Mol. Hum. Reprod. Advance Access originally published online on January 10, 2006
Molecular Human Reproduction 2006 12(1):41-50; doi:10.1093/molehr/gah258
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A novel mechanism of protamine expression deregulation highlighted by abnormal protamine transcript retention in infertile human males with sperm protamine deficiency
1Andrology and IVF Laboratories, 2Department of Surgery, 3Department of Physiology and 4Department of Obstetrics and Gynecology, University of Utah School of Medicine, Salt Lake City, UT, USA
5 To whom correspondence should be addressed at: Andrology and IVF Laboratories, University of Utah School of Medicine, 675 Arapeen Dr Ste 205, Salt Lake City, UT 84117, USA. E-mail: douglas.carrell{at}hsc.utah.edu
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Abstract |
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Sperm protamine deficiency has been associated with human male infertility. However, the aetiology of deregulated protamine expression remains elusive. The objective of this study was to evaluate the underlying aetiology of protamine deficiency in male infertility patients with deregulated protamine expression. Protamine-1 (P1) and protamine-2 (P2) protein concentrations were compared against P1 and P2 mRNA levels in the sperm of 166 male infertility patients and 27 men of known fertility. Protamine protein concentrations were quantified by nuclear protein extraction, gel electrophoresis and densitometry analysis. Semi-quantitative real-time RT–PCR was used to quantify P1 and P2 mRNA levels. P1 mRNA concentrations were significantly increased in patients underexpressing P1 protein versus those with normal and increased P1 levels. In patients with an abnormally low ratio of P1 to P2 (P1/P2 <0.8), there was a significant increase in P1 mRNA retention. Patients underexpressing P2 also had significantly increased mean P2 mRNA levels, although the majority of these P2-deficient patients showed an increased frequency of significantly reduced P2 mRNA levels. This is the first study to concomitantly evaluate P1 and P2 protein and mRNA levels in mature human sperm. Abnormally elevated protamine mRNA retention appears to be associated with aberrant protamine expression in infertile human males. These data suggest that defects in protamine translation regulation may contribute to protamine deficiency in infertile males.
Key words: expression/human/protamines/RT–PCR/sperm
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Introduction |
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During spermiogenesis, the protamine proteins replace the somatic cell histones, a process that results in a highly condensed transcriptionally silent chromatin (Oliva and Dixon, 1991
; Aoki and Carrell, 2003). In humans, there are two protamine proteins, protamine-1 (P1) and protamine-2 (P2), which occur in a strictly regulated one-to-one ratio (Corzett et al., 2002).
The protamines are expressed in the post-meiotic haploid spermatid (Steger et al., 1998, 1999, 2000, 2002; Aoki et al., 2005). An interesting aspect of this expression is that protamine transcription and translation are temporally uncoupled in the developing spermatid (Steger, 1999). Because protamine–DNA binding results in chromatin condensation, and consequently transcription cessation, the protein cannot be transcribed concomitantly at the time it is needed during spermiogenesis (Lee et al., 1995).
P1 and P2 mRNA have been identified in round spermatids, while the proteins remain conspicuously absent at this stage (Steger et al., 1998, 1999, 2002; Steger, 2001). Translation of protamine mRNA is repressed until the elongating stage of spermatid differentiation (Kleene, 2003; Maier et al., 1990; Steger, 1999). It now appears that a subset of these transcripts are completely untranslated, leaving small amounts of protamine mRNA retained in mature human spermatozoa (Wykes et al., 1997, 2000).
Aberrations in protamine expression have been associated with male infertility (Chevaillier et al., 1987; Balhorn et al., 1988; Chevaillier et al., 1990; Belokopytova et al., 1993; de Yebra et al., 1993, 1998; Carrell and Liu, 2001; Aoki et al., 2005). A number of studies have described infertile male populations with abnormally elevated ratios of P1 to P2 (P1/P2) (Aoki et al., 2005; Balhorn et al., 1988; Chevaillier et al., 1990; de Yebra et al., 1993, 1998; Carrell and Liu, 2001). Two of these reports document a small population of infertile men with complete selective absence of P2 (Carrell and Liu, 2001; de Yebra et al., 1993). Recently, another population of infertile males was identified with deregulated P1 expression and abnormally reduced P1/P2 ratios (Aoki et al., 2005). Taken together, these studies indicate abnormal protamine stoichiometry derives from aberrant expression of either P1 or P2.
The aetiology of sperm protamine deficiency in infertile men remains elusive. Protamine expression deregulation may occur at multiple points along the expression pathway, including mutations in the protamine genes, aberrant transcription regulation, unfaithful translation repression or activation, and incomplete post-translational processing. The objective of this study was to identify potential mechanisms of deregulated protamine expression in patients with abnormal protamine protein levels. Concomitant protein and mRNA quantification was used to evaluate the relationship between protamine mRNA levels and protamine protein concentrations in the sperm of fertile men as well as infertility patients with and without protamine deficiency.
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Materials
Unless otherwise noted, all chemicals were obtained from Sigma Chemical Company (St Louis, MO, USA). Reagents for gel electrophoresis were purchased from BioRad Laboratories (Hercules, CA, USA).
Study population and semen processing
Institutional Review Board approval was obtained prior to initiation of this study. Semen was collected from 27 fertile donors and 166 male infertility patients. A single semen sample was used for nuclear protein and mRNA extraction/quantification. Fresh aliquots were used for mRNA extraction within 30 min of semen collection. The remaining sample was divided into aliquots and cryopreserved for nuclear protein extraction.
Nuclear protein extraction
Sperm nuclear proteins were extracted from the cryopreserved semen aliquots as previously described (Carrell and Liu, 2001). The P1/P2 ratio, P1 and P2 concentrations were subsequently quantified in each of the study subjects. All samples were run in duplicate, and the average P1 and P2 concentrations and P1/P2 ratio from the two runs were reported.
Prior to extraction, sperm cell concentrations were evaluated to quantify protamine concentrations (ng/106 cells). Briefly, semen aliquots with a known number of sperm were centrifuged (500g, 5 min, 4°C), and the pellet was washed in 1 mmol/l of phenylmethylsulfonylfluoride (PMSF) diluted in water. After centrifugation (500g, 5 min, 4°C), the pellet was resuspended in 100 µl of Tris buffer (100 mmol/l) containing EDTA (20 mmol/l) and PMSF (1 mmol/l, pH 8.0). One hundred microlitres of guanidine (6 mol/l) and dithiothreitol (DTT, 575 mmol/l) was added to the suspension, followed by the addition of sodium iodoacetate (200 µl, 522 mmol/l).
The suspension was protected from light, kept at room temperature (30 min) and mixed with 100% ethanol (1.0 ml, 4°C). After centrifugation (12 000g, 10 min, 4°C), the ethanol wash was repeated, and the pellet was resuspended in 0.8 ml of 0.5 mol/l HCl, incubated (15 min, 37°C) and centrifuged (10 000g, 10 min). The supernatant was retained, and the nuclear proteins were precipitated by the addition of 100% trichloroacetic acid (TCA) to a final concentration of 20% TCA. After incubation (4°C, 5 min) and centrifugation (12 000g, 10 min), the pellet was washed twice in 500 µl of 1% 2-mercaptoethanol in acetone, dried and stored at –20°C until gel electrophoresis analysis.
P1/P2 quantification
A highly purified human protamine standard was used to quantify the P1 and P2 concentrations ([P1] and [P2]) as previously described (Aoki et al., 2005). The P1 and P2 standard concentrations were calculated from the percent composition of each of the protamines multiplied by the total protamine concentration (determined using the RC DC protein assay kit, BioRad Laboratories).
Acetic acid urea gel electrophoresis was used to evaluate the intensity of the P1 and P2 bands (Aoki et al., 2005). A serial dilution of the standard (1.52, 0.76, 0.38 and 0.19 µg) was loaded in each gel, and a standard regression curve was generated, which afforded quantification of sperm protamine concentrations in the unknowns. Protein quantity is reported as ng protein/106 sperm, and reported values represent the mean ± SEM. The r2 value of the regression curve was 0.96 or better for each gel run. Identities of P1 and P2 bands were verified using Western Blot analysis as reported in a previous study (Carrell and Liu, 2001).
Protamine quantification quality control
Two quality controls were used to ensure protamine quantification could produce valid and reproducible results with respect to the evaluation of the P1/P2 ratio, [P1] and [P2]. First, aliquots of 20 x 106 sperm were made from a common pool of 20 semen samples. One of these aliquots was run with each round of extractions (n = 15). The resulting mean P1/P2 ratio (0.85 ± 0.01), P1 concentration (441.1 ± 3.7 ng/106 sperm) and P2 concentration (522.1 ± 4.5 ng/106 sperm) showed little sample-to-sample variation (CV = 0.8 and 0.9%, respectively) and ensured reproducible results within individual samples.
Second, variations in the P1/P2 ratio, [P1] and [P2] between ejaculates from the same individual were analysed and reported in a previous study from two different ejaculates (6 months apart) in 42 individuals (Aoki et al., 2005). Results indicated no significant differences between ejaculates with respect to the P1/P2 ratio (1.03 ± 0.04 versus 1.11 ± 0.08), P1 concentration (560.4 ± 42.2 versus 571.9 ± 49.6 ng/106 sperm) or P2 concentration (535.5 ± 30.9 versus 527.1 ± 37.2 ng/106 sperm) as assessed by a paired t-test.
Sperm cell RNA extraction
Extraction of RNA from fresh semen samples with a known sperm cell quantity (usually 5 or 10 x 106 cells) was performed using the Trizol® reagent (Invitrogen, Carlsbad, CA, USA). Seminal plasma was removed from the samples after sperm cell sedimentation. Sperm cell washing was avoided to decrease the possibility of mRNA degradation. Sperm cells were lysed by the addition of Trizol® reagent (1 ml), and the homogenates were incubated (15 min) at room temperature to afford complete dissociation of nucleoprotein complexes.
The homogenates were supplemented with 0.1 ml bromochloropropane (BCP) and shaken vigorously for 15 s. The resulting mixture was stored at room temperature for 10 min and centrifuged (12 000g, 15 min, 4°C). The mixtures separated into three layers: a lower phenol–chloroform phase, an interphase layer and a colourless upper aqueous phase. The upper aqueous phase was transferred to a fresh tube, and the RNA was precipitated with isopropanol (0.5 ml), stored at room temperature (10 min) and centrifuged (12 000g, 8 min, 20°C). The RNA formed a gel-like pellet, the supernatant was removed and the RNA pellet was washed once with 75% ethanol and centrifuged (7500g, 5 min, 20°C). The supernatant was removed, and the pellet was allowed to dry (5–10 min at room temperature). RNA was rehydrated in 20 µl of sterile diethylpyrocarbonate (DEPC)-treated water and stored at –80°C until use. The total RNA concentration of each sample was determined by absorbance (A) at 260 nm. The purity of each RNA sample was evaluated using the A260/280 ratio.
First-strand cDNA synthesis
RT was performed using the MMLV RT enzyme (Promega, Madison, WI, USA), following manufacturer’s instructions. Briefly, RNA (1.0 µg) was combined with 0.5 µg of oligo pd(T)12–18 primers (0.28 µg/µl, Amersham, Piscatawa, NJ, USA) and DEPC-treated sterile water in a total volume of 30 µl. Subsequently, the mixture was heated at 70°C (2 min) to denature the RNA and was immediately chilled on ice (no longer than 5 min). Twenty microlitres of a master mix containing 1 µl RNAguard® ribonuclease inhibitor (37 000 U/ml, Amersham), 2 µl MMLV RT enzyme (200 U/µl, Promega), 10 µl x5 buffer supplied with the MMLV RT enzyme (Promega), 6.25 µl dNTPs (12.5 mmol/l each nucleotide, Idaho Technologies, S.L.E. UT, USA) and 0.75 µl DEPC-treated sterile water was added to each sample to in a final volume of 50 µl.
The mixture was incubated at 37°C for 1 h, and the enzyme was subsequently inactivated at 70°C for 10 min. The RT products were diluted (1 : 20) in sterile water and stored at –20°C until use. A negative control was performed during each round of RT by substituting water for the MMLV and using the same reaction conditions.
Semi-quantitative real-time PCR
Semi-quantitative real-time PCR was performed on an ABI 7900HT (Applied Biosystems, Foster City, CA, USA) using previously described intron-spanning primer sets designed to amplify P1 and P2 cDNA (Steger et al., 2000). Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as a housekeeping control gene (Table I). The PCR conditions were as follows: 94°C for 2 min followed by 40 cycles of 94°C for 30 s, 58°C for 30 s, 72°C for 30 s and a final hold for 5 min at 72°C. The reaction mix included cDNA template produced from 50 ng RNA, 0.3 mmol/l of each primer and the fluorescent marker SYBR green (x0.33, Molecular Probes, Eugene, OR, USA) in a total volume of 25 µl. The intron-spanning primer design controlled for genomic DNA amplification with the expected P1 and P2 cDNA amplicon product lengths of 153 and 294 bp, respectively.
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The SDS software package (Applied Biosystems) was used to quantify P1, P2, and GAPDH cDNA levels, based on a standard curve approach (Figure 1). Standard curves were generated using 10-fold dilutions of previously amplified P1, P2 and GAPDH PCR products (Figure 1). Each of these standards were run with every round of real-time PCR amplification and assigned a copy number (101, 102, 103, 104, 105, 106 and 107) that reflected their relative dilution factor. A threshold level was set within the SDS software, which bisected the exponential growth phase of each PCR reaction (Figure 1). The cycle number at which each standard reaction crossed threshold was then plotted against the copy number assigned to that particular standard. The standard curve was then used to quantify the individual P1, P2 and GAPDH cDNA copy numbers in each of the unknowns (reported as copies/106 sperm). Thus, the quantitative methods reported here represent semi-quantitative RT–PCR, since transcript levels represent relative quantities rather than actual quantities. RNA quantities are reported as mean ± SEM.
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A number of quality-control measures were employed to ensure cDNA quantification was valid and reproducible. First, each of the patient samples was run in triplicate. Samples with a CV greater than 10% were repeated. Second, intra-assay variation was assessed by repeated quantification of an identical study sample with every round of PCR (n = 10). The resulting mean P1 mRNA (4217 ± 39 copies/106 sperm), P2 mRNA (4390 ± 67 copies/106 sperm) and GAPDH mRNA (2067 ± 30 copies/106 sperm) showed little sample-to-sample variation (CV = 0.9, 1.5 and 1.5, respectively) and ensured reproducible results within individual samples. Third, a negative water control was used to ensure contamination was not present in the PCR reagents. Fourth, RT-negative controls were evaluated using real-time PCR to ensure genomic DNA contamination was not present. Fifth, melting-curve analysis was performed in concert with gel electrophoresis to ensure that the amplification generated a single product of the proper size.
Statistical evaluation
Patient stratification based on the P1/P2 ratio
On the basis of P1/P2 ratio, study subjects were stratified into four groups: fertile donors, normal P1/P2 patients, low P1/P2 patients and high P1/P2 patients. The values defining abnormally low (<0.8) and high (>1.2) P1/P2 ratios have been established in a previous report, which calculated these critical values from the two-tailed normal distribution for the P1/P2 ratios of fertile donors with 90% confidence limits (Aoki et al., 2005). The fertile population used in this study displayed a similar P1/P2 ratio distribution comparable with that of the previous report. P1 and P2 mRNA levels were compared between these groups using the Kruskal–Wallis test. In addition, correlation between the P1/P2 protein ratio and the P1/P2 mRNA ratio was evaluated using Spearman’s correlation analysis. The Spearman’s correlation coefficient (Rs) defined the magnitude of the relationship between variables. A high correlation was indicated by an Rs with a corresponding P-value >0.01. A moderate correlation was indicated by an Rs with a corresponding P-value >0.05. A low correlation was indicated by an Rs with a non-significant P-value.
Patient stratification based on deregulated P1 and P2 expression
Study subjects were also stratified according to P1 and P2 protein concentrations. The first stratification grouped patients according to P1 concentrations and included three groups: patients significantly underexpressing P1, normally expressing P1 and overexpressing P1. The second stratification grouped patients according to P2 concentrations and included those significantly underexpressing P2, normally expressing P2 and significantly overexpressing P2. P1 and P2 mRNA levels were compared between these groups using the Kruskal–Wallis test. In addition, correlation between the P1 and P2 protein concentrations and the P1/P2 mRNA ratio was evaluated using Spearman’s correlation analysis.
Abnormal expression of P1 and P2 protein is referred to as deregulation, which was quantitatively assessed in another study by comparing patient P1 and P2 protein concentrations against those of the fertile men with normal protamine content (Aoki et al., 2005). P1 and P2 protein concentrations falling outside the critical values for P1 and P2 concentrations in the comparison group were classified as being deregulated. The critical values for normal P1 and P2 quantity define the protein concentration range within a two-tailed normal distribution of the fertile population with a confidence of 95% (483.0–594.4 ng P1/106 sperm and 474.2–556.6 ng P2/106 sperm, respectively). These P1 and P2 concentration confidence intervals, which were reported previously on a larger population of fertile men, are similar to those identified in the smaller fertile population included in this study.
Patient stratification based on P1 and P2 mRNA levels
Study subjects were also stratified according to P1 and P2 mRNA levels. Patients with low, normal and high P1 and P2 mRNA levels were grouped using an approach similar to the one outlined above. P1 and P2 mRNA levels were quantitatively assessed using fertile men with normal protamine protein content as a standard. P1 and P2 mRNA levels falling outside the critical values for P1 and P2 mRNA levels in the fertile group were classified as significantly reduced or elevated. The critical values for normal P1 and P2 mRNA levels define the transcript range within a two-tailed normal distribution of the fertile population with a confidence limit of 95% (3419–5040 P1 copies/106 sperm and 3538–5459 P2 copies/106 sperm).
The frequency of low, normal and high P1 mRNA levels was compared between P1/P2 ratio groups as well as P1 protein concentration groups using Chi-square analysis. The frequency of low, normal and high P2 mRNA levels was compared between P1/P2 ratio groups as well as P2 protein concentration groups using Chi-square analysis.
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Results |
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Protamine protein quantification
Gel electrophoresis revealed that the mean P1/P2 ratio for fertile donors was 1.00 ± 0.01 with a range from 0.78 to 1.27. A similar mean P1/P2 ratio was observed in the infertility patients (0.98 ± 0.03) but with a much broader range (0.19–2.45). In the patient group (n = 166), 61 individuals were identified with an abnormally low P1/P2 ratio (<0.8) and 34 were identified with an abnormally high P1/P2 ratio (>1.2). In the fertile donor group, there was one man with a reduced P1/P2 ratio and another with an elevated P1/P2 ratio. However, the ratios for these fertile men were 0.78 and 1.27, significantly different in comparison with the mean ratios of the low and high P1/P2 ratio patient groups (0.67 ± 0.02 and 1.56 ± 0.05, respectively).
Protamine quantification revealed significantly reduced P1 and P2 protein quantity in the infertility patients (415.2 ± 15.2 and 425 ± 14.6 ng/106 sperm, respectively) versus the fertile population (488.1 ± 30.6 and 500.3 ± 30.8 ng/106 sperm, respectively; P < 0.05). Within P1/P2 ratio groups, marked differences were observed in P1 and P2 quantity. P1 quantity was significantly reduced in the low P1/P2 ratio group (326.7 ± 17.9 ng/106 sperm) versus the normal P1/P2 group (452.9 ± 24.5 ng/106 sperm) and high P1/P2 group (495.2 ± 35.0 ng/106 sperm, P < 0.001). P2 quantity was significantly reduced in high P1/P2 ratio patients (325.6 ± 24.5 ng/106 sperm) versus those with normal (434.8 ± 22.7 ng/106 sperm) and low P1/P2 ratios (471.3 ± 24.2 ng/106 sperm, P < 0.05).
Patients with abnormal P1/P2 ratios experienced an equal frequency of P1 and P2 deregulation (82/95 and 83/95, respectively, NS). However, the frequency of P1 and P2 deregulation differed significantly between the low and high P1/P2 ratio groups. P1 underexpression accounted for the majority of low P1/P2 ratio cases. In this group, 72% (44/61) of patients displayed P1 underexpression versus 28% (17/61) with P2 overexpression (P < 0.001). There were five patients who showed low P1 concurrent with high P2. Meanwhile, P2 underexpression accounted for the majority of elevated P1/P2 ratio cases. In this group, 88% (30/34) of patients underexpressed P2 versus 35% (12/34) who overexpressed P1 (P < 0.001). Eight of these patients showed high P1 concentrations concurrent with low P2.
Relationship between sperm protamine protein and mRNA quantity
Semi-quantitative real-time PCR revealed similar mean P1 and P2 mRNA levels in the patient (4487 ± 431 and 3621 ± 381 copies/106 sperm, respectively) and fertile populations (4230 ± 394 and 4499 ± 467 copies/106 sperm, respectively, NS). Mean P1 mRNA was significantly increased in patients underexpressing P1 protein (5853 ± 606 copies/106 sperm) and significantly reduced in patients overexpressing P1 (1500 ± 404.2 copies/106 sperm) versus those normally expressing P1 (2818.7 ± 615.3 copies/106 sperm, P < 0.001, Figure 2A). There appeared to be a tendency towards increased mean P2 mRNA levels in patients underexpressing P2 protein (4197 ± 500.12 copies/106 sperm) versus those with normal and elevated P2 expression (1773 ± 324.2 and 2703 ± 775 copies/106 sperm, respectively, P < 0.05, Figure 2B).
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Mean P1 and P2 mRNA levels were compared between P1/P2 ratio groups (Figure 3). In patients with low P1/P2 ratios, P1 mRNA was significantly increased versus fertile men and patients with normal and high ratios (7172 ± 746 versus 4230 ± 394, 3035 ± 645 and 2699 ± 506 copies/106 sperm, respectively, P < 0.001; Figure 3). P2 mRNA was increased in fertile men (4499 ± 467 copies/106 sperm) versus patients in each of the three ratio groups (low P1/P2, 3761 ± 432 copies/106 sperm; normal P1/P2, 3599 ± 764 copies/106 sperm; and high P1/P2, 3413 ± 593 copies/106 sperm), although not to the level of statistical significance (Figure 3). GAPDH mRNA quantity was statistically similar within all four groups (low P1/P2, 2180 ± 69 copies/106 sperm; normal P1/P2, 2138 ± 57 copies/106 sperm; high P1/P2, 2295 ± 99 copies/106 sperm; fertile men, 2006 ± 9 copies/106 sperm, Figure 3).
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Aberrant protamine mRNA frequency analysis
The frequency of abnormal protamine mRNA levels was compared between P1/P2 ratio groups (Table II). Patients with low P1/P2 ratios displayed a significantly increased frequency of abnormally high P1 mRNA levels (61%, 37/61) compared with the frequency of normal (23%, 14/61) and low P1 mRNA levels (16%, 10/61, P < 0.001). Within the high P1/P2 ratio group, there was a significant increase in the frequency of patients with abnormally low P2 mRNA levels (58%, 20/34) versus those with normal (12%, 4/34) and elevated P2 mRNA (20%, 10/34, P < 0.001).
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The frequency of abnormal protamine mRNA levels was compared between protamine protein deregulation groups (Table III). The majority of patients with normal P1 and P2 protein concentrations displayed normal P1 and P2 mRNA levels (84 and 95%, respectively). Patients underexpressing P1 protein displayed an equal frequency of abnormally low and high P1 mRNA levels (44%, 47/106; 45%, 48/106, respectively). Meanwhile, there was an increased frequency of patients overexpressing P1 protein, who displayed abnormally low P1 mRNA levels (82%, 28/34, P < 0.001). The majority of patients underexpressing P2 protein displayed abnormally low P2 mRNA levels (58%, 67/115, P < 0.005). However, 25% (29/115) of these low P2 protein patients also displayed abnormally elevated P2 mRNA levels, accounting for the significant increase in mean P2 mRNA levels in patients underexpressing P2 protein.
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Protamine protein and mRNA correlation analysis
Correlation analysis was used to evaluate the relationship between protamine mRNA and protein levels (Figure 4). A significant relationship was detected between P1 and P2 mRNA levels (Rs 0.77, P < 0.001, Figure 4A) as well as P1 and P2 protein concentrations (Rs 0.68, P < 0.001, Figure 4B). Significant negative correlations were detected between P1 protein and P1 mRNA levels (Rs –0.36, P < 0.001, Figure 4C) and between P2 protein and P2 mRNA levels (Rs –0. 1792, P < 0.05, Figure 4D).
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Correlation analysis was also used to explore the relationship between the P1/P2 protein ratio, the P1/P2 mRNA ratio and protamine mRNA levels (Figure 5). Two separate correlations were performed for each comparison. The first included patients with low and normal P1/P2 ratios and the second included patients with normal and high P1/P2 ratios. The P1/P2 mRNA ratio showed a significant negative correlation with the P1/P2 protein ratio in patients with low and normal P1/P2 ratios (Rs –0.37, P < 0.001, Figure 5A). However, in patients with normal and high ratios there was no significant correlation between P1/P2 mRNA and protein ratios (Rs –0.14, NS, Figure 5A).
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P1 mRNA levels showed a significant negative correlation with the P1/P2 ratio in an analysis of low and normal ratio patients (Rs –0.44, P < 0.001, Figure 5B). However, no correlation was detected between P1 mRNA levels and the P1/P2 ratio in patients with normal and high ratios (Rs –0.12, NS, Figure 5B). No significant correlations were detected between the P1/P2 ratio and P2 mRNA levels either in the low-to-normal P1/P2 ratio group (Rs –0.04, NS) or in the normal-to-high P1/P2 ratio group (Rs 0.05, NS, Figure 5C and D).
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Discussion |
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This is the first study to concomitantly evaluate protamine protein and mRNA levels in mature ejaculated spermatozoa. A number of studies have now established a relationship between deregulated protamine expression and male infertility (Balhorn et al., 1988
; de Yebra et al., 1993; Khara et al., 1997; Carrell and Liu, 2001; Mengual et al., 2003; Aoki et al., 2005). In particular, these reports highlight populations of infertile males with abnormal P1/P2 stoichiometry, which now appears to be a result of diminished P1 and P2 expression (Aoki et al., 2005; de Yebra et al., 1993; Carrell and Liu, 2001). However, the aetiology of protamine deficiency remains elusive. The data presented here offer significant contributions to our understanding of the underlying mechanisms of protamine deficiency in these infertile men. The population of infertility patients included in this study consisted of patients with normal sperm protamine stoichiometry as well as patients with abnormally low and high P1/P2 ratios. Significant P1 underexpression was shown to underlie abnormally low P1/P2 ratios, whereas the majority of patients with abnormally elevated P1/P2 ratios displayed a significant reduction in P2 expression. The objective of this study was to evaluate protamine mRNA levels to identify possible deregulated points in the expression pathway that may foster P1 or P2 deficiency.
In the case of diminished P1 expression, mRNA quantification strongly suggests that there may be a disruption in normal P1 translation regulation. In particular, it appears that in cases where P1 protein is reduced, there is an abnormally high level of P1 transcript retention. The mRNA quantification data consistently support this conclusion in four ways. First, in patients with diminished P1 concentrations and significantly reduced P1/P2 ratios, there was a significant increase in mean P1 mRNA levels. Second, there was a significant increase in the frequency of patients with low P1/P2 ratios who displayed abnormally high levels of P1 mRNA. Third, a negative correlation was identified between P1 mRNA levels and P1 protein levels. Fourth, in patients with low-to-normal P1/P2 ratios, there was a significant negative relationship between P1 mRNA levels and the P1/P2 ratio.
During spermiogenesis, protamine transcription and translation are temporally uncoupled. Translational regulation is one of the more important aspects of protamine biology and accounts for this delay in protamine protein production (Aoki et al., 2005). If protamine transcription and translation are allowed to occur concurrently, the chromatin undergoes precocious compaction and sperm development is arrested (Lee et al., 1995).
Protamine translation regulation begins immediately with RNA processing via intron splicing and mRNA polyadenylation (Steger, 1999). Polyadenylation serves a dual function to both protect the mRNA from degradation and provide a binding site for translation repressor proteins. As the poly-A mRNA enters the cytoplasm, it is translationally repressed via storage in messenger ribonucleoprotein particles (mRNP) and binding by specific translation repressor proteins, which target the 5'-UTR, 3'-UTR and poly-A sequences (Aoki and Carrell, 2003).
Translation repression is removed a few days later during the elongating spermatid stage, by covalent modification of the mRNP, release of translatable mRNA and removal or migration of the translation repressor proteins, leaving the poly-A tail susceptible to degradation. Subsequently, the protamine proteins are translated, phosphorylated and incorporated into the chromatin. The increased P1 mRNA retention observed in patients underexpressing P1 protein may arise because of defects in any of these translation repression/activation steps.
In the case of P2 underexpression, the mRNA data suggest a multifaceted deregulation pattern. The significantly increased mean P2 mRNA levels in patients underexpressing P2 protein suggest deregulated expression via retention of translation repression, similar to what was observed in P1-deficient patients. Correlation analysis demonstrating a negative relationship between P2 protein and mRNA levels further supports this conclusion. However, upon closer inspection of the data, the mean P2 mRNA levels were similar in patients with low, normal and high P1/P2 ratios. This juxtaposition in the P2 mRNA data casts doubt to the conclusion that P2 deficiency arises due to an abnormal retention of translation repression.
The P2 mRNA frequency data serve to clarify this apparent discrepancy, demonstrating that the majority of patients (58%) with abnormally low P2 concentrations and high P1/P2 ratios actually display a significant reduction in P2 mRNA levels. Thus, the significantly increased mean P2 mRNA level observed in patients with P2 deficiency derives from the 25% of patients with P2 deficiency who have significantly increased levels of P2 mRNA. Careful inspection of the data revealed that the P2 mRNA levels in these patients were significantly large enough to push the mean data to significance.
Thus, the majority of cases involving P2 protein deficiency are associated with decreased mRNA levels. However, 25% of the P2-deficient patients do appear to have an abnormal retention of P2 transcripts, similar to what was observed in P1-deficient patients. This dichotomy in P2 mRNA levels within P2-deficient patients is further exemplified by correlation analysis, which indicates that no significant correlation exists between the P1/P2 ratio and P2 mRNA levels.
A number of possibilities may explain why P2 is underexpressed in patients with low and normal levels of P2 mRNA. The strongest possibility is abnormal post-translational processing of the P2 protein. After translation, P2 is rapidly phosphorylated by the Ca2+/calmodulin-dependent protein kinase IV (Camk4) (Wu et al., 2000). This phosphorylation step is an absolute requirement for proper binding of P2 to the DNA (Wu et al., 2000). After the full-length phosphorylated form of P2 is bound to the DNA, it undergoes proteolytic cleavage to produce a mature shortened P2 protein (Aoki and Carrell, 2003). Thus, P2-deficient patients with abnormally elevated P1/P2 ratios may possess defects in the Camk4 protein itself or defective signalling pathways that serve to activate the kinase. Indeed, support for this hypothesis is provided by a study demonstrating an abnormal accumulation of P2 precursors in patients with reduced P2 levels (de Yebra et al., 1998). Future studies should evaluate the accumulation of these P2 precursors in P2-deficient patients with normal and abnormally low levels of P2 transcripts.
The decreased P2 levels associated with low P2 mRNA levels may also derive from diminished levels of P2 transcription. Intuitively, this does not appear a likely scenario, given that P1 and P2 are transcribed from a single coordinately expressed gene cluster with similar upstream regulatory elements located on human chromosome 16p13.3 (Domenjoud et al., 1990, 1991; Oliva and Dixon, 1991; Nelson and Krawetz, 1994). However, a variable-length GA-repeat specific to the P2 promoter has now been identified which may serve to modulate transcription efficiency (Nelson and Krawetz, 1994; Tanaka et al., 2003). Future studies should examine the relationship between this dinucleotide repeat and P2 transcript levels.
To conclude, the concomitant protamine protein and mRNA evaluation conducted in this study provides a necessary first step to elucidating the underlying aetiology of protamine deficiency in infertile human males. Future studies should explore the possible mechanisms of protamine expression deregulation highlighted by these data. In particular, the 5'-and 3'-UTR regions of the protamine genes should be sequenced in protamine-deficient patients with abnormal accumulation of P1 and P2 transcripts. In addition, evaluation of the expression and function of the translation regulator proteins in these patients may yield valuable insights into protamine deficiency in infertile human males.
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The authors wish to thank Steve Hamblin and Michael P. Klein at the University of Utah for their technical expertise and assistance with the quantitative real-time PCR.
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Submitted on November 7, 2005; accepted on December 7, 2005.
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