Distribution of a- and d-Tocopherols in Seminal Plasma andSperm Fractions of Men With Normal and AbnormalSemen Parameters
ELENA MORETTI,*{ CESARE CASTELLINI,{ EVANGELIA MOURVAKI,{ SERENA CAPITANI,{§
MICHELA GEMINIANI,* TOMMASO RENIERI,*{ AND GIULIA COLLODEL*{
From the *Department of Biomedical Sciences, Applied Biology Section, the �Interdepartmental Centre for Research
and Therapy of Male Infertility, and the §Department of Physiopathology, Experimental Medicine and Public Health,
University of Siena, Siena, Italy; and the `Department of Applied Biology, Section of Animal Science, University of
Perugia, Perugia, Italy.
ABSTRACT: The; aim of this study was to investigate the presence
of the different isoforms of tocopherol (Ts) in the seminal plasma (P)
and in the sperm (S) fractions< of individuals with abnormal (group 1)
and normal (group 2) sperm parameters; the relationships between
these isoforms and conventional sperm parameters were also
explored. Two vitamin E homologues, a-T and d-T, were identified
in the semen of all participants. Although a-T and d-T concentrations
were similar in the semen of the 2 groups, group 1 showed a lower a-
T ratio (S/P) (0.90 vs. 1.20, P , .001) and d-T ratio (0.86 vs 1.13, P
5 .007) than group 2. In addition, both T ratios were correlated with
the percentage of viable cells, detected by eosin staining. These
results suggested that a-T and d-T are not homogeneously
distributed in the semen fractions; in normal semen they are more
concentrated in the sperm membrane, whereas in abnormal semen
the damaged sperm cells may release both Ts in the plasma. To
verify whether sperm membrane breakage could alter a-T and d-T
distribution between the seminal plasma and the spermatozoa,
normal sperm samples were sonicated; after sonication a consistent
sperm plasma membrane fragmentation, highlighted by transmission
electron microscopy, and a concomitant release of a-T and d-T were
observed. In conclusion, the Ts coupled directly with the sperm
membrane seem to play the main protective role in the semen, and
the release of a-T and d-T in the plasma fraction is probably an index
of lower antioxidant power and sperm quality.
Key words: HPLC, spermatozoa, sperm sonication, TEM.
J Androl 2011;32:000–000
O xidative stress is a condition that is associated with
an imbalance between the production and the
removal of reactive species (ROS) and free radicals by
antioxidants. An excess of ROS and free-radical
generation has frequently been detected in the seminal
plasma and the sperm of infertile men (Nakamura et al,
2002; Baker and Aitken, 2005; Pasqualotto et al, 2008).
The biological role of spermatozoa is, in part, assured
by the intrinsic properties of their plasma membrane,
which is particularly rich in polyunsaturated fatty acids
(PUFA) able to increase membrane fluidity (Conquer et
al, 2000) and the responsiveness of the acrosome to
exogenous stimuli. This high concentration of PUFA
concomitant with low antioxidant power and the
inability of the sperm, a highly differentiated cell, to
repair the damages make the sperm membrane vulner-
able to the harmful effect of ROS.
To ensure proper sperm membrane function, the
equilibrium between the insaturation level and oxidative
stability must be maintained (Aitken and Fisher, 1994).
Semen contains a variety of antioxidant compounds and
defense mechanisms including uric acid, taurine, thiols,
ascorbic acid, tocopherols (Ts), catalase, superoxide
dismutase, and glutathione peroxidase/reductase system
to counteract the detrimental effects of ROS (Alvarez et
al, 1987; Alvarez and Storey, 1989). Most of these
compounds are found in the seminal plasma, whereas
spermatozoa show very few antioxidant molecules.
Vitamin E is one of the most powerful exogenous
antioxidants (Bjørneboe et al, 1990) and it includes a-,
b-, c-, and d-T and tocotrienols, each consisting of a
chromanol ring with a side chain of 16 carbon atoms
(Schneider, 2005); a-T is the most powerful of these.
The effects of vitamin E and its derivatives on semen
characteristics have been extensively investigated. Gen-
erally, 2 kinds of approaches have been adopted: dietary
supplementation of vitamin E in case of male infertility
(Agarwal et al, 2004) or in animal studies (Castellini et
al, 2007) and direct addition to cryopreservation media
in order to improve postthaw motility of human or
Journal of Andrology andr-32-03-07.3d 22/12/10 19:49:47 1 Cust # JANDROL/2009/009936
Correspondence to: Dr Giulia Collodel, Department of Surgery,
Biology Section, University of Siena, Policlinico Le Scotte, Viale
Bracci, 14, 53100 Siena, Italy (e-mail: [email protected]).
Received for publication January 2, 2010; accepted for publication
September 23, 2010.
DOI: 10.2164/jandrol.109.009936
Journal of Andrology, Vol. 32, No. 3, May/June 2011Copyright E American Society of Andrology
0
animal spermatozoa (Castellini et al, 2007; Jeong et al,
2009; Taylor et al, 2009).
The T (a-T, c[+b]-T, and d-T) profile in rabbit semen
has recently been reported (Mourvaki et al, 2008), but
no information is available regarding these tocol-derived
compounds in human semen. Many studies revised by
Castellini et al (2007) showed that dietary incorporation
of a-T in rabbit sperm membranes greatly enhances the
oxidative stability of semen. d-T was found to be present
in a considerable amount in rabbit droplets, lipid-rich
products of the prostate gland (Castellini et al, 2006),
but the biological role is not yet clear.
In humans, despite the numerous studies concerning
the role of vitamin E intake in male infertility (Agarwal
et al, 2004), the efficacy of this antioxidant is still being
debated, and only a few reports have measured a-T
concentration in human ejaculates. Lewis et al (1997)
found that a-T was present in the seminal plasma of
fertile and infertile men, although at low concentrations
(0.08–0.69 mmol/L) and only traces were detected in
spermatozoa. On the other hand, Bhardwaj et al (2000)
reported a significantly decreased level of vitamin E in
the seminal plasma of oligozoospermic and azoospermic
men compared with normozoospermic samples.
This study was aimed at investigating the presence of
the different isoforms of T in human seminal plasma
and in the sperm fractions. The next step was to attempt
to understand the correlations among the sperm
characteristics and the distribution of measured Ts in
the fractions of semen from individuals with normal and
abnormal sperm parameters.
Materials and Methods
Patients
From December 2008 to June 2009 we analyzed 150 semen
samples from men attended at the Interdepartmental Centre for
Research and Therapy of Male Infertility, University of Siena.
Semen samples were obtained from both men with fertility
problems and those who just wanted to get tested for fertility. For
this investigation 27 men (aged 25–36 years) were selected and the
inclusion criteria consisted in non azoospermic patients with a
normal 46,XY karyotype evaluated by conventional cytogenetic
analysis, with a normal hormonal profile and no history of
radiotherapy, chemotherapy, chronic illness, or medication,
including supplementation with antioxidant compounds. More-
over, these patients did not show varicocele or other anatomic
injury, leukocytospermia (leukocyte concentration .1 6 106
cells/mL) and chronic genitourinary infections diagnosed by
bacteriologic analyses performed in seminal plasma.
All of the subjects who participated in the study gave
informed consent for this research.
Patients were divided into 2 groups: group 1 (n 5 12
individuals) showed one or more altered semen parameters
(sperm concentration ,20 6 106, rapid [a] and slow [b]
progressive motility [a + b] ,50%, sperm morphology ,30%)
and group 2 (n 5 15 individuals) had normal semen
parameters evaluated following World Health Organization
(WHO, 1992, 1999) guidelines.
Semen Analysis
Semen samples were collected by masturbation after 3–5 days
of sexual abstinence and examined after liquefaction for
30 minutes at 37uC. Volume, pH, concentration, and motility
were evaluated according to WHO (1999) guidelines. Sperm
morphology was assessed by the Papanicolaou (Pap) test
modified for spermatozoa. Ejaculated human spermatozoa
were washed twice in phosphate-buffered saline (PBS),
smeared on precleaned glass slides, air dried, and fixed in an
equal volume of ethanol 95% and ether for 5–15 minutes. The
fixed slides were stained using the protocol in the WHO
guidelines (1992).
In order to evaluate sperm viability, semen samples were
stained with 0.5% eosin Y (CI 45380) in 0.9% aqueous sodium
chloride solution. A few minutes after staining, the samples
were observed under a light microscope (Leica, Wetzlar,
Germany); a total of 200 sperm cells were scored for each
sample, and stained (unviable) and unstained (viable) cells
were categorized.
Leukocytes were identified by peroxidase stain; a concen-
tration of .1 6 106 cells/mL was considered out of range
(WHO, 1999). Moreover, May-Grunwald–Giemsa staining
(J. T. Baker; =Sigma Chemical, St Louis, Missouri) was
routinely performed in order to obtain a general picture of
the cell types, such as erythrocytes, granulocytes, or germinal
cells, that could be present in a semen sample.
Samples with a leukocytes concentration out of range or
with germinal cells were excluded from this investigation.
For electron microscopy, sperm samples were fixed in cold
Karnovsky fixative and maintained at 4uC for 2 hours. Fixed
semen was washed in 0.1 mol/L cacodylate buffer (pH 7.2) for
12 hours, postfixed in 1% buffered osmium tetroxide for 1 hour at
4uC, then dehydrated and embedded in Epon Araldite. Ultrathin
sections were cut with a Supernova ultramicrotome (Reickert
Jung, Vienna, Austria), mounted on copper grids, stained with
uranyl acetate and lead citrate, and then observed and photo-
graphed with a Philips EM208 transmission electron microscope
(TEM; Philips Scientifics, Eindhoven, The Netherlands).
Evaluation of Vitamin E in Whole Semen,Seminal Plasma, and Sperm Samples
Vitamin E was extracted from whole semen, plasma, and
sperm separately with hexane (Mourvaki et al, 2008).
In details, for each sample 200 mL of whole semen was
recovered 1 hour after semen collection and 200 mL of semen
was fractioned by centrifugation (800 6 g for 15 minutes at
4uC) into the seminal plasma (P, supernatant) and spermato-
zoa (S, pellet) and maintained at 280uC until the analysis was
performed.
The vitamin E liquid-liquid extraction was carried out in
200 mL of whole semen, and separately in the P and S fractions.
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0 Journal of Andrology N May �June 2011
The aliquots of each fraction (whole semen, P, and S) was
deproteinized with 500 mL of ethanol and Ts were extracted
twice with 1 mL of n-hexane (Mourvaki et al, 2008). The
hexane extracts were evaporated to dryness under nitrogen
flow and reconstituted with 100 mL acetonitrile. Fifty
microliters were injected into a high-performance liquid
chromatography (HPLC) system.>The chromatographic separation of Ts in the 3 fractions
(whole semen, P and S) was performed by a Jasco HPLC (PU-
1520 equipped with a 7125 Rheodyne injector) system (Jasco
Corporation, Tokyo, Japan) using a reversed-phase Beckman
Ultrasphere-ODS column (5 mm particles size, 4.6 6250 mm;
Beckman Coulter, Milan, Italy). The mobile phase consisted of
acetonitrile/tetrahydrofuran/methanol/ammonium acetate 1%
(684/220/68/28, vol/vol/vol/vol). The flow rate was 1.5 mL/min.
Concentrations of a-T, c (+b)-T, and d-T were quantified by
fluorescence detection (FP-1525; Jasco) using, respectively,
excitation and emission wavelengths of 295 and 330 nm.
Quantification was performed by reference to external
calibration curves prepared with increasing amounts of pure
T standards in ethanol. HPLC results of Ts from P and S are
expressed as nmol/mL to permit comparison between the
semen fractions. The S/P ratio was calculated by dividing the
concentration of T in the sperm fraction by the concentration
of T in the seminal plasma fraction. This parameter was the
most significant one for the correlation with sperm membrane
damage.
Sonication of Sperm Samples
To verify the potential effects of sperm membrane breakage on
vitamin E distribution between the P and the S fractions, 3
pooled semen samples from men with normal semen param-
eters (group 2) were sonicated (IKASONIC, model U-50
control) in PBS for 0, 10, 30, 60, and 180 seconds at maximum
amplitude (100%) and the vitamin E content was measured
by HPLC as previously described (results are given as peak
area of Ts). Twenty million spermatozoa were used for each
treatment run in duplicate. The resultant damages on sperm
membrane structure were observed by transmission electron
microscopy as previously described.
Statistical Analysis
Statistical analysis was performed using an SPSS (version 13)
statistical package.
The Kolmogorov-Smirnov test was used to verify normal or
nonnormal distribution of values.
The Mann-Whitney test was performed to evaluate the
statistical differences between the variables because the
conditions of normality of distribution and of homogeneity
of variance were not satisfied.
Spearman’s coefficient of correlation of ranked data was
calculated for detecting the statistical correlation between 2
variables. P , .05 was considered significant.
Results
The results of semen analysis of both groups expressed
as medians and 95% confidence intervals are reported in
Table 1.
Semen volume, sperm concentration, and rapid (a) +slow (b) progressive motility were determined following
WHO guidelines (1999). Sperm morphology was eval-
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Table 1. Medians and 95% confidence intervals of semen parameters, and values of a-tocopherol (a-T) and d-tocopherol (d-T)in whole semen samples, sperm fraction (S), plasma fraction (P), and S/P ratio of 27 men divided into 2 groups according tovalues of semen parametersa
Variables
Studied Patients
P Value
Group 1 (n 5 12) Group 2 (n 5 15)
Median
95% Confidence
Interval Median
95% Confidence
Interval
Volume, mL 4 3.17–5.74 3 2–3.87 .056
Sperm/mL 34.25 14.63–69.02 76 28.53–102.35 .067
Motility % 27.5 21.17–35.83 52 51–53.47 ,.001
Morphology % 20.05 19.17–22 31 30–33 ,.001
Viable cells % 50 47–56 79 77.5–82.5 ,.001
aT whole semen 8.15 7.20–9.50 8.91 6.92–11.99 .558
aT S 3.72 2.72–6.47 5.14 3.83–5.94 .367
aT P 4.45 4.31–7.06 3.80 2.22–4.73 .088
aT S/P 0.90 0.88–0.94 1.20 1.06–2.08 ,.001
dT whole semen 0.79 0.51–2.97 0.81 0.67–1.35 .942
dT S 0.33 0.33–0.62 0.42 0.40–0.84 .981
dT P 0.46 0.48–0.84 0.38 0.26–0.73 .18
dT S/P 0.86 0.79–1.02 1.13 1.02–1.88 .007
a Motility % is the percentage of rapid + slow progressive motility. Morphology % is the percentage of normal sperm evaluated by Papanicolaou
test). Viable cells % is the percentage of unstained sperm after eosin Y treatment. aT and dT were evaluated in whole semen and S and P
fractions by high-performance liquid chromatography (nmol/mL). Patients in group 1 show one or more altered semen parameters; those in
group 2 show normal semen parameters evaluated following World Health Organization (1992, 1999) guidelines. The P values reported in the
last column were obtained by applying the Mann-Whitney U test among the groups.
Moretti et al N Vitamin E Distribution in Human Semen 0
uated by Pap staining, and the viability of sperm cells
was assessed by eosin Y test. HPLC was used to
determine the concentration of T in the whole semen,sperm (S) and seminal plasma (P) fractions separately.
In all 3 analyzed fractions, a-T was present in higher
concentrations compared with d-T (Table 1).
In the chromatogram shown in Figure 1, the vitamin
E pure standard is reported (Figure 1a): a-T and d-T
were identified as 2 separate peaks, whereas b-T and c-T
coeluted, giving a single peak in an intermediate position
between those of a-T and d-T; in Figure 1b achromatogram of the whole human semen sample was
shown: the peaks related to a-T and d-T were evident,
whereas the peak of c (+b)-T was not revealed;
moreover, no tocotrienols were detected.
The S/P ratio was calculated in order to identify the
different distributions of a-T and d-T in the 2 fractions.
Individuals in group 1 showed one or more altered
semen parameters among sperm concentration, motility,and morphology, whereas those in group 2 had normal
semen parameters.
Comparing the considered variables between the 2
groups, the percentage of progressive motility (a+b), the
percentage of normal morphology, the a-T and d-T S/P
ratios, and the percentage of eosin Y negative (viable)
spermatozoa were significantly higher in group 2
compared with the values observed in group 1 (Table 1).It is important to stress that the a-T and d-T values
measured in the whole semen samples and separately in
S and in P fractions did not appear to be different in the
2 groups, even changing the unit of measurement in
nmol/108 sperm, in S fraction (a-T: group 1 12.42 [2.20–
22.65] vs group 2 22.65 [11.22–34.08]; d-T group 1 1.37
[0.23–2.49] vs group 2 2.62 [1.35–3.88]).
However, men with abnormal semen parametersgenerally showed higher levels of a-T and d-T in the P
fraction compared with those found in the S fraction; an
inverted trend was observed in men with normal semen
parameters (Figure 2). This situation is clearly shown by
the S/P ratio: for a-T 0.90 in group 1 and 1.20 in group
2; for d-T 0.86 in group 1 and 1.13 in group 2 (Table 1).
Table 2 shows the correlations among the considered
semen parameters and the T values in the 2 groups. Itappears that progressive sperm motility is positively
correlated with a-T and d-T values measured in the
whole semen (P , .01), in the S fraction (P , .05 for a-T
sperm in group 1; P , .01 for all the other variables) and
in the P fraction (P , .05 for a-T plasma in group 2; P
, .01 for all the other variables) in both groups. The
percentage of viable sperm is correlated with the a-T
and d-T S/P ratios (P , .01) in both groups.In order to verify whether plasma membrane break-
age could be the cause of a-T and d-T release in the
seminal plasma of patients with altered semen param-
eters, human spermatozoa were sonicated and then
observed by TEM. The complete release of d-T was
detected after 10 seconds of sonication, whereas a-T was
released after 60 seconds (Figure 3). This trend was
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Figure 1. (a) High-performance liquid chromatography (HPLC)chromatogram of pure standard Vitamin E. toc3 indicates tocotrie-nols; toc, tocopherols. (b) HPLC chromatogram of the whole semensample. Conditions are reported in ‘‘Materials and Methods.’’
0 Journal of Andrology N May �June 2011
concomitant with plasma membrane fragmentation
detected at the ultrastructural level (Figure 4).
Discussion
Vitamin E is a recognized exogenous, lipophilic antiox-
idant of the semen, and it plays a key role in male
reproduction by protecting sperm membrane PUFA
against oxidation by ROS (Bjørneboe et al, 1990).
The present study was aimed at investigating the
distribution of different T homologues in spermatozoa
and seminal plasma from men with normal and
abnormal semen parameters.
Strict criteria were applied for patient selection in
order to obtain 2 homogeneous groups, avoiding the
most common risk factors for male infertility, such as
varicocele, infections, altered karyotype and hormonal
imbalance, which could interfere with the present
experiment and possibly with the bioavailability of Ts.
Independently of the group of patients considered, the
major vitamin E homologue detected was a-T, the most
potent antioxidant against peroxyl and alkoxyl radicals.
In addition, for the first time we demonstrated that
human semen contains significant amounts of another
tocol-derived compound, d-T. d-T differs from a-T as it
lacks 2 methyl groups on the chromanol ring, which is
responsible for its more polar nature. Because of the
coelution of b-T and c-T we were unable to separately
quantify concentrations of b-T and c-T.
In this study, the statistical analysis confirmed that
sperm quality, except for sperm concentration, was
significantly reduced in the group with altered semen
parameters, and in particular in this group the
percentage of sperm with broken plasma membrane
was significantly increased.
The a-T and d-T values detected in whole semen were
very similar in the 2 populations analyzed. Our results
are not in agreement with those from Omu et al (1999)
and Bhardwaj et al (2000) who showed higher a-T level
in semen of men with normal semen parameters than
those with oligozoospermia, asthenozoospermia, and
azoospermia. This discrepancy could be due to the
selection of the patient population, but the low number
of analyzed patients could be a factor involved. In
addition, it could be that these studies did not consider
the fact that Ts, because of their lipophilic properties,
show affinity to plasma membrane and can display
different distributions in the cells and in the seminal
plasma.
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Figure 2. Distribution of a-tocopherol level in seminal plasma fractionin the 2 examined groups.
Table 2. Correlations (Spearman’s coefficient) between variables related to a-T and d-T and evaluated semen parameters inthe 2 analyzed groups
Group Variable Volume Sperm/mL Morphology % Viability % Motility %
1 aT whole 0.039 0.189 0.214 20.063 0.958a
aT sperm 0.075 0.182 0.295 0.275 0.643b
aT plasma 0.075 20.014 0.238 20.148 0.727a
aT S/P 20.568 0.49 20.064 0.838a 20.413
dT whole 0.046 20.091 0.068 20.007 0.769a
dT sperm 0.263 20.273 0.164 20.039 0.760a
dT plasma 0.111 20.238 0.210 20.366 0.713a
dT S/P 20.363 0.308 0.071 0.754a 20.014
2 aT whole 20.203 20.227 20.166 0.068 0.928a
aT sperm 20.381 20.113 0.013 0.46 0.666a
aT plasma 20.415 0.154 20.268 20.455 0.533b
aT S/P 20.295 20.157 0.27 0.653a 20.029
dT whole 20.017 20.121 20.251 0.014 0.795a
dT sperm 20.374 20.099 0.092 0.466 0.688a
dT plasma 20.085 0.039 0.116 20.027 0.803a
dT S/P 20.386 20.161 0.060 0.657b 20.335
Abbreviation: S/P, ratio of sperm fraction to plasma fraction.a P , .01.b P , .05.
Moretti et al N Vitamin E Distribution in Human Semen 0
Regarding the distribution of Ts in the different
considered fractions, the amounts of a-T and d-T in
sperm samples were higher in group 2 compared with
group 1; a-T and d-T S/P ratios were significantly higher
in the group with normal semen parameters compared
with those of the group with abnormal semen parameters,
suggesting a release of these tocol-derived compounds
from sperm to the surrounding aqueous medium.
To the best of our knowledge, very few studies related
to the distribution of T isoforms in human semen are
available. Lewis et al (1997) observed concentrations of
a-T ranging from 0.08 to 0.69 mmol/L in seminal plasma
of fertile and infertile normozoospermic men and
asthenozoospermic individuals, but the differences
among these groups were not significant; thus, they
did not consider the detection of a-T as an important
predictive element. They also highlighted traces of a-T
directly in sperm cells, although they did not consider
this observation to be an important finding. The first
evidence for the presence of a-T in human spermatozoa
was reported by Therond et al (1996) and the a-T
concentration in spermatozoa was related to sperm
motility, viability, and morphology.
Concerning the detection of the other vitamin E
isoforms in human semen and spermatozoa, no data are
yet available, and this is the first report related to d-T
distribution.
In the present study, the values of a-T and d-T
determined in whole semen samples, in sperm, and in
seminal plasma fractions were positively correlated with
progressive motility in both groups. These data could be
consistent with the report showing a positive effect of in
vitro a-T supplementation in human sperm motility and
viability (Verma and Kanwar, 1999).
Another pivotal point regards the observation that in
rabbits the antioxidant efficiency of a-T depends mainly
on its incorporation into the sperm membranes, whereas
the a-T plasma fraction does not seem to play a role,
confirmed by the fact that the antioxidant protection of
semen is not greatly reduced after dilution (Castellini et
al, 2007). Even in humans, Therond et al (1996) didn’t
find any correlation between a-T in seminal plasma and
conventional semen parameters, concluding that the
measurement of a-T in seminal plasma was not very
useful; in contrast, the a-T concentration in spermato-
zoa is significantly related to the morphofunctional
characteristics of sperm. This issue is deeply related to
our observation in humans, indicating that the speci-
mens from men with abnormal semen parameters were
characterized by an increased percentage of sperm with
broken plasma membranes and decreased T S/P ratios,
giving experimental support to the altered vitamin E
distribution in the 2 semen fractions.
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Figure 3. The a-tocopherol and d-tocopherol levels measured byhigh-performance liquid chromatography (HPLC) of pooled spermsamples from men with normal semen parameters sonicated for 0,10, 30, 60, and 180 seconds at maximum amplitude. The results aregiven as peak area of tocopherols. The conditions of HPLC arereported in ‘‘Materials and Methods.’’
Figure 4. Transmission electron microscopy micrograph of longitu-dinal sections of human sperm after 60 seconds of sonication. Theplasma membranes are clearly fragmented (arrows); N indicatesnucleus. Scale bar 5 1 mm.
0 Journal of Andrology N May �June 2011
This remark has led us to assume that the fragmen-
tation of plasma membrane could be responsible for the
release of a-T and d-T in the seminal plasma. In order toconfirm this hypothesis, normal semen samples were
sonicated and the a-T and d-T levels were measured in
seminal plasma at different times. About 60 seconds of
sonication are adequate to obtain a consistent release of
a-T in plasma, whereas d-T requires only 10 seconds,
but it appears to be quickly destroyed after sonication.
This observation suggests that d-T seems to be less
closely coupled with membranes than a-T, and thiscould be in agreement with the more polar nature of the
d-T isoform. TEM analysis of sonicated semen samples
confirmed that spermatozoa showed broken plasma
membranes.
In the analyzed patients with poor semen quality, the
observed high percentage of broken plasma membranes
could be an effect of the presence of ROS, known to be
elevated in patients with altered semen parameters(Pasqualotto et al, 2008). Unfortunately, ROS levels
were not measured in the semen of the study partici-
pants, making this explanation speculative.
For instance, because the a-T and d-T levels in whole
semen are found to be very similar in the 2 analyzed
groups, it seems that Ts associated with sperm
membranes are the main source of nonenzymatic
antioxidants in semen and the release of a-T and d-Tin the plasma fraction is probably an index of lower
antioxidant power and lower sperm quality as well.
Enzymatic antioxidants, such as superoxide dismutase
and glutathione peroxidase, may play a major role in
protecting sperm against ROS-induced damage, and
they would be the primary defense systems (Griveau et
al, 1995), whereas a-T would act as the last barrier to
maintain the integrity of the sperm membrane (Therondet al, 1996).
In addition to the possibility that a-T and d-T may be
released into the seminal plasma by plasma membrane
fragmentation, we cannot exclude that this transport
could occur, at least in part, as a function of lipophilic
protein concentration in seminal plasma. To this
purpose, albumin is demonstrated to be involved in
the transfer of Ts between semen fractions in rabbit
(Mourvaki et al, 2010), and variations in its concentra-tion in semen may probably affect such a T transfer. The
hypothesis that the variations in albumin concentration
might have been responsible for the different distribu-
tion of Ts between the seminal plasma and the sperm
fractions observed in group 2 with respect to group 1
cannot be ruled out, because we did not dose the
albumin concentration in the semen samples. However,
it is important to underline that the sonicationexperiment was carried out in an albumin-free medium
(PBS) suggesting that membrane breakage can be a real
phenomenon inducing the release of Ts in seminal
plasma.
Further investigations focused on the a-T and d-T
fractions present at the plasma membrane level will be
necessary in order to understand the exact function of
this lipophilic compound and in order to examine the
behavior of these fractions during vitamin E supple-mentation.
Thus, in the near future it could be interesting to
study whether dietary vitamin E supplementation could
be useful for adjusting vitamin E distribution in the
semen fractions of individuals showing altered semen
parameters.
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0 Journal of Andrology N May �June 2011
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Journal: Journal of AndrologyPaper: andr-32-03-07Title: Distribution of a- and d-Tocopherols in Seminal Plasma and Sperm Fractions of Men With Normaland Abnormal Semen Parameters
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