertile (Yu et al, 2000).

	Materials and Methods

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Participants

Japanese infertile subjects (N = 282) were divided into sub-groups according to the degree of defective spermatogenesis: 192 (68%) of these patients had nonobstructive azoospermia, while 90 (32%) had severe oligospermia (<5 x 10⁶ cells/mL). The subjects had primary idiopathic infertility based on a genetic study (Birmingham, 2004). The control group of fertile males (N = 266) consisted of men who had fathered children born to pregnant women at the maternity clinic. The donors gave permission for their blood to be used for the analysis of genomic DNA in this study.

Identification of SNPs in the TNP1 and TNP2 Genes by Direct Sequencing of PCR-Amplified DNA

Genomic DNA was isolated from the blood samples using protease treatment and phenol extraction (Sambrook et al, 1989). Two polymerase chain reaction (PCR) primer sets, TNP1A-TNP1B and TNP1C-TNP1D, were designed to amplify the TNP1 gene (Figure 1). The TNP1A-TNP1B primer set comprised TNP1A (5'-CACAGTATCTACTGTGTTTATCCTCCAC-3') from nucleotide (nt) -785 to nt -757 upstream of the transcription start site (Luerssen et al, 1990), and TNP1B (5'-GTGCAGCTCAAGGGCTGCCC-3') from nt -152 to nt -171 downstream of the transcription start site. The TNP1C-TNP1D primer set comprised TNP1C (5'-GGCTGGGATTCAGTTTCTCAATAACACC-3') from nt -244 to nt -217 upstream of the transcription start site, and TNP1D (5'-TACGGTGGTGGGAGGGAATTGGAGG-3') from nt 724 to nt 748 upstream. The following PCR conditions were used: 35 cycles of denaturation at 98°C for 10 seconds, annealing at 62°C for 30 seconds, and extension at 72°C for 1 minute for TNP1A-TNP1B; and 35 cycles of denaturation at 98°C for 10 seconds, annealing at 60°C for 30 seconds, and extension at 72°C for 1 minute for TNP1C-TNP1D. The PCR-amplified fragments were purified using the SUPREC PCR spin column (Takara, Shiga, Japan). The fragments of TNP1A-TNP1B and TNP1C-TNP1D DNA were independently sequenced from both ends using the same PCR primers with thermal-cycle sequencing kits purchased from Applied Biosystems (Foster City, Calif). The DNA sequences of TNP1A and TNP1B were compared based on the results of sequencing from both directions. The reaction products were analyzed using an ABI-PRISM 310 Genetic Analyzer (Applied Biosystems).

View larger version (61K): [in this window] [in a new window]   Figure 1. Genomic DNA sequences of the transition nuclear protein genes (TNP1s) and primers used for polymerase chain reaction (PCR) amplification and sequencing. The deduced amino acid sequences of the TNP1 open-reading frames (ORFs) are shown below the DNA sequence (Luerssen et al, 1990). The recognition site for CRE, Sp1, and TATA nucleotide sequences are underlined. The single nucleotide polymorphisms (SNPs) are shown by shadowed letters, and minor alleles are indicated underneath the nucleotide. The numbers in the right margins indicate the nucleotide positions (with the transcription start site designated as +1); the arrows below the DNA sequence, the primers used for PCR amplification and sequencing; the stars below the DNA sequence, the transcription start site and polyA-additional signal (the polyA signal is added from the straight arrow to the TNP1 mRNA); a box, the intron; and the shadowed sequence at nt -106 to -91, the region that is deleted found in the infertile male patients.

View larger version (69K): [in this window] [in a new window]   Figure 2. Schematic representation of the protamine gene cluster and genomic DNA sequences of the TNP2 genes. a) The PRM1, RPM2, and TNP2 genes are clustered within approximately 10 kb of genomic DNA on 16p13.13 (Engel et al, 1992). Previously, we identified SNPs in the ORFs of PRM1 and PRM2 (Tanaka et al, 2003). The GenBank accession numbers are listed. b) The deduced amino acid sequences of the TNP2 ORFs are shown below the DNA sequence (Nelson et al, 1993). Several of the SNPs (nt 613, 721, 816, 946, 947, and 950) that are registered in the NCBI dbSNPs were not found in this study (see Table 2). Comparisons with the DNA sequences registered in GenBank indicate deletions at nt 1036 and nt 1046 (see Table 2). The SNPs are shown as shadowed letters, and minor alleles are indicated below the nucleotide. The numbers in the right margins indicate the nucleotide positions (with the transcription start site designated as +1); the arrows below the DNA sequence, the primers used for PCR amplification and sequencing; the stars below the DNA sequence, the transcription start site; the bar below the DNA sequence; the polyA-additional signal (the polyA signal was added from the straight arrow to the TNP1 mRNA); and a box, the intron.

	Results

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Analysis of TNP1 Gene SNPs

Two PCR primer sets were used to analyze the TNP1 gene sequences (EMBL/DDBJ/GenBank accession number M29704; Keyeux et al, 1989) of the infertile and proven-fertile subjects. Direct sequencing of the PCR-amplified DNA was performed using genomic DNA from blood samples. The PCR-amplified 1533-bp DNA fragment included the promoter region and 200-nt intron of TNP1 (Figure 1). Thus, we could identify SNPs that were located within 1480 bp of the primer sequence in the 1533-bp DNA fragment. The SNP prevalences were compared for infertile males and proven-fertile males. We found 5 SNPs or mutations in the promoter region at nt -694 to -689, nt -364, nt -258, and nt -222, a deletion of the nt -91 to -106 region in the recognition site for the CRE transcription factor, 2 SNPs in the 5'-untranslated region of the TNP1 mRNA, and 1 SNP in the intron (Table 1; Figure 1). One of the SNPs registered in the NCBI dbSNPs (rs1179733) was not found in this study. The nt -245 to -239 region in the sequence registered in GenBank (M59924) differed from that found in this study. None of these SNPs resulted in changes to the amino acid-coding region. The 3 SNPs found in this study, at -222A>G, 54A>C, and 55G>A (Figure 1), were either major homozygous or heterozygous SNPs; no minor homozygous SNPs were observed (Table 1). The SNPs did not show significantly higher prevalences in infertile patients than in proven-fertile volunteers (Table 1). Similarly, the SNPs of nucleotide sequence AAAAA (A x 5) or AAAAAA (A x 6) at nt -694 to -698, -364C>T, and -258G>A in the 3'-promoter region (Figure 1) did not show significantly higher prevalences in infertile patients than in proven-fertile volunteers (Table 1). The heterozygous deletion at nt -91 to -106, which introduced a deletion in the recognition site for the CRE transcription factor for haploid-specific expression of TNP1, was observed in only one of the azoospermic patients and was absent in the 266 fertile controls (Table 1). Because the recognition site for the CRE transcription factor is important for the expression of TNP1, this deletion may cause azoospermia, even in the hemizygous condition.

	Discussion

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During spermiogenesis, chromatin is changed by the replacement of the somatic-type histones with PRMs, which results in the sperm nucleus being compacted. Drastic alteration of chromatin during spermiogenesis is achieved through systematic expression of spermiogenesis-specific nuclear proteins (Tanaka and Baba, 2005). Because deficiencies in the genes that code these nuclear proteins lead to male infertility in mice, it appears that the essential event in chromatin transformation via the replacement of the somatic-type histones with PRMs is the systematic expression of spermiogenesis-specific nuclear proteins (Zhao et al, 2004). To examine PRM gene expression in relation to human male infertility, we previously assessed the prevalence of these SNPs in infertile patients and proven-fertile volunteers and found that the 248C>T mutation of the PRM2 gene induced a nonsense codon under conditions of heterozygosity in 1 infertile patient (Tanaka et al, 2003). This nonsense mutation in PRM2 may cause male infertility due to haploinsufficiency of PRM2. In the present study, we assessed the prevalence of TNP gene SNPs in male patients who were undergoing fertility evaluation.

Two TPs have been identified as abundantly expressed basic nuclear proteins that participate in chromatin condensation by the replacement of somatic-type histones with protamines during spermiogenesis (Meistrich et al, 2003). The TP1 amino acid sequence is highly conserved among various mammals (Kremling et al, 1989). In contrast, the TP2 sequence is poorly conserved (Alfonso and Kistler, 1993), and the levels of TP2 expression and TP2 protein vary among species (Steger et al, 1998). The relative molecular mass (Mr) of TP1 is approximately 6200, with about 20% arginine and 20% lysine distributed uniformly, and without cysteine residues (Kistler et al, 1975). TP2 (Mr 13 000) is composed of about 10% arginine, 10% lysine, and 5% cysteine residues (Grimes et al, 1975). It has a highly basic C-terminal domain and an N-terminal domain that forms zinc fingers (Meetei et al, 2000). The TNP2 gene is closely linked to the 2 PRM genes (Engel et al, 1992), which suggests that they arose by gene duplication and have retained common functions. In contrast, TNP1 exists on a separate chromosome (Heidaran et al, 1989). Biochemical analyses have shown that TP1 decreases the melting temperature of DNA (Akama et al, 1998). In contrast, TP2 increases the melting temperature of DNA and compacts the DNA into nucleosomal cores, which indicates that it is a DNA-condensing protein (Baskaran and Rao, 1990). These analyses of TPs show that these proteins play different roles in chromatin remodeling during spermiogenesis. TP2 contains cysteine residues, as do the PRMs, and it is duplicated in the protamine gene cluster, which suggests that it might have a similar role to the PRMs.

In general, the loss of the TNP1 or TNP2 gene does not lead to infertility in mice, although some TNP1-deficient mice are infertile (Yu et al, 2000). The loss of both TNP1 and TNP2 results in reduced litter sizes (Zhao et al, 2004). The idea that these 2 genes compensate each other functionally evolved from the biochemical characterization and expression kinetics of TP1 and TP2. However, individual TNP1-null mice cannot compensate completely for the TP1 deficiency with TP2 (Meistrich et al, 2003), which is probably due to differences in the expression levels of some other proteins. It is clear that TPs, especially TP1, play important roles in mouse spermiogenesis. However, the functions of TPs in human sperm formation are not fully understood. We need to study the roles of TPs in human sperm formation.

In this study, we found that the ORF of TNP2 included 5 different SNPs. TNP2 localizes to the protamine gene cluster, and the transcription of each of these genes is regulated by association with the nuclear matrix and attachment region on the genomic DNA (Martins et al, 2004). A previous study found 4 PRM1 SNPs, which did not cause any amino acid substitutions, in an ORF of almost the same nucleotide length. The frequency of SNPs in the protamine gene cluster is almost the same (average, 1 SNP/190 bp; PRM1, 1 SNP/106 bp; PRM2, 1 SNP/299 bp; TNP2, 1 SNP/219 bp). However, the amino acid sequence of PRM1 is more highly conserved than that of TP2. These results indicate that TP2 functions may not be stringent, in that a few amino acid changes are tolerated. Therefore, other proteins with similar characteristics might readily compensate for TP2.

SNPs in the region surrounding the TNP1 gene, including the promoter unit, were analyzed. We noted a deletion of the recognition site for the CRE transcription factor in the promoter region of 1 of the infertile patients. The recognition site for the CRE transcription factor plays an important role in the transcription of TNP1 (Kistler et al, 1994). The TNP1 promoter region is conserved and is considered to be important in mammals (Kistler et al, 1994). This mutation decreases the expression of TNP1 mRNA dramatically; otherwise, the mutational promoter that includes the recognition site for the Sp1 transcription factor might disturb the expression timing of TNP1 during spermiogenesis. Moreover, it is possible that this mutation influences spermiogenesis by modulating the expression of TNP1 in somatic cells, such as Sertoli cells. In humans, the amino acid sequences of TP1 are highly conserved. TP1 may be more important for spermiogenesis in humans than in mice. The mutation in the recognition site of the CRE transcription factor of the promoter region of TNP1 is thought to be a cause of male sterility. The prevalences of other SNPs in the region 5'-upstream of the transcription start site of TNP1 did not differ for the infertile and fertile control populations. The A x 5 or A x 6 stretch was identified in the region 5'-upstream of the transcription start site. This region may not have a specific function, and the easily deleted adenine stretches or DNA structures of A x 5 and A x 6 are very similar to each other. Some of the SNPs that appear in the NCBI dbSNPs were not found in this study. These unidentified SNPs may reflect the ethnicity of the loci registered in the NCBI dbSNPs.

Previously, Schlicker et al (1994) did not detect any mutation in PRM1, PRM2, or TNP1 in 36 infertile men whose spermatozoa showed the presence of histones in the nuclei. In our analysis of the TNP1 DNA sequences of a total of 552 men, we detected a deletion of the recognition site for the CRE transcription factor in the TNP1 promoter region in 282 idiopathic infertile men, but not in 270 fertile men. The human TNP1 gene is highly conserved, as are the protamine genes. By contrast, the TNP2 gene in the protamine gene cluster appears to have many more SNPs than the protamine genes. These results imply that TP1 has a more important role than TP2 in the formation of human sperm nuclei. Further investigations using a larger population of infertile cases and defined SNP pedigrees should confirm the causal link between TNP gene polymorphisms and male infertility.

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This Article Abstract Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Miyagawa, Y. Articles by Tanaka, H. Search for Related Content PubMed PubMed Citation Articles by Miyagawa, Y. Articles by Tanaka, H.