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From the * University of Pennsylvania, Center for
Research on Reproduction and Women's Health, Philadelphia, Pennsylvania; the
Departamento de Química
Biológica, IIMHNO (UBA) and IQUIFIB, (CONICET-UBA), Facultad de
Farmacia y Bioquímica, Universidad de Buenos Aires, Buenos Aires,
Argentina; the Laboratorio de Estudios en
Reproducción (LER), Buenos Aires, Argentina; and
CONRAD, Department of Obstetrics and Gynecology,
The Jones Institute for Reproductive Medicine, Eastern Virginia Medical
School, Norfolk, Virginia.
| Correspondence to: Dr Gustavo F. Doncel, CONRAD Program, Department of OB/GYN, Eastern Virginia Medical School, 601 Colley Avenue, Norfolk, VA 23507 (e-mail: doncelgf{at}evms.edu). |
| Received for publication August 10, 2008; accepted for publication March 3, 2009. |
Poor-quality sperm show reduced capacity to undergo capacitation-induced
protein tyrosine phosphorylation and hyperactivation. Given that these
deficiencies can be overcome by membrane-permeant stimulators of the
cAMP-dependent kinase system, we hypothesize that the main defect underlying
these deficiencies resides on the sperm plasma membrane. Spermatozoa from
semen samples obtained from 15 consenting healthy donors were separated in 2
subpopulations, L45 (first interface) and L90 (pellet), using a 45:65:90
ISolate gradient centrifugation method. These sperm fractions were studied
before and after a 6-hour capacitating incubation for sperm motion parameters
(computer-assisted analysis), including hyperactivation, protein tyrosine
phosphorylation (immunofluorescence), membrane fluidity (Laurdan
fluorescence), and sterol and phospholipid content (high-performance
thin-layer chromatography). In summary, data indicate that L45 (poor-motility)
spermatozoa present an excess of cholesterol and desmosterol, which impairs
the normal increase in membrane fluidity during capacitation and its
consequent activation of protein tyrosine phosphorylation and hypermotility.
Therefore, a defect in membrane composition and dynamics is underlying human
sperm biochemical and functional deficiencies related to inadequate
capacitation.
Key words: Capacitation, hyperactivation, plasma membrane
From a functional point of view, human spermatozoa are notoriously heterogeneous. With Percoll/ISolate gradient centrifugation, they can be separated in distinct subpopulations showing different functional qualities (Ollero et al, 2000; Calamera et al, 2003; Buffone et al, 2004). The upper portion, the one isolated from the interface of the 45% and 90% Percoll layers, has been demonstrated to produce more reactive oxygen species and have less motility and more DNA damage than the cells under the 90% layer. This subpopulation, so-called pellet sperm, on the contrary, displays the best functional quality and is the one used for assisted reproductive technologies (Mortimer and Mortimer, 1992).
We previously demonstrated that sperm isolated from the 45:65 interface (poor-quality sperm) show an inability to improve motion parameters and develop hyperactivation under capacitating conditions, which is associated with an incapacity to increase protein tyrosine phosphorylation. This deficiency, however, does not appear to be at the kinase level because stimulation with permeable cAMP overcomes the impairment (Buffone et al, 2004). We therefore postulate that the main defect resides on the plasma membrane, a structure that undergoes multiple changes during capacitation and is critically involved in the acquisition of sperm-fertilizing ability.
In this investigation, we examine the sterol and phospholipid (PL) content of human sperm subpopulations and their impact on membrane fluidity, hyperactivation, and protein tyrosine phosphorylation before and after a capacitating incubation.
Materials and Methods
Preparation of Spermatozoa
Semen samples were obtained from 15 consented healthy donors by
masturbation after 3 to 5 days of sexual abstinence. The protocol was approved
by the LER Institutional Review Board. Spermatozoa from individual ejaculates
were not pooled. All samples had normal semen parameters, as evaluated from a
sperm concentration >40 x 106 spermatozoa/mL, percentage
of progressive cells 
50%, percentage of viable spermatozoa >80%, and
percentage of normal forms 14% as assessed by Kruger strict criteria
(Kruger et al, 1986).
Samples were allowed to liquefy for 1 hour at room temperature, and sperm concentration and motility were assessed using a computer-assisted semen analysis (IVOS V10.8s; Hamilton Thorne Research, Danvers, Massachusetts). Sperm viability was assessed in the original semen samples as well as in the isolated fractions at all subsequent experimental conditions by light microscopy using the Eosin Y assay (World Health Organization, 1999).
Sperm Fractionation and Incubation
Aliquots of semen (1 mL) were loaded onto a 45%, 65%, and 90% discontinuous
ISolate (Irvine Scientific, Santa Ana, California) gradient. Density gradients
were performed by layering 1 mL of each ISsolate concentration into a 15-mL
conical tube and centrifuging for 20 minutes at 400 x g. The
resulting interfaces between the layers of 45% and 65% (L45) and the pellet of
the 90% layer (L90) were aspirated and transferred to separate tubes. Sperm
suspensions were then diluted with Ham F10 medium containing 3 mg/mL bovine
serum albumin (Ham/BSA) and centrifuged twice for 10 minutes at 400 x
g. An aliquot of each interface was used to assay sperm
concentration, motility, and morphology. Washed spermatozoa were resuspended
in 1 mL of Ham/BSA at a concentration adjusted to 1 x 107
spermatozoa/mL. Cells were used either immediately after washing (T0) or after
incubation for 6 hours (T6) at 37°C in a 5% CO2 atmosphere.
Some experiments were performed incubating spermatozoa in the presence of
cholesterol sulfate (CHO-SO4)–saturated BSA (20 µM)
(Sigma, St Louis, Missouri). In this case, the BSA was preincubated with the
CHO-SO4 for 30 minutes prior to its addition to the sperm
suspension. Because of the need to perform the functional assays with as many
cells as possible, we did not measure CHO content after incubation with
CHO-SO4–saturated BSA. Instead, we assumed effective loading
of the sterol based on previously reported results by Cross
(2003). As a control, we
evaluated the addition of CHO-saturated BSA to the T0 cells. We did not find
any difference in the values with or without CHO (data not shown). At each
time point, cells were assessed for motility, hyperactivation, sterol content,
protein tyrosine phosphorylation, and membrane fluidity. Because of the number
of spermatozoa of each sample, not all parameters were assessed in all
samples. The sample size used for each study is stated in its corresponding
table or figure.
Measurement of Sterol and PL Content
The CHO and desmosterol content of sperm fractions was analyzed by micro
high-performance thin-layer chromatography (HPTLC) as described
(Alvarez and Storey, 1995).
Following extraction, the lower phases were evaporated to dryness and
resuspended in 50 µL of chloroform/methanol (1:1 vol/vol). Aliquots of 4
µL of the different samples and of CHO and desmosterol standards at a
concentration of 0.1 mg/mL were applied to AgNO3-impregnated HP-K
plates, developed in chloroform/acetone (95:5 vol/vol), and stained with a
CuSO4 reagent. The resulting bands were scanned at 400 nm in the
reflectance mode using a Shimadzu CS-9000U spectro-densitometer (Shimadzu
Scientific Inc, Columbia, Maryland).
Sperm Motility Parameters and Hyperactivation
Aliquots of each sperm suspension were loaded into 37°C prewarmed,
20-µm deep disposable chambers (Microcell; Conception Technologies, San
Diego, California). Computer-assisted sperm motion analysis was performed
using the Hamilton-Thorne digital image analyzer (HTR-IVOS v 10.8s; Hamilton
Thorne Research). At least 300 spermatozoa and 5 fields were assessed.
Eight motion parameters were assessed in this study: 1) motility (%), 2) average path velocity (VAP, µm·s–1), 3) track speed or curvilinear velocity (VCL, µm·s–1), 4) progressive or straight-line velocity (µm·s–1), 5) straightness (%), 6) beat cross frequency (Hz), 7) linearity (LIN, %), and 8) lateral head amplitude (ALH, µm). The following settings were used during the analysis: frames acquired, 30; frame rate, 60 Hz; minimum contrast, 85; minimum cell size, 4 pixels; straightness threshold, 80%; low VAP cut-off, 5 µm·s–1; medium VAP cut-off, 25 µm·s–1; head size—nonmotile, 12 pixels; head intensity—nonmotile, 130 AU; static head size, 0.68 to 2.57 pixels; static head intensity, 0.31 to 1.21 AU; and static elongation, 23 to 100%. The playback function was used to accurately identify motile cells. Hyperactivated motility (HA, %) was defined as motility with starspin or high-amplitude thrashing patterns and short trajectory distances (Burkman, 1984). This percentage represents the portion of motile spermatozoa displaying HA movement. The criterion for detecting HA spermatozoa was VCL >150 µm·s–1, ALH >7.0 µm, and LIN <50% (Mortimer and Mortimer, 1992).
Protein Tyrosine Phosphorylation: Indirect Immunofluorescence
Immunofluorescence was used to examine the subcellular localization of
proteins phosphorylated in tyrosine residues, as well as the incidence of this
process in the sperm subpopulation. Immunolabeling was performed in the
absence of phosphatase inhibitors, which could preclude biologically relevant
differences between the assessed sperm subsets. Spermatozoa from the different
ISolate fractions were incubated under capacitating conditions for 6 hours and
washed twice with phosphate-buffered saline (PBS). Sperm concentration was
adjusted to 5 x 106 cells/mL. An aliquot of 15 µL of the
sperm suspension was spotted onto 8-well glass slides, air-dried, fixed, and
permeabilized with methanol for 30 minutes at room temperature. Slides were
next incubated for 1.5 hours at room temperature in a humidified chamber in
the presence of anti-phosphotyrosine antibody PY20 (50 µg/mL in PBS/0.1%
BSA) (ICN Biomedicals Inc, Aurora, Ohio). After 2 washings with PBS, slides
were incubated for 30 minutes at room temperature in a humidified chamber with
50 µg/mL goat anti-mouse IgG fluorescein isothiocyanate conjugate (ICN
Biomedicals). After incubation, slides were washed with PBS 3 times,
air-dried, and mounted with Antifade (Molecular Probes, Eugene, Oregon).
Spermatozoa were examined using a fluorescence microscope (BX40F; Olympus
America, Mellville, New York). At least 200 cells were counted in different
fields, and the percentage of spermatozoa showing fluorescence in their tails
was calculated. Negative controls were performed by blocking PY20 with
ortho-D, L phosphotyrosine (Sigma).
Membrane Fluidity
Sperm membrane fluidity was evaluated using the fluorescent probe
6-dodecanoyl-2-dimethylaminonaphthalene (Laurdan; Molecular Probes). Laurdan
is a probe that spontaneously incorporates into membranes at the glycerol
backbone of PLs, distributing itself evenly among the different lipid domains
(Parasassi and Gratton, 1995).
Laurdan responds to variations in the number of water molecules accessible to
the probe, which in turn depends on both lipid packing and CHO content. This
alteration in the polarity of Laurdan's microenvironment is visualized as an
alteration in its fluorescence excitation and emission spectra. Spermatozoa
were mixed with 0.2 µM Laurdan and incubated for 15 minutes at 37°C to
allow the incorporation of the probe into the plasma membrane. After
incubation, membrane fluidity was evaluated by changes in Laurdan generalized
polarization (GP) calculated as:
|
|
excitation: 350 nm)
(Parasassi et al, 1995),
measured at 37°C in a Kontron SFM-25 spectrofluorometer with temperature
control (Kontron Instruments SpA, Milan, Italy). The lower the GP ratio, the
higher the fluidity of the membrane.
Statistical Analysis
Results are expressed as means ± SEM. Statistical differences
between 2 groups were evaluated by unpaired Student's t tests.
Comparisons of more than 2 groups of sperm fractions were evaluated by 1-way
analysis of variance (ANOVA) followed by Bonferroni's multiple comparison
test. All tests were 2-tailed with a statistical significance assessed at
P < .05. Statistical analyses were performed using GraphPad Prism
4.0 (GraphPad Software, San Diego, California).
Results
As previously reported, spermatozoa recovered from the 45:65 ISolate interface (L45) showed significantly less motility and poorer sperm movement than those collected from the pellet (L90) (Table 1).
|
Testing our hypothesis that sperm phosphorylation and functional deficiencies are linked to membrane lipid alterations, we first analyzed the membrane lipid profile of spermatozoa in both subsets (L45 and L90). Prior to incubation under capacitating conditions (T0), L90 cells contained significantly lower amounts of CHO and desmosterol than L45 cells, displaying a lower CHO to PL ratio (Table 2). Upon incubation under capacitating conditions (T6), both L45 and L90 subsets decreased their content of CHO and desmosterol. However, the decrease in CHO in L90 sperm was higher in magnitude (42%) than that of L45 spermatozoa (25%). On the other hand, the decrease in desmosterol content was similar in both cell fractions, and no changes were observed in PL level (Table 2). As a result, although not statistically significant, after 6 hours under capacitating conditions, L90 cells decreased their CHO to PL ratio by 31%, whereas this change was only 14% in L45 cells (Table 2).
|
To assess if the membrane CHO excess found in L45 spermatozoa translated into a membrane dynamic impairment, we compared the fluidity of the sperm membrane in both subpopulations, using changes in Laurdan fluorescence emission. L45 sperm had less fluid membranes than L90 sperm from the beginning of the incubation and regardless of the time point of study (Figure 1). Unlike their L90 counterpart, L45 sperm were unable to increase membrane fluidity with capacitation. In the presence of CHO-SO4 in the medium, L45 sperm membrane fluidity also remained unchanged. Conversely, the presence of CHO in the incubation medium abolished the capacitation-associated increase in membrane fluidity in spermatozoa from the L90 subset (Figure 1).
|
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In a similar fashion, addition of CHO-saturated BSA to the medium significantly reduced the number of L90 spermatozoa undergoing tyrosine phosphorylation (Figure 3). Excess CHO in the medium did not change the percentage of tyrosine phosphorylated sperm in the L45 subset in a statistically significant manner.
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Human spermatozoa recovered from the upper layers of a Percoll gradient have been shown to be of poorer quality than those recovered from the bottom of the gradient (pellet) (Ollero et al, 2000; Gil-Guzman et al, 2001; Calamera et al, 2003). These spermatozoa show a variety of functional deficiencies. The cause(s) for such deficiencies, however, has not been fully established.
We previously reported impairment in tyrosine phosphorylation as a potential mechanism underlying the functional deficiencies observed in Percoll-separated human sperm subpopulations (Buffone et al, 2004). Treating poor-quality sperm recovered from the upper layers of a Percoll gradient (L45) with activators of the cAMP-dependent kinase (PKA) pathway (dbcAMP + pentoxifylline), we were able to overcome both their functional and protein tyrosine phosphorylation deficiencies. Given that the membrane-permeant activators bypassed the sperm plasma membrane and directly acted on intracellular molecules, these findings indicated that the main kinase systems implicated in capacitation-associated sperm protein tyrosine phosphorylation were intact. Similar results were obtained when asthenozoospermic samples showing deficient tyrosine phosphorylation were treated with PKA activators (Buffone et al, 2005).
The defect, therefore, should be upstream from the kinases. In a hypothetic model for regulation of sperm protein tyrosine phosphorylation proposed by Visconti and Kopf (1998), the earliest capacitation-associated events occur at the level of the sperm plasma membrane. Efflux of CHO increases membrane fluidity, allowing for conformational changes and relocation of receptors and channels of which activation stimulates the intracellular kinase system. CHO acceptors like serum albumin, β-cyclodextrins, and high-density lipoproteins have been implicated in these changes in several species (Osheroff et al, 1999, Visconti et al, 1999; Flesch and Gadella, 2000).
Considering our previous data on human sperm subpopulations and the information existing in the literature, we hypothesized that the defect leading to tyrosine phosphorylation and functional deficiency in poor-quality human sperm resided on the sperm plasma membrane. We further speculated that such inability to increase membrane fluidity was due to an excess of membrane CHO. In this work, measurement of sterols by HPTLC showed 2.4- and 6.6-fold more CHO and desmosterol, respectively, in L45 sperm than in L90 spermatozoa. The CHO/PL ratio was also higher (0.69 vs 0.35) for L45 sperm, a fact that remained unchanged after a 6-hour capacitating incubation (0.59 vs 0.24).
Using the variation in the generalized polarization of the fluorescent hydrophobic probe Laurdan, we then determined the lipid order of sperm membranes in both L45 and L90 subpopulations. Results clearly showed that the membranes of L45 (poor-quality) sperm were less fluid (ie, higher lipid packing and rigidity) than those of L90 (good-quality) spermatozoa. Furthermore, unlike L90 sperm, spermatozoa from L45 subpopulations could not increase membrane fluidity during the 6-hour capacitating incubation. This deficiency was associated with an impairment in protein tyrosine phosphorylation and an incapacity to develop hyperactivation. An association between inability to increase membrane fluidity during capacitation and tyrosine phosphorylation and functional deficiencies was also observed in pathologic semen samples of patients with asthenozoospermia and varicocele (Buffone et al, 2005; Buffone et al, 2006). Thus, a common theme connecting defects in membrane dynamics and sperm functional quality is evident.
Because sperm have an intrinsically high membrane CHO content, addition of CHO-SO4 to the incubation medium, which prevents the efflux of sterols from the plasma membrane (Cross, 2003), did not change the membrane fluidity or the functionality of L45 sperm. Conversely, incubated in the presence of CHO-saturated BSA, L90 sperm displayed decreased ability to modulate their membrane fluidity and a concomitant reduction in protein tyrosine phosphorylation and hyperactivation.
Loss of sperm sterols, CHO and desmosterol, is an obligatory step in human sperm capacitation. Removal of these sterols likely accounts for the change in membrane fluidity observed during capacitation and the consequent decrease in the membrane CHO/PL ratio (Travis and Kopf, 2002). Steady-state fluorescence anisotropy of the membrane probe diphenyl hexatriene decreases during capacitation in a sterol-loss–dependent manner, suggesting a diminution of sterol-mediated PL ordering (Cross, 2003). Another fluorescent probe, merocyanine 540, showed that increased membrane lipid disorder in macaque spermatozoa was associated with caffeine and cAMP-stimulated capacitation (Baumber and Meyers, 2006).
CHO efflux during capacitation has been linked to both increased membrane fluidity and signal transduction. Sperm incubated with β-cyclodextrins, potent CHO-binding heptasaccharides, initiate transmembrane signaling, leading to increased tyrosine phosphorylation, capacitation, and acquisition of fertilizing ability (Osheroff et al, 1999; Visconti et al, 1999). It has been proposed that CHO efflux from membrane subdomains called lipid rafts might initiate transmembrane signaling by allowing previously partitioned integral membrane proteins to interact with one another (Travis and Kopf, 2002). Another group, however, suggested that the preferential loss of CHO from the nonraft pool may be the stimulus that promotes raft clustering and activates signal transduction (Shadan et al, 2004). Regardless of the precise place in the membrane from where CHO is coming, it is clear that high concentrations of CHO can inhibit capacitation by indirectly diminishing the conformational freedom and hence the biologic activity of sperm surface proteins. Alternatively, CHO might directly affect specific membrane proteins (eg, adenylyl cyclase) that function in transmembrane signaling (Travis and Kopf, 2002).
Flesch et al (2001) demonstrated that bicarbonate-stimulated PL scrambling induces CHO redistribution and enables CHO depletion from the plasma membrane of porcine sperm. Interestingly, the authors noted that only 1 subpopulation of spermatozoa responded to bicarbonate-induced capacitation with changes in lipid architecture and CHO loss. They concluded that the subpopulation differences were caused by variable efficiencies in epididymal maturation as judged by cell morphology. Studying ejaculates from 10 different boars, they found a straight-line relationship between the percentage of cells that showed bicarbonate-induced PL scrambling and CHO loss and the percentage of cells with no cytoplasmic droplet.
Studying human sperm separated by a discontinuous density gradient in 4 fractions, as well as mouse testicular and epididymal sperm, Ollero et al (2000) demonstrated a net loss of docosahexaenoic acid, the major polyunsaturated fatty acid in human spermatozoa, saturated fatty acids, CHO, and desmosterol during the process of sperm maturation. Fraction 1, containing the highest proportion of immature and defective spermatozoa, showed the highest content of CHO and saturated fatty acid mass. Similar results were observed when sperm from pathologic semen samples were fractionated using an ISolate gradient (Ollero et al, 2001).
It is plausible, therefore, that the observed low membrane fluidity as well as the high CHO/desmosterol content of L45 sperm may be due to insufficient loss of sterols during the process of epididymal maturation. Because the length of sperm capacitation in different species has been directly related to their membrane CHO content (Yanagimachi, 1994), it would be possible to hypothesize that L45 spermatozoa might suffer only a temporary, time-dependent inability to undergo capacitation-induced changes. If true, it would be in agreement with observations by Benoff (1993), who showed that prolonged incubation and further loss of CHO of sperm from infertile patients who had failed in vitro fertilization correlated with increased expression of sperm surface receptors and successful fertilization. However, as previously demonstrated by our group (Buffone et al, 2004), L45 spermatozoa are unable to achieve normal levels of protein tyrosine phosphorylation and hyperactivation even after 18 hours of incubation under capacitating conditions. It is highly likely, therefore, that poor-quality sperm such as those isolated from the lower-density Percoll layers (L45) may possess more than one defect that impairs their ability to capacitate adequately. At the membrane level, for instance, these spermatozoa are known to suffer more lipid peroxidation (Ollero et al, 2000; Aitken et al, 2007). This susceptibility could be explained, in part, by their membrane composition, which is responsible for the reported higher oxidation coefficient of these cells (Calamera et al, 2003). Because the generation of membrane lipid hydroperoxides has been associated with membrane fluidity reduction, peroxidative damage could be another cause for the membrane-associated functional deficiencies in this population of spermatozoa (Aitken et al, 1993, 1994; Windsor et al, 1993). Furthermore, increased oxidative damage may also be responsible for altered ion fluxes and membrane hyperpolarization by affecting membrane transporters, cotransporters, and enzymes linked to signal transduction and capacitation (Wertheimer et al, 2008). In this article, however, we focused on the impact of excess sterols in the membranes of poor-quality spermatozoa isolated from normozoospermic samples and the biochemical and functional consequences of such defects.
Our data, together with previous findings reported in the literature, suggest that functional deficiencies in capacitation-induced protein tyrosine phosphorylation and hyperactivation may be linked to defects in membrane lipid composition and dynamics owing to defective sperm maturation. Membrane defects entailing excess sterols and decreased fluidity would therefore be a causative factor underlying poor functional quality in human spermatozoa.
Acknowledgments
The authors wish to thank Bayard Storey for critically reviewing this manuscript prior to submission. The authors also wish to thank CONRAD (USAID) for supporting Dr Doncel's work.
Footnotes
Supported by Laboratorio de Estudios en Reproducción (internal research funds to J.C.C.).
The views of the authors do not necessarily reflect those of CONRAD or USAID.
References
Aitken RJ, Harkiss D, Buckingham DW. Analysis of lipid peroxidation mechanisms in human spermatozoa. Mol Reprod Dev. 1993; 35: 302 –315.[CrossRef][Medline]
Aitken RJ, Krausz C, Buckinham D. Relationships between biochemical markers for residual sperm cytoplasm, reactive oxygen species generation, and the presence of leukocytes and precursor germ cells in human sperm suspensions. Mol Reprod Dev. 1994; 39: 268 –279.[CrossRef][Medline]
Aitken RJ, Wingate JK, De Iuliis GN, McLaughlin EA. Analysis of
lipid peroxidation in human spermatozoa using BODIPY C11. Mol Hum
Reprod. 2007;13: 203
–211.
Alvarez JG, Storey BT. Differential incorporation of fatty acids into and peroxidative loss of fatty acids from phospholipids of human spermatozoa. Mol Reprod Dev. 1995; 42: 334 –346.[CrossRef][Medline]
Baumber J, Meyers SA. Changes in membrane lipid order with
capacitation in rhesus macaque (Macaca mulatta) spermatozoa.
J Androl. 2006;27: 578
–587.
Benoff S. Preliminaries to fertilization. The role of cholesterol
during capacitation of human spermatozoa. Hum Reprod. 1993; 8: 2001
–2006.
Buffone MG, Brugo-Olmedo S, Calamera JC, Verstraeten SV, Urrutia F, Grippo L, Corbetta JP, Doncel GF. Decreased protein tyrosine phosphorylation and membrane fluidity in spermatozoa from infertile men with varicocele. Mol Reprod Dev. 2006; 73: 1591 –1599.[CrossRef][Medline]
Buffone MG, Calamera JC, Verstraeten SV, Doncel GF.
Capacitation-associated protein tyrosine phosphorylation and membrane fluidity
changes are impaired in the spermatozoa of asthenozoospermic patients.
Reproduction. 2005; 129: 697
–705.
Buffone MG, Doncel GF, Marin Briggiler CI, Vazquez-Levin MH,
Calamera JC. Human sperm subpopulations: relationship between functional
quality and protein tyrosine phosphorylation. Hum
Reprod. 2004;19: 139
–146.
Burkman LJ. Characterization of hyperactivated motility by human spermatozoa during capacitation: comparison of fertile and oligozoospermic sperm populations. Arch Androl. 1984; 13: 153 –165.[Medline]
Calamera J, Buffone M, Ollero M, Alvarez J, Doncel GF. Superoxide dismutase content and fatty acid composition in subsets of human spermatozoa from normozoospermic, asthenozoospermic, and polyzoospermic semen samples. Mol Reprod Dev. 2003; 66: 422 –430.[CrossRef][Medline]
Cross NL. Decrease in order of human sperm lipids during
capacitation. Biol Reprod. 2003; 69: 529
–534.
Flesch FM, Brouwers JF, Nievelstein PF, Verkleij AJ, van Golde LM,
Colenbrander B, Gadella BM. Bicarbonate stimulated phospholipid scrambling
induces cholesterol redistribution and enables cholesterol depletion in the
sperm plasma membrane. J Cell Sci. 2001; 114: 3543
–3555.
Flesch FM, Gadella BM. Dynamics of the mammalian sperm plasma membrane in the process of fertilization. Biochim Biophys Acta. 2000;1469: 197 –235.[Medline]
Gil-Guzman E, Ollero M, Lopez MC, Sharma RK, Alvarez JG, Thomas
AJJr, Agarwal A. Differential production of reactive oxygen species by subsets
of human spermatozoa at different stages of maturation. Hum
Reprod. 2001;16: 1922
–1930.
Kruger TF, Menkveld R, Stander FS, Lombard CJ, Van der Merwe JP, van Zyl JA, Smith K. Sperm morphologic features as a prognostic factor in in vitro fertilization. Fertil Steril. 1986; 46: 1118 –1123.[Medline]
Mortimer D, Mortimer ST. Methods of sperm preparation for assisted reproduction. Ann Acad Med Singapore. 1992; 21: 517 –524.[Medline]
Ollero M, Gil-Guzman E, Lopez MC, Sharma RK, Agarwal A, Larson K,
Evenson D, Thomas AJJr, Alvarez JG. Characterization of subsets of human
spermatozoa at different stages of maturation: implications in the diagnosis
and treatment of male infertility. Hum Reprod. 2001; 16: 1912
–1921.
Ollero M, Powers RD, Alvarez JG. Variation of docosahexaenoic acid content in subsets of human spermatozoa at different stages of maturation: implications for sperm lipoperoxidative damage. Mol Reprod Dev. 2000;55: 326 –334.[CrossRef][Medline]
Osheroff JE, Visconti PE, Valenzuela JP, Travis AJ, Alvarez J, Kopf
GS. Regulation of human sperm capacitation by a cholesterol efflux-stimulated
signal transduction pathway leading to protein kinase A-mediated up-regulation
of protein tyrosine phosphorylation. Mol Hum Reprod. 1999; 5: 1017
–1026.
Parasassi T, Giusti AM, Raimondi M, Gratton E. Abrupt modifications of phospholipid bilayer properties at critical cholesterol concentrations. Biophys J. 1995; 68: 1895 –1902.[Medline]
Parasassi T, Gratton E. Membrane lipid domains and dynamics as detected by Laurdan fluorescence. J Fluorescence. 1995; 5: 59 –69.[CrossRef]
Shadan S, James PS, Howes EA, Jones R. Cholesterol efflux alters
lipid raft stability and distribution during capacitation of boar spermatozoa.
Biol Reprod. 2004; 71: 253
–265.
Travis AJ, Kopf GS. The role of cholesterol efflux in regulating the fertilization potential of mammalian spermatozoa. J Clin Invest. 2002;110: 731 –736.[CrossRef][Medline]
Visconti PE, Galantino-Homer H, Ning X, Moore GD, Valenzuela JP,
Jorgez CJ, Alvarez JG, Kopf GS. Cholesterol efflux-mediated signal
transduction in mammalian sperm. Beta-cyclodextrins initiate transmembrane
signaling leading to an increase in protein tyrosine phosphorylation and
capacitation. J Biol Chem. 1999; 274: 3235
–3242.
Visconti PE, Kopf GS. Regulation of protein phosphorylation during
sperm capacitation. Biol Reprod. 1998; 59: 1
–6.
Wertheimer EV, Salicioni AM, Liu W, Trevino CL, Chavez J,
Hernandez-Gonzalez EO, Darszon A, Visconti PE. Chloride is essential for
capacitation and for the capacitation-associated increase in tyrosine
phosphorylation. J Biol Chem. 2008; 283: 35539
–35550.
Windsor DP, White IG, Selley ML, Swan MA. Effects of the lipid
peroxidation product (E)-4-hydroxy-2-nonenal on ram sperm function.
J Reprod Fertil. 1993; 99: 359
–366.
World Health Organization. Laboratory Manual for the Examination of Human Semen and Sperm-Cervical Mucus Interaction. Cambridge, United Kingdom: Cambridge University Press; 1999 .
Yanagimachi R. Fertility of mammalian spermatozoa: its development and relativity. Zygote. 1994; 2: 371 –372.[Medline]
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) and L90
(
) subsets of human
spermatozoa before (T0) and after (T6) incubation under capacitating
conditions either in the absence (T6 – CHO) or the presence (T6 + CHO)
of CHO-SO4-saturated bovine serum albumin (20 µmol/mL). Results
are shown as means ± SEM (n = 5). * indicates statistically different
in comparison with values obtained from L45 at T0 (P < .05); **,
statistically different than values obtained in the same cell subset at T0
(P < .005); CHO, cholesterol.
) and L90
(
) subsets of human
spermatozoa before (T0) and after (T6) incubation under capacitating
conditions either in the absence (T6 – CHO) or the presence (T6 + CHO)
of CHO-SO4-saturated bovine serum albumin (20 µmol/mL). Results
are shown as means ± SEM (n = 12). * indicates statistically different
than the values obtained at T0 in the same cell subset (P < .005);
**, statistically significant compared with cells from the same fraction
incubated in the absence of CHO (P < .05); CHO, cholesterol.
) and L90
(
) subsets of human
spermatozoa before (T0) and after (T6) incubation under capacitating
conditions either in the absence (T6 – CHO) or the presence (T6 + CHO)
of CHO-SO4-saturated bovine serum albumin (20 µmol/mL) and
evaluated by immunofluorescence. Results are shown as means ± SEM (n =
12). * indicates statistically different than values of cells from the same
subset at T0 (P < .005); **, statistically significant compared
with cells from the same subfraction incubated in the absence of CHO
(P < .05); CHO, cholesterol.