Sperm nuclear proteins, the protamines and transitional proteins play a crucial role in sperm nuclear condensation. The compact packaging of sperm DNA by protamines maintains sperm genome integrity, which is prerequisite for normal sperm function. However the effect of nucleotide variations in P and TP genes on sperm DNA integrity and male fertility is not clear. This case-control study was planned to analyse P and TP gene nucleotide variations and sperm DNA integrity in 100 oligozoospermic infertile men and 100 fertile controls. P and TP genes were amplified by polymerase chain reaction and sequenced. Flow cytometry- sperm chromatin structure assay (FC-SCSA) was applied to measure DNA fragmentation index (DFI) in sperm. Semen analysis was performed as per WHO, 1999 guidelines with slight modification. In total, 7 nucleotide variations including two novel changes, a non-synonymous mutation in the exon-2 of P2 gene (c.443C>A) and a novel insertion of T (c.396_397InsT) at the 3' UTR region of T1 was detected. None of the SNPs were observed at risk frequency in the oligozoospermic infertile men compared to the controls. Though overall DFI was significantly (p<0.0001) higher in infertile men compared to controls (36.31 Â± 7.25 Vs 26.49 Â± 2.78) irrespective of nucleotide changes, no such difference was observed between infertile men (100) or pooled population (200) with and without mutations. However it was observed that that two cases with novel nucleotide changes (P2 c.443C>A and TP1 c.396_397InsT) had higher DFI value. In conclusion, our study for the first time in Indian population revealed two rare novel mutations in sperm nuclear protein genes that are associated with higher sperm DNA fragmentation. Further large number of samples and functional studies are required to find out the effect of these novel variations on male fertility.
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Couples with higher risk for infertility are increasing in number, where idiopathic cases are observed more frequently than other causes. Approximately half of these cases are due to male factor [Poongothai et al. 2009]. Men with idiopathic infertility often present with compromised semen quality. Though several genetic factors are involved in the regulation of spermatogenesis, their role in impaired semen quality is still not clearly understood. Spermiogenesis is a crucial event in the maturation of sperm where sperm nuclear proteins tend to play an important role in chromatin condensation. Moreover, compact packaging of sperm chromatin is essential for maintaining the sperm DNA integrity, which is associated with many physiological events like sperm motility, capacitation, acrosome reaction, normal embryogenesis and birth of healthy offspring [Giwercman et al. 2003; Moskovtsev et al. 2005]. Therefore, the role of sperm nuclear proteins, the protamines (P) and transitional proteins (TP) in sperm function has been the focus of many recent studies [Hammoud et al. 2009; Haraguchi et al. 2009; Tavalaee et al. 2009; Tseden et al. 2007; Zhao et al. 2001]. P1 and P2 are present in equal proportions but unlike P1, P2 gene is translationally regulated in a species specific manner [Bianchi et al. 1992] and unlike TP1, TP2 amino acids are highly conserved among various mammals [Kremling et al. 1989]. Altered P1/P2 ratios in sperm have also been reported as one of the important cause of male infertility [Mengual et al. 2003; Zhang et al. 2006]. The reason for this altered ratio may be an interrupted post translation modification or mutation in the P/TP genes. Though various population studies initially showed mutations in these genes was a rare cause of infertility, studies also have reported novel mutations and high frequency of single nucleotide polymorphisms (SNPs) in infertile men [Adham et al. 2001; Carrell and Liu 2001; Cho et al. 2003; Cho et al. 2001; Mengual, et al. 2003]. Our study was the first to report the effect of both P and TP DNA sequence variations on sperm DNA integrity in Indian population. Since there are no studies available on these genes in Indian population, this would help to explore the role of P and TP gene variants on male infertility in Indian population. Therefore the study was planned to address the controversial results of sperm nuclear protein gene mutations on male infertility and also to study the effect of these mutations on sperm DNA integrity for first time in Indian men with idiopathic infertility.
The cDNA nucleotide positions were numbered according to their position in the genomic sequence from the starting codon (ATG), where A is considered as +1. In protamine (P1&2) genes, a total of four nucleotide changes were identified (Figure 1). It includes one synonymous variant c.230 C>A in exon 2 of P1 and two variants (c.298 G>C, c.373 C>A) in intron 1 of P2 and one novel non-synonymus change (c. 443C>A - Gene bank ID- GU190363) (Figure 2) in exon 2 of P2 (Table 1). By polyphen prediction analysis c. 443C>A novel mutation was found to be benign and it resulted in amino acid change from polar threonine to polar asparagine (p.T94N). This patient was an oligo-asthenozoospermic male with sperm count of 17 million/ml and contained only 10% motile sperms. No difference in the frequency of P nucleotide changes between infertile men and controls was observed (Table 1).
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In TP gene, totally 3 variants and one novel insertion were identified (Figure 3). A variant c.214T>C in the intron 1 of TP1 gene was observed at no risk frequency in the infertile men compared to controls (Table 1). A novel insertion of nucleotide T between 396 and 397 in 3'UTR [T (c.396_397InsT) - Gene bank ID- GU190364] of TP1 exon 1 (Figure 4) was identified in one patient with severe oligozoospermia (sperm count of 2.3 million/ml). Another variant c.391C>T changing amino acid polar arginine (R) to non-polar tryptophan (W) (p.R131W) in TP2 was identified in both controls and infertile men. By Polyphen prediction analysis this change was found to be benign. All the nucleotide changes in TP gene were found at no risk frequency in infertile men compared to controls.
Sperm chromatin structure assay revealed that the overall DFI in infertile men was found to be significantly (P<0.0001) higher as compared to controls (36.31 Â± 7.25 Vs 26.49 Â± 2.78). The lowest DFI in the study population was found in control (19.82 %) and the highest DFI was found in an infertile patient (43.85%). When the subjects were pooled (200) into cases and controls with and without variations (31.27 Â± 7.32 Vs 31.99 Â± 5.62) no significant (p=0.592) difference in the DFI was observed. Similarly non significance difference (p=0.751) in the DFI was observed between infertile men with and without nucleotide variations (36.19 Â± 7.22 Vs 36.83 Â± 4.57). However, a case with novel C443A mutation in P2 gene showed DFI - 34.82 % (Figure 5), whereas the second case with novel insertion in the non-coding 3'UTR of (c.396_397InsT) TP1 gene showed highest DFI (43.85%) in the study (Figure 6).
Sperm has unique function of transmitting the paternal genome to the egg and thus integrity of sperm genomic DNA is a prerequisite for normal embryogenesis and fetal well being. Gradual replacement of histones by transitional proteins followed by protamines play a crucial role in sperm nuclear condensation. However, studies have shown altered P1/P2 levels in infertile men against equal proportion in spermatozoa of normal men [Balhorn et al. 1988; de Yebra et al. 1993]. These altered levels could be either due to pathogenic mutations in P/TP genes or aberrant post translation modification of protamines/transitional protein mRNAs. Mutations in P/TP genes and its effect on male infertility remain controversial. However, studies are lacking in showing the effect of such mutations on sperm DNA integrity.
This study, which was first in Indian population and has analysed P/TP nucleotide changes and DFI. In our study, previously reported synonymous SNP c.230C>A (rs737008) in the exon 2 of P1 gene was identified in both infertile men and controls with no significant difference in their frequency. Other SNPs reported by Ravel et al., 2007 such as c.102G>T and c.119G>A, Aoki et al., 2006 (c. C230A), Gázquez et al., 2008 (c.-190 C>A), Imken et al., 2009 (c.65G>A), Tanaka et al., 2003 (A133G, C160A, G320A, C321A, A431G) in the P1 gene were not identified in this study population which may be due to different geographical origin and ethnicity [Aoki et al. 2006; Gazquez et al. 2008; Imken et al. 2009; Ravel et al. 2007; Tanaka et al. 2003]. However, three nucleotide changes including one novel mutation were observed in P2 gene. A heterozygous c. 373C>A and the homozygous c.298G>C in P2 gene which has been previously reported were observed at no significant differences in the frequency between infertile men and controls. Moreover none of the previously reported P2 SNPs (-C67T, G398A) and translation termination induced C248T were identified in this study [Imken, et al. 2009; Tanaka, et al. 2003]. But, a novel non-synonymous mutation (c.443C>A) in the exon 2 of P2 was observed in an oligo-asthenozoospermic man with high DFI (34%), which may be due to the amino acid change that altered P2 protein conformation leading to deficient protamine packaging. This may require screening of large number of cases with functional studies to explore its pathogenicity, though computational polyphen analysis predicted it's a non-pathogenic change with no alteration in the polarity of the amino acid. Therefore SNPs in P gene which consists of highly conserved amino acids may not contribute to male infertility in this study population.
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Since TPs are the initiators of sperm chromatin condensation, disruption in TP expression and binding may also impair sperm DNA integrity. Most of the studies have screened only P genes for SNPs [Iguchi et al. 2006; Kichine et al. 2008; Ravel, et al. 2007] and very few have screened TP genes [Adham, et al. 2001; Miyagawa et al. 2005]. Since TPs are equally important in sperm DNA packaging, we have screened both P and TP genes in this study. Studies in mice have shown lack of TP or null mutant for TP genes lead to infertility [Yu et al. 2000; Zhao et al. 2004; Zhao, et al. 2001]. In our study, a previously reported heterozygous SNP c.214T>C (rs62180545) in intron 1 of TP1 gene was observed at no risk frequency between infertile and control population. Moreover the identified novel insertion of T between 396 and 397 in 3'UTR region of the TP1 gene in a severe oligozoospermic man (sperm count-2.3 million/ml) showed higher DFI (44%). The change in the non-coding region (5' and 3' UTR) may have strong effect on protein expression due to disruption of their regulatory mechanism [Hammoud et al. 2007]. This mutation in 3'UTR may alter mRNA stability, localization, and translational efficiency during expression. A previously reported homozygous c.391C>T change in exon 1 of TP1 gene was observed at equal frequency in both infertile men and controls which is in accordance to Aoki et al., 2006. Therefore similar to P genes, TP gene nucleotide variants also have no role in disrupting spermatogenesis in this study population. Though the effect of these SNPs on gene expression and translation is not well known, but the coordinate control of P1, P2 and TP2 genes in the protamine cluster are essential during spermatogenesis [Wykes et al. 1995]. Similar to most of the previous reports, no risk frequency of sperm nuclear protein gene nucleotide changes is associated with male infertility in our population too [Aoki, et al. 2006]. However, the detected two novel mutations may require further screening in large number cases and different populations.
Infertile men with protamine deficiency may have poor sperm DNA packaging that makes the sperm genome highly susceptible to toxic environmental stimulus such as mutagenic agents, chemical, mechanical stimuli and is more susceptible to free radicals induced damage and higher DNA fragmentation [Nili et al. 2009; Venkatesh et al. 2009]. When the pooled (200) population were grouped into men with and without nucleotide changes, our results failed to support the effect of the observed mutations on sperm DNA integrity. Similar results were achieved when infertile men were grouped into men with and without mutations. However it is important to notice that the observed two cases with novel mutations c.443C>A and 3'UTR c.396_397InsT had higher degree of sperm DNA fragmentation of DFI 34.82% and 43.85% respectively. The DFI of the oligoasthenozoospermia case with c.443C>A is higher than the average controls (26.49%) and the effect of amino acid change on the protein structure cannot be overruled. This case also harboured the P1 c.230 C>A silent mutation. In the second case with novel 3'UTR c.396_397InsT in the TP1 gene, the sperm cells showed highest DFI (43.85%) in this study. The case also harboured previously reported P2 intronic c.373 C>A variant. As TP2 proteins are necessary to initiate the protamine binding to the sperm DNA, mutation in the 3'UTR may alter the mRNA stability and subsequently results in aberrant translation. Both these two novel variants were not observed in the control population and would be interesting to screen these novel variants in large number of samples. It is well known that compact packaging of sperm chromatin is essential for maintaining the sperm DNA integrity, which is essential for normal sperm motility, capacitation, acrosome reaction, successful fertilization, placentation and embryogenesis as sperms are not mere vectors of paternal DNA but are important determinants of development competence of embryo [Wykes, et al. 1995]. Moreover, in our study the variants observed in the intronic regions such as c.298G>C, c.373C>A of P2 and c.391C>T, c.396_397InsT of TP1 gene are in the highly conserved region of eutherian mammals. Though earlier studies have not explained about these SNPs in the conserved region, altered nucleotide changes in conserved regions may have significant role in impaired gene expression and translation. Though protamine deficiency may lead to aberrant DNA packaging in the sperm, polymorphisms in the sperm nuclear protein gene mutation may not have significant effect on sperm DNA integrity. Altered epigenetic mechanism and impaired post translation modification of P and TP genes may need more attention. However 92% of infertile men in the current study had high DFI (>30%) and it is necessary to elucidate other important factors involved in the impairment of sperm DNA integrity.
In conclusion, our study for the first time in Indian population revealed two rare novel mutations in sperm nuclear protein genes that are associated with higher sperm DNA fragmentation.
Materials and methods
After approval from institute ethical committee (IEC), AIIMS, informed consent was obtained from the subjects enrolled in the study. The study included 100 oligozoospermic men with average duration of infertility, 5.60 Â± 3.77 years and 100 controls (fathered a child in last one year) were included in the study. After thorough examination and questionnaire evaluation, oligozoospermic men (<20 million sperm/ml) with normal 46, XY chromosomal complement, absence of Yq microdeletion, no varicocele and no hydrocele, were only included in the study. After 4 days of sexual abstinence, semen samples were collected in a wide mouth sterile plastic container and delivered to the laboratory immediately. Semen was allowed to liquefy at room temperature and the semen parameters were analyzed as per WHO, 1999 guidelines [WHO 1999]. For morphological evaluation, 10Âµl of semen smear was made in a clean microscope slide and fixed with 90% ethanol and stained with Giemsa. Minimum of 100 sperms per sample were evaluated for the morphological defects.
Separation of spermatozoa and sperm DNA isolation
After semen analysis, the sample was layered in Isolate sperm separation medium, (Irvine Scientific Co., Santa Ana, CA) and centrifuged at 300 rpm for10 minutes to separate sperm cells from other cells like leukocytes, epithelial cells and debris. After confirming the absence of other cells by examining the smear under microscope, sperm cells were washed with sperm washing media (Irvine Scientific Co., Santa Ana, CA). The washed cells were subjected to DNA isolation by the following method. The sperm pellet was incubated at 55Â°C overnight with the sperm lysis medium (60mM DTT, 4% SDS and 350Âµg/ml protienase K made in lysis buffer). After complete digestion, sperm DNA was precipitated by adding equal amount of chilled isopropanol. The DNA pellet was then washed with 70% ethanol, dried at 37Â°C and dissolved in Tris-EDTA (TE) buffer.
Identification of SNPs in the P and TP genes
Four primer pairs were used and PCR of 25Âµl reaction mixture was set up as described by Aoki et al., 2006 [Aoki, et al. 2006] with slight modification: 94Â°C for 4 minutes followed by 35 cycles of 94Â°C for 30s; annealing temperature for 30s [P1-62.5Â°C, P2-88.5Â°C,TP1-55.5Â°C,TP2-59Â°C], extension of 72Â°C [P1-60s,P2-80s,TP1-50s,TP2-70s] and a final hold for 5 minutes at 72Â°C. The amplified PCR products were confirmed by running 2Âµl of the products in 2% agarose gel. The amplified products were then purified by guanidium-Hcl method. The purified products were directly sequenced after the samples were dissolved in 10 Âµl of 50% Hi-Di formamide and analyzed in automated DNA analyzer (Applied Biosystems). The obtained sequences were analyzed using chromas software and compared with the reference sequence to find out the nucleotide changes using Genebee multiple alignment (http://www.genebee.msu.su).
Sperm chromatin structure (SCSA) assay
Preparation of samples
The SCSA was performed according to the procedure described elsewhere [Evenson et al. 2002] with slight modification. The aliquot from each ejaculate was thawed in a water bath at 37Â°C for 30s and diluted to a concentration of 2 Ã- 106 sperm/ml in TNE buffer to a total of 200 Î¼l in a Falcon tube. Immediately, 0.4 ml of acid detergent solution (0.08 M HCl, 0.15 M NaCl, 0.1% v/v Triton X-100, pH 1.2) was added to the Falcon tube. After exactly 30s, 1.2 ml of AO-staining solution (6 Î¼g AO (chromatographically purified) (Polysciences, Inc. - USA) per ml citrate buffer (0.037M citric acid, 0.126M Na2HPO4, 1.1mM EDTA disodium, 0.15M NaCl, pH 6.0) was added. For every six test samples, one reference sample was analysed to ensure instrument stability.
Flow cytometric measurements
The samples were analysed using a FAC Scan flow cytometer (BD Biosciences), with an air-cooled argon laser operated at 488 nm and a power of 15 mW. The green fluorescence (FL1) was collected through a 515-545 nm bandpass filter, and the red fluorescence (FL3) was collected through a 650 nm longpass filter. The sheath/sample was set on 'low', adjusted to a flow rate of 200 events/s when analysing a sample containing 2Ã-106 sperm/ml. immediately after the addition of the AO staining solution, the sample was placed in the flow cytometer and run through the flow system. After complete analysis of sample, the X-mean (red fluorescence) and Y-mean (green fluorescence) values were recorded manually after selecting gate for sperm cells using Cyflogic software version 1.2.1 (CyFlo Ltd, Finland).
The sperm cells are gated after excluding debris and high DNA stainability (HDS) cells. The DNA fragmentation index was calculated by the formula, DFI = mean red fluorescence/ mean red fluorescence+ mean green fluorescence. The study population including both infertile men and controls were categorized into group with one or more mutation and without mutation. DFI was compared between these two groups to find the effect of these mutations on sperm DNA integrity.
Computational assessment of missense mutations
The homology-based program PolyPhen (polymorphism phenotyping; Division of Genetics, Department of Medicine, Brigham and Women's Hospital/Harvard Medical School, Boston, MA) was used in this study to predict the functional impact of missense mutation. PolyPhen scores of >2.0 indicate the polymorphism is probably damaging protein function. Scores of 1.5-2.0 are possibly damaging, and scores of <1.5 are likely benign.
DFI between infertile men and controls were compared by Mann-Whitney test. Genonotypic and allelic frequency between infertile men and controls were analyzed by Fischer's exact test. Statistical analyses were performed using MedCalc trial version for Windows, (MedCalc Software, Mariakerke, Belgium).
Table 1. Sperm P and TP genotypic and allelic frequency in oligozoospermic infertile and control men
Figure 1. Schematic representation of P1 and P2 gene nucleotides showing SNPs. Blue-coloured regions are introns. Nucleotide changes are green coloured and the variant is shown just above in brown colour with their respective position. The corresponding amino acid change is shown below the wild type in red colour.
Figure 2. Sequence electrophoreogram showing novel nucleotide variant SNP c443 Câ†’A in P2 gene: A. control sequence showing homozygous wild type (C/C), B. Sequence showing homozygous mutant (A/A)
Figure 3. Schematic representations of TP1 and TP2 genes showing SNPs. Blue-coloured regions are introns. Nucleotide changes are green coloured and the variant is shown just above in brown colour with their respective position. The corresponding amino acid change is shown below the wild type in red colour. Non coding exon region is shown underlined
Figure 4. Sequence electrophoreogram showing insertion c. Ins T 396-397 3'UTR TP1 gene: A. control sequence showing homozygous wild-type (GGTG), B. Sequence showing insertion (GGTTG)
Figure 5. SCSA pseudocolour dot plot of semen sample of case with c443 Câ†’A mutation. FL 3-H -axis represents fragmented DNA and FL 1-H- axis represents native DNA. HDS- High DNA stainability cells in the semen.
Figure 6. SCSA pseudocolour dot plot of semen sample of case with c.396_397InsT mutation. FL 3-H -axis represents fragmented DNA and FL 1-H- axis represents native DNA. HDS- High DNA stainability cells in the semen.