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A Common Protamine 1 Promoter Polymorphism (–190 CA) Correlates With Abnormal Sperm Morphology and Increased Protamine P1/P2 Ratio in Infertile Patients

JOSÉ LUIS BALLESCÀ

From the ^* Human Genetics Research Group, IDIBAPS, Faculty of Medicine, University of Barcelona and Genetics and Biochemistry Service, Hospital Clínic i Provincial, and the Institut Clínic de Ginecologia, Obstetricia i Neonatologia, Hospital Clínic i Provincial, Barcelona, Spain.

Correspondence to: Dr Rafael Oliva, Human Genetics Research Group, IDIBAPS, Faculty of Medicine, University of Barcelona, Casanova 143, 08036, Barcelona, Spain (e-mail: roliva{at}ub.edu).

Received for publication October 19, 2007; accepted for publication March 3, 2008.

	Abstract

Top Abstract References

 
It is known that targeting the protamine 1 gene in mice leads to infertility, abnormal chromatin packaging, and abnormal sperm morphology. Because many infertile patients also have an abnormal sperm morphology and chromatin packaging, the human protamine 1 gene (PRM1) is an important candidate to screen for potential mutations. In this work, we have screened the PRM1 gene in search of potential mutations and determined the sperm morphology and the ratio between protamine 1 and protamine 2 (P1/P2 ratio). Direct sequencing of the PRM1 promoter led to the identification of a common single-nucleotide polymorphism (SNP; –190 CA). The –190 AA genotype was detected at a higher frequency (13.8%) in patients with markedly altered sperm morphology (9% normal forms) compared with other patients (4.5%; P < .05) or compared with controls (2.97%; P < .005). The allelic frequency of the PRM1 –190 CA change was also consistently higher (.331) in infertile patients with a markedly altered morphology compared with population controls (.178; P < .01). Additionally, we have determined that the P1/P2 ratio is significantly increased in patients with the PRM1 –190 AA genotype compared with patients with the CA or CC genotypes (P = .006, Mann-Whitney). These findings indicate that the common PRM1 –190 CA polymorphism identified is associated with abnormal sperm head morphology and abnormal P1/P2 ratio in infertile patients.

     Key words: Haplotype block, infertility, sperm, reproductive genetics, risk factor

As soon as these differences in the protamine content were identified, it was postulated that potential mutations in the corresponding genes could be present (de Yebra et al, 1993). However initial mutational analysis of the protamine genes suggested that the presence of pathogenic mutations in the protamine genes was a rare cause of infertility (de Yebra et al, 1993; Queralt and Oliva, 1993; Schlicker et al, 1994; Schnulle et al, 1994; Queralt et al, 1995; Kramer et al, 1997; Tanaka et al, 2003; Aoki et al, 2006a). Subsequently it was shown in mice that protamine or transition protein haplo-insufficiencies resulted in infertility, abnormal chromatin packaging, DNA damage, and altered sperm morphology (Cho et al, 2001; Zhao et al, 2004; Suganuma et al, 2005). More recently, a single-nucleotide polymorphism (SNP; G197T) resulting in an arginine to serine change in the protamine 1 protein has been detected in 3 of 30 unrelated infertile patients on the basis of a spermatozoan phenotype similar to that present in protamine knockout mice (Iguchi et al, 2006).

We initiated this work with the goal to search for potential mutations in the coding region of the protamine 1 gene in patients with altered morphology of the spermatozoa and to determine whether any potential changes detected could correlate with the presence of an altered ratio between protamine 1 and protamine 2 (P1/P2 ratio). We also included in our analysis the promoter region up to –375 bp upstream from the translation start site of the human protamine 1 gene (PRM1), which so far had not been described as screened in previous studies.

Subjects, Materials, and Methods

     Study Design— An exploratory study was based on a cohort of infertile male patients that were analyzed for mutations in the PRM1 gene. Patients from the cohort were subgrouped by their PRM1 genotype, and the incidence of low abnormal sperm forms was analyzed. This cohort of patients and their subgroups were then used as the case group in a case population control study. The population control group comprised blood donors (men) from the general Spanish population.

     Subjects and Sample Collection— Sperm samples (220) corresponding to infertile patients undergoing intracytoplasmatic sperm injection or in vitro fertilization treatment at the Assisted Reproduction Unit of the Hospital Clinic of Barcelona were included in this study. Semen samples were collected in specific sterile containers after at least 3 days of sexual abstinence and were allowed to liquefy. After liquefaction of the semen, sperm parameters were evaluated according to published recommendations (World Health Organization, 1999). An aliquot of the sperm samples was removed for isolating DNA (see next paragraph), another aliquot was smeared onto a microscope slide to determine the sperm morphology, and the rest of the sample was centrifuged, washed twice with HamF10 to remove the seminal fluid, and finally frozen in HamF10 supplemented with 20% glycerol for later protamine analysis. Sperm morphology was evaluated by the Kruger strict criteria (Kruger et al, 1987), and at least 100 cells were examined per slide. Additionally, DNA samples from 208 blood donors (101 males and 107 females) were also isolated and included as controls to determine the population allelic frequency of the identified polymorphism. The average age of patients and controls was 43 and 27 years, respectively. Both the population controls and the patients were Caucasian and from the same geographical area (Catalonia, Spain). This project was approved by the bioethics committee of the hospital, and informed consent was obtained from the participants.

     Isolation of DNA— DNA from an aliquot of semen samples was extracted by a modified guanidinium thiocyanate method (Hossain et al, 1997). The spermatozoa were separated from seminal plasma with a 10-minute centrifugation (3000 x g) at 4°C and washed with 1x phosphate-buffered saline. The sperm pellet was resuspended (6 x 10⁶ spermatozoa/mL) in lysis buffer containing 6 M guanidinium, 30 mM sodium citrate (pH 7.0), 0.5% sarkosyl, 0.20 mg/mL proteinase K, and 0.3 M β-mercaptoethanol and incubated at 55°C for 3 hours. Isopropyl alcohol (half of the volume of lysate) was added directly to the lysate, and the tube containing the mix was inverted back and forth until the DNA fibers clumped together. After a 10-minute centrifugation (12 000 x g) at room temperature, the DNA pellet was washed in 1 mL of ethanol 70% followed by a second 10-minute centrifugation (12 000 x g) at room temperature. The recovered DNA was finally dissolved in 50 mM Tris-EDTA buffer.

     Sequencing of the PRM1 Gene— The promoter and coding regions of the protamine 1 gene were amplified by polymerase chain reaction (PCR). Two oligonucleotides have been used to amplify these 2 regions. The sequence of the primers were 5'-TCACTATATACCAGGGCCTAGGCC-3' (forward; designed specifically for the present work) and 5'-TCAAGAACAAGGAGAGAAGAGTGG-3' (reverse; Tanaka et al, 2003). The PCR conditions were: 1.25 µL of each primer (10 pmol/µL), 2.5 µL of 1.25 mM deoxynucleotide triphosphates (dNTPs), 0.5 units of Taq polymerase (Invitrogen, Carlsbad, California), 2.5 µL of 10x Taq buffer, and 1 µL of DNA in a final volume of 25 µL. The cycling conditions were: DNA denaturation at 94°C for 5 minutes, 35 cycles of 94°C for 30 seconds, 60°C for 30 seconds, 72°C for 30 seconds, and a final extension of 10 minutes at 72°C. Residual dNTPs and primers were removed from the PCR products with ExoSAP-IT (USB, Cleveland, Ohio). In brief, 2 µL of ExoSAP-IT was added to 5 µL of PCR product, and the mixture was incubated at 37°C for 20 minutes and at 80°C for 10 minutes. After that, we added 0.5 µL (20 pmol) of the specific primer and 2 µL of the sequencing Mix (BigDye Terminator v3.1 cycle sequencing kit, Applied Biosystems, Foster City, California). The reactions were run in a 3100 automated DNA sequencer (Applied Biosystems).

     Restriction Fragment Length Polymorphism Detection of the –190 CA— To facilitate fast screening of additional patients and controls, we designed a strategy to PCR amplify and detect the polymorphism with a restriction endonuclease (PCR-RFLP; Figure 1). We realized that the –190 CA polymorphism would destroy a restriction enzyme site (HaeIII) present at position –190. However, other HaeIII sites were present at positions –372, –345, –233, and –183. A primer adjacent to the –190 polymorphic site was designed (5'-GACCT CACAA ACCAT AGCCA GGTG-3') with a mismatch at position –181 so that after PCR it would suppress the internal HaeIII site normally present at the –183 position. After this procedure, the resulting PCR product would be cleaved by HaeIII into several fragments, including the 42-bp fragment specific for the –190 C allele and the 67-bp fragment specific for the –190 A allele (Figure 1).

View larger version (29K): [in this window] [in a new window]   Figure 1. Detection of the –190 CA protamine 1 (PRM1) gene promoter polymorphism. (A) Electropherograms corresponding to a homozygous –190 CC individual, to a heterozygous –190 AC individual, and to a homozygous –190 AA individual. (B) Diagram of the promoter and transcribed region of the PRM1 gene indicating the position of the –190 CA polymorphism and the potential regulatory elements present at this site. (C) Design of a polymerase chain reaction restriction fragment length polymorphism (PCR-RFLP) strategy to genotype the PRM1 –190 CA polymorphism. The results of the electrophoretic separation after HaeIII digestion of the PCR products corresponding to the 3 possible genotypes of the PRM1 –190 CA polymorphism are shown. The different genotypes and the relevant bands (67 and 42) are indicated. At the right side of this figure a schematic representation of the PCR amplification product and the HaeIII sites present in the C allele and in the A allele is shown. Abbreviations: SRE, serum response element; SEP, SRE extended protection.

View larger version (20K): [in this window] [in a new window]   Figure 2. Box plot of the distribution of the sperm morphology in each of the patient groups according to the protamine 1 (PRM1) –190 CA promoter polymorphism. The proportion of spermatozoa with normal morphology in the AA genotype group was significantly lower compared with the AC and CC groups (P = .012, Mann-Whitney).

View larger version (17K): [in this window] [in a new window]   Figure 3. Box plot of the distribution of the ratio between protamine 1 and protamine 2 (P1/P2 ratio) in each of the patient groups according to the protamine 1 (PRM1) –190 CA promoter polymorphism. The P1/P2 ratio was significantly higher in the AA genotype group compared with the AC and CC groups (P = .006, Mann-Whitney).

     SNP Analysis of the Candidate Region— The HapMap (release 21; The International HapMap Consortium, 2003) and the SNP Browser (Applied Biosystems LD Map, database version 3.1.29) were used to determine whether any other polymorphisms located in nearby genes were in linkage disequilibrium with the –190 CA polymorphism (rs2301365). By this procedure, the haplotype block map containing the genes and their SNPs in the region were obtained (Figure 4). The SNPs detected in the coding region of each gene included in the haplotype block were analyzed for amino acid changes. Also, the SNPs located in the promoter regions of each gene included in the haplotype block were analyzed by SNPInspector (Cartharius et al, 2005) to detect the creation or deletion of potential transcription factor binding sites.

View larger version (32K): [in this window] [in a new window]   Figure 4. Chromosome 16 (Ch 16) haplotype blocks containing the protamine 1(PRM1) –190 CA polymorphism. (A) Genomic region of the PRM1 gene showing the haplotype block details. (B) Gene structure, single nucleotide polymorphism (SNP) location, and possible functional changes of the gene. Black boxes indicate the coding regions, and the grey boxes indicate the untranslated region. The asterisks (*) indicate the potential promoter elements detected by SNPInspector.

     RFLP Detection of the –190 CA Polymorphism in Patient and Control Groups— To allow rapid screening of additional patients and controls, we designed a PCR-RFLP method to genotype the –190 CA change (Figure 1C). The results of the genotyping of 220 patients and 208 controls are reported in Figure 2 and in the Table. When the patient sperm morphology was plotted according to the –190 promoter polymorphism genotype, a significantly lower number of morphologically normal spermatozoa were detected in the patient AA genotype group compared with the AC or the CC groups (Figure 2; Mann-Whitney; P = .012). The patients were divided into 2 groups according to their sperm morphology determined by Kruger strict criteria according to the median value (9%) present in the group of homozygous –190 AA patients. With the use of this criterion, 65 patients had 9% of spermatozoa with normal forms (NF) and 155 patients had >9% normal forms in their sperm (Table). The morphological abnormalities consisted mostly in sperm head defects. The –190 A allelic frequency in the patient group with 9% NF was markedly higher (.331) compared with infertile patients with >9% NF (.255; P < .001). Also, the –190 A allelic frequency in the overall patient group was significantly higher (.277) compared with the male population control group (.178; P < .01) or the male plus female population control group (.183; P < .001). No significant differences were detected in the genotype or allelic frequencies in the control group when comparing males with females (not shown). The AC and AA genotypes also consistently increased in the patient group with 9% NF compared with controls (Table).

View this table: [in this window] [in a new window]   Table. Genotype and allelic frequencies of the protamine 1 promoter – 190 CA polymorphism in infertile patients and in controls

     Protamine P1/P2 Ratio— A box plot of the P1/P2 ratio in each of the patient groups that stratified according to the PRM1 –190 CA promoter polymorphism indicated the presence of an increased value in the AA patient group compared with the CA or CC groups (Figure 3). Statistical analysis indicated that the P1/P2 ratio was significantly higher in the AA genotype group compared with the AC and CC groups (P = .006, Mann-Whitney).

     SNP Analyses of the Candidate Region— The PRM1 –190 CA polymorphism (rs2301365) is located in a haplotype block present in the Caucasian population in chromosome 16, approximately between positions 11 277 000 and 11 372 000 (Human Genome build NCBI-35), according to the results of the HapMap and SNP Browser. This haplotype block contains completely the PRM1 gene, the Human protamine 2 gene (PRM2), and the C16orf75 gene and contains partially the LOC388210 gene (Figure 4A). The PRM1 gene contains 3 SNPs in the promoter region (rs7206749, rs2550479, and rs2301365). All of these SNPs destroy potential binding sites for their transcription factors (neuron-restrictive silencer element, transforming growth factor beta–inducible early gene, basonuclin [BNC], and survival interacting protein 1 [SIP1]; Figure 4B). Exon 2 of the PRM1 gene also contains a SNP (rs737008), which causes a synonymous change in the amino acid sequence (Figure 4B).

Discussion

In this work, we have identified a common polymorphism in the promoter region of the PRM1 gene (–190 CA) that is present at a significantly higher frequency in infertile patients with altered morphology of the spermatozoa (9% normal forms) compared with other patients or compared with a population control group (Figures 1 and 2; Table). Additionally, we have determined that a significantly higher P1/P2 ratio is also present in the PRM1 –190 AA homozygous patients.

The proposal that protamine gene mutations could be involved in infertility had its origins in the detection of differences in the protamine content in the sperm cells of some infertile patients (de Yebra et al, 1993). This idea was supported additionally because the lack of P2 in the sperm nucleus of some mammals, such as the pig or the bull, was due to the presence of mutations in the corresponding genes (Maier et al, 1990). However initial mutational analysis in the genes encoding the protamines P1 and P2 or transition protein 1 did not reveal any pathogenic mutations (de Yebra et al, 1993; Queralt et al, 1993; Schlicker et al, 1994; Schnulle et al, 1994). In a subsequent study, a postulated role for a candidate mutation in a region of contact in the nuclear matrix close to the protamine genes was presented in 2 of 5 individuals (Kramer et al, 1997). More recently, mutations in the P1 (PRM1) and P2 (PRM2) genes have been characterized in 226 Japanese sterile patients and in 270 controls (Tanaka et al, 2003). In this case, 4 synonymous polymorphisms (SNPs) were found in the coding region of the PRM1 gene and 1 SNP (C248T) in the PRM2 gene, creating a premature stop codon. Also in this work, 1 SNP in the 3' region of the PRM1 gene and 2 SNPs in the intron of the PRM2 gene were identified. Several nonpathogenic coding region polymorphisms have been reported in infertile patients (Aoki et al, 2006a). Recently, 1 SNP (G197T) resulting in an arginine to serine change in the protamine 1 gene has been detected in 3 of 30 unrelated infertile patients (Iguchi et al, 2006). It is interesting to note that these patients were selected in the base of a spermatozoon phenotype similar to that present in P1 or P2 knockout mice (Cho et al, 2001; Iguchi et al, 2006). However, we have not detected this mutation (G197T) in any of our 32 infertile patients with <9% normal forms in their sperm initially screened by direct sequencing. Therefore, on the basis of the previous, as well as on the present, mutational study, it can be concluded that PRM1 gene mutations are a rare cause of infertility.

Unexpectedly however, in this study, we have identified a common promoter polymorphism in the protamine 1 gene (PRM1 –190 AC; Figure 1). This part of the promoter region had not been reported as screened in previous mutational studies (Tanaka et al, 2003; Aoki et al, 2006a; Iguchi et al, 2006). We have determined that this common polymorphism is present at a frequency significantly higher in the patient group compared with the population frequency (Table). The frequency detected is consistent with that described in a recent independent study in which the sperm morphology was not measured (Ravel et al, 2007). Of relevance, we have detected that the allelic frequency of the PRM1 –190 CA polymorphism is higher in the group of infertile patients with abnormal morphology (9% normal forms) compared with the patient group with >9% normal morphology (Table). The homozygous AA genotype also has been detected consistently at a higher frequency in the patient group with 9% normal forms compared with the patient group with >9% normal forms (Table). Therefore, we conclude that we have identified a common SNP associated with abnormal sperm morphology and male infertility. A potential explanation for the detected association between the PRM1 –190 CA polymorphism, abnormal sperm morphology, and infertility could be found, in that the region containing the presently described polymorphism resulted in a clear DNAse I footprint protection in vitro, which spanned an adjacent serum response element (SRE) element and a region called SEP (SRE extended protection; Queralt and Oliva, 1995; Figure 1). Also, this polymorphism site is located within a potential E-alpha regulatory element and binding sites for BNC and SIP1 present in the promoter region in the PRM1 gene (Figures 1 and 4; Queralt and Oliva, 1995). Therefore, a reasonable hypothesis is that this –190 CA polymorphism could result in changes in the expression of the PRM1 gene, resulting in an abnormal sperm morphology and infertility. This hypothesis is consistent with the results of the protamine determination in the different patient groups stratified by their PRM1 –190 genotype, indicating the presence of an increased P1/P2 ratio in the homozygous AA group (Figure 3). Our overall interpretation of these results is that we have identified a common P1 promoter polymorphism that is associated with an altered expression of the protamine resulting in abnormal P1/P2 content, abnormal sperm morphology, and infertility. The increase in the P1/P2 ratio could be due to an increase in the expression of the protamine 1 gene or to an overall altered expression of the protamine 1 and 2 genes. Of potential relevance, both genes are located in the same chromatin loop and therefore could be subjected to a coordinate regulation (Kramer et al, 1997; Aoki et al, 2006c; Martins and Krawetz, 2007).

Additionally, the detected –190 CA polymorphism could be in linkage disequilibrium with other polymorphisms located in nearby genes that could also further contribute to alter the P1/P2 ratio and the sperm head morphology. We have determined that the haplotype block that contains the detected PRM1 –190 CA polymorphism also contains the PRM2 gene, the C16orf75 gene, and, partially, the LOC388210 gene (Figure 4A). In fact, different SNPs are indeed present in these 4 genes, which results in potential amino-acid changes in the protein or changes in the promoter sequence elements (Figure 4B). Even though the function of the C16orf75 gene is unknown at present, it is interesting to point out that its promoter region contains consensus elements for different transcription factors expressed during spermatogenesis. It is also interesting that the LOC388210 gene contains a polymorphism in a potential regulatory element site and a missense mutation in exon 2, both present in the same haplotype block as the PRM1 –190 CA polymorphism (Figure 4B). Thus, the opportunity is now open for subsequent genome-based association studies with these additional genes present in the haplotype block in which the PRM1 –190 CA polymorphism is included. Finally, it will also be interesting to determine how genetic variation in other gene loci or environmental factors interact with the presence of the polymorphisms described in this work.

In this work, we identified a common PRM1 promoter polymorphism (–190 CA) that is associated with altered sperm morphology, altered P1/P2 ratio, and infertility. The opportunity is now open to look further into the mechanisms of these associations and to determine whether this common PRM1 polymorphism might also correlate with the results of assisted reproduction.

	Acknowledgments

 
The authors thank Professor Dr Cristóbal Mezquita for critical review.

	Footnotes

 
This study was supported by grants from Ministerio de Ciencia y Tecnología BMC2003-03937, fondos FEDER, to R.O.

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