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From the Departments of * Physiology, Human
Genetics Research Group, and Public Health,
Biostatistics Unit, IDIBAPS, Faculty of Medicine, University of Barcelona,
Barcelona, Spain; and Genetics Service and
Institut Clínic de Ginecologia,
Obstetricia i Neonatología, Hospital Clínic i Provincial,
Barcelona, Spain.
| Correspondence to: Dr Rafael Oliva, Department of Physiology, Human Genetics Research Group, Faculty of Medicine, University of Barcelona, Casanova 143, 08036 Barcelona, Spain (e-mail: oliva{at}medicina.ub. es). |
| Received for publication November 13, 2002; accepted for publication December 4, 2002. |
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Abstract |
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Key words: Chromatin condensation, male infertility, Percoll gradients, sperm chromatin, sperm morphology, sperm motility
The goals of this study were 1) to determine the quality of subpopulations of spermatozoa isolated from different fractions of a 3-layer discontinuous Percoll gradient by determining their motility, morphology, and nuclear maturity in terms of P1/P2 ratios and P1 + P2 relative amounts, and 2) to compare all of the above sperm parameters in the different fractions obtained from control fertile men, oligozoospermic samples, and asthenozoospermic samples in search for potential correlations.
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Materials and Methods |
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Sperm Cell Fractionation
A stock Percoll (Pharmacia Biotech AB, Uppsala, Sweden) solution was
prepared by mixing 8.7 parts of the Percoll product, 1 part of Ham F10
10x medium (Gibco BRL, Life Technologies Ltd, Paisley, United Kingdom),
and 0.3 parts of 7.5% NaHCO3 (Merk, Farma y Quimica, Spain). This
solution was designated the 100% Percoll-Ham F-10 stock solution.
Subsequently, the 100% Percoll-Ham stock solution was diluted with Ham F10
supplemented with 3% NaHCO3 7.5% to obtain the 90%, 70%, and 50%
Percoll working solutions. A step Percoll gradient was prepared in a conical
polypropylene tube (NUNC; Brand Products, Roskilde, Denmark) by first
introducing 1 mL of the 90% dilution; this was then followed by carefully
layering with 1 mL of the 70% dilution and finally with 1 mL of the 50%
dilution. Between 1 and 2 mL of the semen sample was carefully placed on the
top of the step Percoll gradient and centrifuged at 300 x g for
20 minutes at room temperature. After centrifugation, the different layers of
the gradient were recovered. The first fraction of the gradient corresponding
to the top of the gradient (seminal fluid and nonsedimented cell debris) was
discarded. The second fraction (labeled F2) corresponding to the interface
between the 50% Percoll and the sample was recovered, as well as the third
fraction (F3) corresponding to the interface between 50% and 70% dilution of
Percoll, the fourth fraction (F4) corresponding to the interface between 70%
and 90% dilution of Percoll, and the last fraction (fifth, F5) corresponding
to the material present at the bottom of the tube.
Aliquots of the each of fractions were taken to measure standard sperm parameters using a CASA and a Makler counting chamber (count and percentage of motility) and Diff-Quik–stained slides (morphology). The rest of the fraction was used for the extraction of nuclear sperm proteins and DNA.
Sperm Morphology
Smears of each of the factions were prepared for the examination of sperm
morphology. Smears were fixed with Labofix (Labonort, Templemars, France) and
stained using the Diff-Quik kit (Baxter Healthcare Corporation Inc, McGraw,
Ill). Smears were rinsed immediately with water after staining to remove the
excess of dye and air dried. Sperm morphology was evaluated using strict
criteria (Kruger et al, 1986),
and at least 100 cells were examined per slide.
Extraction of Sperm Proteins From Percoll Fractions
The fractions were washed twice with Ham F10 1x supplemented with 3%
of a 7.5% NaHCO3 solution. The sediment was resuspended in 200
µL of 20 mM EDTA, 1 mM phenylmethyl sulfonylfluoride (PMSF; Sigma
Chemicals, St Louis, Mo), and 100 mM Tris HCl (pH 8) and then processed as
described in De Yebra and Oliva
(1993), with the variant that
no iodoacetate treatment was performed (an equal volume of H2O was
added instead and incubated for 10 minutes). The sediment obtained after an
extraction of the proteins with 0.5 M HCl was used to quantify the DNA using
0.5 N perchloric acid hydrolysis (80°C for 30 minutes) and the
diphenylamine reaction (De Yebra and
Oliva, 1993).
Preparation of the Human Protamine Standard
A pool of 72 semen samples was made in order to extract and quantitate the
sperm protamines. The cells were pelleted at 60 x g at 4°C
for 10 minutes. The supernatant was discarded, and the pellet was washed twice
with 3 mM MgCl2, 1 mM PMSF, and 0.25% Triton X-100 at 60 x
g for 10 minutes (4°C). The sediment was then resuspended in 0.5
M HCl and incubated between 5 and 10 minutes at 37°C before centrifugation
at 2000 x g for 20 minutes (4°C). The supernatant was
discarded, and the pellet was washed twice more with 0.5 M HCl, incubated
between 5 and 10 minutes at 37°C, and centrifuged at 2000 x
g for 20 minutes (4°C). The sediment was finally resuspended in
20 mM EDTA, 1 mM PMSF, and 100 mM Tris HCl (pH 8)and then processed as
described in De Yebra and Oliva
(1993) to extract the
protamines, with the variant that no iodoacetate treatment was performed (an
equal volume of H2O was added instead and incubated for 10 minutes)
(De Yebra and Oliva,
1993).
An amino acid analysis of the solution obtained after processing was performed to quantitate the protamines in the standard using an Alpha Plus (Pharmacia LKB Biotechnology, Piscataway, NJ) autoanalyzer. Subsequently, 0.22, 0.36, 0.44, and 0.58 µg of human sperm protamine standard were loaded in each of the gels (see below), and a regression curve was made to calculate the amount of protamines contained in each sample.
Separation and Analysis of Proteins
Nuclear proteins were analyzed in acid-urea polyacrylamide gels. The
composition of the polymer gels was 0.9 M acetic acid, 2.5 M urea, 15%
acrylamide, 0.09% bis-acrylamide, 0.53% ammonium persulfate, and 0.53%
N,N,N',N-tetramethylenediamine. Electrophoresis was
performed on a Miniprotean System (Bio-Rad, Life Science Group, Hercules,
Calif). Following the polymerization, the gel was preelectrophoresed for 1
hour at 150 V before loading the samples. Following gel loading, the gel was
electrophoresed for 1 hour at 150 V in 0.9 M acetic acid buffer.
Staining of the gels was performed with 1.1 g of Coomassie blue R-250 (Bio-Rad) dissolved in 250 mL methanol, 250 mL H2O, and 50 mL acetic acid for 30–45 minutes, destained for 5 minutes in 50% methanol and 10% acetic acid, and then destained overnight in 10% methanol and 10% acetic acid. The gels were then dried between 2 sheets of cellophane film for 48 hours. Lastly, the gels were scanned, and the intensity of the bands was quantified with Quantity One software (Bio-Rad).
Statistical Analysis
Data are expressed as the mean plus or minus the standard deviation of the
mean. For normally distributed variables, analysis of variance with repeat
measures was used. For non-normal variables, nonparametric analysis was used;
the Kendall test was used for comparisons between fractions, the
Kruskal-Wallis test was used for comparisons between groups, and the
Mann-Whitney U test was used for 2-to-2 comparisons (with Bonferroni
correction). Sperm concentration was not included in the analysis, as this
semen parameter was used in the initial selection of the groups. Statistical
analyses were performed by SPSS software, version 10.0 (SPSS Corp, Chicago,
Ill), and statistical tests have been evaluated by a significance level of
.05.
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Results |
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TopAbstractMaterials and MethodsResultsDiscussionReferences |
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We also determined whether the F5 fraction recovered from the Percoll separation had increased motility parameters compared to the total sperm sample. Table 1 shows how the Percoll gradients significantly increase the quality of many of the sperm parameters measured in each group. For the fertile men control, only count, percentage of cellular motility, and percentage of WHO grade D motility are not improved. In the oligozoospermic samples, there are improvements in the percentage of cellular motility, the percentage of WHO grade A motility, and the percentage of WHO grade D motility. Finally, in asthenzoospermic males, there are the same improvements detected as in oligozoospermic males but, in addition, the means of progressive motility and the linearity index are improved.
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Sperm Nuclear Basic Proteins
Figure 2 shows an example of
the pattern obtained from the extracted protamines from each fraction
separated by polyacrylamide gel electrophoresis and subsequent staining with
Coomassie blue. The analysis of the nuclear sperm proteins and the DNA content
present in the cells recovered from the different Percoll fractions revealed a
uniform distribution of the P1/P2 ratio among all of the fractions in the
group of control fertile males (P = .948) and in the group of
oligozoospermic males (P = .95) as well as in the asthenozoospermic
males (P = .59). No differences were detected in the protamine 1 or
protamine 2 content or in the total content of protamines between each
fraction in the control fertile group, in the oligozoospermic group, or in the
asthenozoospermic group (Table
2). However, we found statistically significant differences when
comparing equivalent fractions between groups in the P1/P2 ratio, µg
P2/µg DNA, and µg P1 + P2/µg DNA (Tables
2 and
3). Particularly high
statistically significant differences were found in the P1/P2 ratio between
the control males and the oligozoospermic males (P < .001;
Table 3). This difference is
also confirmed when compared with each fraction from the control fertile males
and the corresponding fraction from the oligozoospermic males
(Table 2). The lack of
statistically significant differences between the control males and the
asthenozoospermic males is most likely due to the high heterogeneity in the
ratio and content of protamines found in this group of patients (range,
0.78–4.92; Table 3).
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We also found a different amount of P2 in oligozoospermic and asthenozoospermic males compared to controls (P = .006 and .002, respectively). When we analyzed the total amount of protamines for each group, we found statistically significant differences between the normal control males and the asthenozoospermic males (P = .008; Table 3). We did not find statistically significant differences in the total amount of protamines between the control males and the oligozoospermic males, although the lack of differences could be considered borderline (P = .038; not significant because of the Bonferroni correction) and could be because of the low number of samples analyzed.
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Discussion |
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Centrifugations through Percoll-based (or other alternatives such as PureSperm or ISolate) density gradients are the most routinely used methods to select morphologically normal spermatozoa with good motile characteristics in assisted reproduction techniques. Although there are many types of Percoll gradients used for sperm separation, ranging from 1 layer to 10, the most commonly used are those formed by 2–3 layers (Chen and Bongso, 1999). From our results (Figure 1), it becomes clear that motion and morphological characteristics of spermatozoa recovered from each of the gradient fractions are different and that the sperm characteristics can be improved by selecting the appropriate gradient fraction, as has been described (Centola et al, 1998; Chen and Bongso, 1999; Gil-Guzman et al, 2001). We have found that the quality of spermatozoa, in terms of the motility and normal forms, recovered from the bottom of the tube (F5) is significantly improved compared to the lower density layers (Figure 1) or to the total semen sample (Table 1), in agreement with previously reported data (Chen and Bongso, 1999; Gil-Guzman et al, 2001). This fact is important because selected spermatozoa from the bottom of the tube, with an improved morphology and motility, are currently being used for oocyte insemination in assisted reproduction techniques. This procedure is justified in light of the large number of reports stating that male infertility is due to abnormal morphology or motion of the ejaculated spermatozoa. In addition, the improvement throughout the gradient results in comparable motility parameters from equivalent fractions for normal or abnormal samples (Table 1). Consistently, Gil-Guzman et al (2001) found that spermatozoa recovered from the bottom of the tube of a discontinuous ISolate gradient (47%, 70%, and 90%) had a percentage of motility of around 70%–80%, both in samples with normal or with abnormal semen parameters—values that are very similar to those obtained in our study (Table 1; Figure 1).
However, in contrast to the abundant data on the morphology and motion characteristics of sperm cells separated through Percoll gradients, little is known about their nuclear maturity. Thus, we have analyzed the protamine content, which is indicative of the stage of maturation, of subpopulations from spermatozoa with different morphological and motion characteristics. The P1/P2 ratio and the relative amount of protamines in the isolated Percoll fractions are easily measured by densitometry of the protamine bands present in the polyacrylamide gels (Figure 2). One of the most relevant findings of this part of the study is the fact that sperm cells from different fractions of the same patient show a similar P1/P2 ratio and a similar relative amount of these proteins (Table 2). Thus, we have not found a correlation between the motility and the morphology of spermatozoa and the levels of protamines in the sperm nuclei. Although sperm chromatin integrity improves after preparation by density gradient centrifugation (Tomlinson et al, 2001), we have demonstrated that the altered levels of protamines in oligozoospermic patients are present even in the selected spermatozoa from F5. Consistent with this finding is the observation of a significant number of mature spermatozoa with decondensed DNA (measured by sperm chromatin structure assay) in the spermatozoa recovered from the 90% fraction of a discontinuous 3-layer ISolate gradient from infertile patients (Ollero et al, 2001). Experiments with the fluorochrome chromomycin A3, which is indicative of protamine deficiency, detected staining in morphologically normal spermatozoa (Bianchi et al, 1993; 1996).
An increased proportion of the protamine P2 content of 90%–100% Percoll selected spermatozoa compared to semen and spermatozoa selected from 60% Percoll has been previously reported (Colleu et al, 1996). Here, we have extended observations to 4 different sperm fractions and, contrary to the above observations, we do not detect any differences between sperm protamines in the different subpopulations of spermatozoa. Perhaps slight methodological differences (such as the presence or absence of iodoacetate in the extraction of protamines) (De Yebra et al, 1998) or differences in the type of samples used could result in different results.
There are differences in the P1/P2 ratios and the relative amount of protamines between the group of control fertile men and the infertile group. A remarkable increase in the P1/P2 ratio from 1.01 (range, 0.68–1.43) in the control group to 1.51 (range, 0.88–2.92) in the oligozoospermic group or 1.23 (range, 0.78–4.92) in the asthenozoospermic group is detected (Table 3). The normal P1/P2 ratio in normozoospermic samples has been described to range between 0.79 and 1.27, with the average being 0.98 (Balhorn et al, 1988; Corzett et al, 2002). Here, we report a wider range in the P1/P2 ratio (0.68–1.43) and in the relative amount of P1 and P2 in normal fertile men in a series of 10 independent samples. An altered P1/P2 ratio in infertile patients had been described to range between 1.2 and 1.94, with the average being 1.58 (Balhorn et al, 1988). Subsequently, more marked increases in the P1/P2 ratio were found after analysis of a larger proportion of samples (De Yebra et al, 1993, 1998; Carrell and Liu, 2001). It has been suggested that the increased P1/P2 ratio could be due to a decrease in the P2 content caused by deficient processing of this protamine (De Yebra et al, 1998). Here, we demonstrate that the relative amount of P2 is decreased in samples with an altered ratio, while P1 content remains constant in all of the samples, as has been described previously (De Yebra et al, 1993).
We have also detected large variations in the P1/P2 ratio and the amount of protamines in different indiviuals from the asthenozoospermic group; while some patients have a normal P1/P2 ratio (n = 8; P1/P2 = 1.006), others have very high P1/P2 ratios (n = 5; P1/P2 = 1.672). This is also consistent with previous findings in our lab when analyzing sperm nuclear proteins from 116 unselected infertile patients (De Yebra et al, 1993). This heterogeneity in the infertile group and the fact that the number of patients studied is relatively low could influence statistical analysis, resulting in the lack of detection of statistically significant differences in some cases when analyzing all samples from a group. This heterogeneity in the P1/P2 ratio in independent samples also points to different origins for the infertility. In the future it should be interesting to look at the P1/P2 and the histone/protamine ratios in different additional subgroups of patients, such as patients with varicocele, Jacuzzi users, professions at risk, or consanguineous patients affected by potential recessive mutations.
Many studies have demonstrated that ejaculated spermatozoa have anomalies in their nucleus (Evenson, 1999; Sakkas et al, 1999, 2000; Ollero et al, 2001; Tomlinson et al, 2001; Colleau et al, 1996). An alteration in the condensation state of the sperm head has been previously correlated with an increase in DNA fragmentation in the mature sperm (Gorczyca et al, 1993). In addition, a correlation has also been established between low levels of protamines and nicking of DNA (Bianchi et al, 1993). Different mechanisms have been proposed to explain the presence of these anomalies in human ejaculate. The first possibility is that some spermatozoa could have escaped programmed cell death (apoptosis), which could be linked to defects in remodeling of the cytoplasm during spermiogenesis (Sakkas et al, 2002). The second possibility is that the presence of damaged DNA could arise from problems in nuclear remodeling resulting directly from defective protamine deposition during spermiogenesis (Sakkas et al, 1999, 2002). Finally, the coexistence of mature and immature spermatozoa during migration from the seminiferous tubules to the epididymis could result in oxidative DNA damage of mature spermatozoa (Ollero et al, 2001). Since we have not found statistically significant differences from different fractions in the same group of patients, our findings could be consistent with the second possibility described above. Thus, the abnormally low protamine levels detected in functionally immature sperm from the high density fractions may be related to incomplete maturation during spermiogenesis leading to a less efficient protamine packaging (with increased DNA fragmentation). However, it is difficult to determine which of the above possibilities is related to the nuclear alterations detected.
The consequences of the damaged or decondensed sperm chromatin in the fertilization and subsequent development of the embryo are not clear. It has been postulated that morphological sperm parameters are important up to the fertilization step, while the DNA integrity becomes the most important sperm parameter related to the establishment and continuation of a pregnancy (Tomlinson et al, 2001). Poor chromatin packaging and possible DNA damage may also contribute to failure of sperm decondensation after intracytoplasmic sperm injection and subsequently result in fertilization failure (Bianchi et al, 1996). It is possible that protamine 2 acts after the fertilization process in the species that contains it, initiating pronucleus formation and the correct development of the embryo (Corzett et al, 2002).
Spermatozoa used for assisted reproductive techniques are selected on the basis of their motility and their morphology, but as has been demonstrated, these samples could carry altered levels of protamines and damaged DNA. From this work, it becomes clear that the P1/P2 ratio and the amount of protamines are relatively independent of the morphology and motility of the spermatozoa in the different Percoll fractions. Thus, if alterations in nuclear maturity are present, this should be taken as an alteration in the whole sample, not only in the abnormally motile or morphologically abnormal sperm cells.
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Footnotes |
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