Biol Reprod
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Marín-Briggiler, C. I.
Right arrow Articles by Miranda, P. V.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Marín-Briggiler, C. I.
Right arrow Articles by Miranda, P. V.
Agricola
Right arrow Articles by Marín-Briggiler, C. I.
Right arrow Articles by Miranda, P. V.
Biology of Reproduction 61, 673-680 (1999)
©Copyright 1999 Society for the Study of Reproduction, Inc.

Articles

Strontium Supports Human Sperm Capacitation but Not Follicular Fluid-Induced Acrosome Reaction1

Clara I. Marín-Briggilera, Mónica H. Vazquez-Levina, Fernanda Gonzalez-Echeverríab, Jorge A. Blaquierb, Jorge G. Tezóna, and Patricia V. Mirandaa

a Instituto de Biología y Medicina Experimental, (1428) Buenos Aires, Argentina Fertilab, b (1116) Buenos Aires, Argentina


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The ability of strontium (Sr2+) to replace calcium (Ca2+) in maintaining human sperm function has still not been completely characterized. In the present study, acrosome reaction (AR) inducibility in response to human follicular fluid (hFF) was compared in spermatozoa incubated in either Ca2+- or Sr2+-containing media. Other events related to sperm capacitation, such as protein tyrosine phosphorylation and hyperactivation as well as zona pellucida (ZP) recognition under both conditions, were also analyzed. Spermatozoa incubated overnight in the presence of Sr2+ were unable to undergo the AR when exposed to hFF. Nevertheless, when spermatozoa were incubated under this condition and then transferred to medium with Ca2+, sperm response to hFF was similar to that of cells incubated throughout in the presence of Ca2+. The sperm protein tyrosine phosphorylation patterns and the percentages of sperm motility and hyperactivation were similar after incubation in Ca2+- or Sr2+-containing media. Under both conditions, the same binding capacity to homologous ZP was observed. Similar results were obtained when EGTA was added in order to chelate traces of Ca2+ present in Sr2+ medium. From these results, it can be concluded that Sr2+ can replace Ca2+ in supporting capacitation-related events and ZP binding, but not hFF-induced AR of human spermatozoa.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Mammalian spermatozoa are not able to fertilize an oocyte immediately after ejaculation. In vivo, spermatozoa are deposited in the female reproductive tract where they undergo several metabolic and structural changes, becoming fertilization-competent. These events, whose nature is still not completely elucidated, are known as sperm capacitation. Modifications in sperm plasma membrane composition and fluidity, changes in intracellular ion concentrations, an increase in protein tyrosine phosphorylation, and alterations in the oxidative metabolism have been associated with capacitation. As a result of this process, spermatozoa are able to undergo acrosomal exocytosis or acrosome reaction (AR) in response to physiological stimulants such as zona pellucida (ZP) or follicular fluid components. In addition, they develop a distinct pattern of motility known as hyperactivation (for review see [1–4]).

Capacitation and AR can be achieved in vitro by incubating spermatozoa under defined conditions. Balanced salt solutions supplemented with appropriate concentrations of electrolytes, metabolic energy sources, and serum albumin are fundamental to complete such complex processes.

Regarding ionic requirements, it is widely accepted that the presence of calcium (Ca2+) is essential for the induction of the AR by physiological stimuli. An influx of this cation is one of the first events in the signal transduction cascade leading to acrosomal loss in response to follicular fluid, progesterone, or ZP [5–10]. Nevertheless, the requirement of Ca2+ for capacitation is not clear [3, 4]. There is evidence that micromolar concentrations of Ca2+ are necessary to achieve capacitation in the mouse [11]. On the other hand, millimolar concentrations of this ion seem to be required for human sperm capacitation [12, 13]. Studies done in a variety of species have also shown that an increase in intracellular concentrations of Ca2+ occurs when spermatozoa are incubated under capacitating conditions [14–18]. However, it has been reported that Ca2+ can be replaced by strontium (Sr2+) in triggering hyperactivation and spontaneous AR of guinea pig [19] and mouse spermatozoa [20–22] as well as A23187-induced AR of ram spermatozoa [23]. Additionally, Sr2+ has been shown to maintain ZP penetration and sperm-egg fusion in the mouse [22] and to support fertilization of homologous zona-free eggs by hamster spermatozoa [24]. In humans, incubation in medium in which Sr2+ replaced Ca2+ did not modify the percentage of motile and spontaneously acrosome-reacted spermatozoa [12, 25]. However, their ability to penetrate zona-free hamster oocytes was improved in comparison to spermatozoa incubated in the presence of Ca2+ [26, 27]. On the other hand, it was later reported that Ca2+ is required to develop human sperm fertilizing capacity [25]. In any case, the specific requirement of Ca2+ for human sperm capacitation, induced AR, and/or interaction with the oocyte has not been completely determined.

In the present study, the ability of Sr2+ to replace Ca2+ in supporting sperm function was evaluated. Acrosome reaction inducibility in response to human follicular fluid (hFF) was compared in spermatozoa incubated in Ca2+- or Sr2+-containing media. In addition, sperm protein tyrosine phosphorylation, sperm motility and hyperactivation, and ZP interaction under both conditions were also analyzed.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Culture Media

Modified Tyrode's medium, HSM [28], was used throughout the study. It consisted of 117.5 mM NaCl, 0.3 mM NaH2PO4, 8.6 mM KCl, 2.5 mM CaCl2, 0.49 mM MgCl2, 2 mM glucose, 19 mM sodium lactate, 25 mM NaCO3H, 0.25 mM sodium pyruvate, 50 µg/ml penicillin, and 75 µg/ml streptomycin. In the present report, this medium was named HSM Ca2+. When calcium was replaced by 2.5 mM SrCl2, resulting in similar osmolarity (319 mOsm for HSM Ca2+ and 317 mOsm for HSM Sr2+), the medium was called HSM Sr2+. Control experiments with medium prepared without the addition of either Ca2+ or Sr2+, defined as HSM (-) (308 mOsm), and with HSM Sr2+ plus 0.1 mM EGTA were also carried out.

Semen Samples and Sperm Processing

Semen samples were obtained from normospermic donors according to World Health Organization (WHO) standards [29]. After complete liquefaction, samples were split into two aliquots, diluted with HSM Ca2+ or HSM Sr2+ supplemented with 0.3% globulin and fatty acid-free BSA (Sigma Chemical Co., St. Louis, MO; cat. #A7030), and centrifuged twice at 300 x g for 10 min. Washed spermatozoa were resuspended in the corresponding medium supplemented with 2.6% BSA, and filtered through glass wool columns (Microfibre Manville, Denver, CO) to recover highly motile cells. Sperm concentration was adjusted to 1.5 x 106 cells/ml, and 2-ml aliquots were incubated at 37°C, 5% CO2 in air for different periods of time (see below). Postincubation motility was determined by observation under light microscopy (x400 magnification; Alphaphot-2 YS2; Nikon, Tokyo, Japan). Only samples with more than 75% progressive motility were included in the study. Sperm vitality was assessed using 0.5% Eosin Y in aqueous sodium chloride solution as indicated [29].

In the experiments comparing sperm capacitation in HSM (-) and HSM Sr2+ + EGTA with capacitation in the presence of either Ca2+- or Sr2+-containing media, motile spermatozoa were separated using HSM (-) supplemented with 2.6% BSA and then resuspended in the corresponding media as previously described.

Follicular Fluid Preparation

Human follicular fluid (hFF) was obtained from women undergoing assisted fertilization. Multiple follicular development was induced by sequential FSH (Metrodine; Serono, Buenos Aires, Argentina) and human menopausal gonadotropin (Pergonal; Serono) injections in patients receiving GnRH analogues as described [30]. All follicles were aspirated, and fluids were centrifuged for 15 min at 1500 x g to remove cellular debris. Aliquots from ten samples of hFF were pooled and stored at -20°C until further use. Throughout the study, three different pools of hFF were used, which proved to be equally capable of inducing a significant increase in the AR.

AR Induction

To determine the ability of Sr2+ to support human sperm capacitation and hFF-induced AR, three sets of experiments were conducted. First, aliquots of 0.75 x 106 spermatozoa, incubated for 18 h in HSM Ca2+ or HSM Sr2+, were exposed to 10% hFF (hFF-induced AR) or buffer (control of spontaneous AR) for 45 min at 37°C and 5% CO2 in air. Second, in order to further characterize the requirement of both cations on capacitation and/or AR, spermatozoa incubated overnight in either HSM Ca2+ or HSM Sr2+ were divided into two aliquots, centrifuged, resuspended in media containing Ca2+ or Sr2+, and incubated with hFF as previously described. In this case, four experimental conditions were obtained, according to the divalent cation present in the capacitation/AR medium: Sr2+/Sr2+, Sr2+/Ca2+, Ca2+/Ca2+, and Ca2+/Sr2+. Finally, a similar crossover design was followed, but with HSM Sr2+ supplemented with 0.1 mM EGTA in order to chelate traces of Ca2+.

After the induction procedure, sperm suspensions were fixed for 4 min with 2% formaldehyde in PBS, washed twice with PBS, placed on polylysine-coated slides, and air-dried. The slides were immersed for 20 sec in methanol at 4°C for cell permeabilization [31]. Acrosome reaction was evaluated by sperm staining with 50 µg/ml fluorescein isothiocyanate (FITC)-labeled Pisum sativum agglutinin (Sigma Chemical Co.) [32] in duplicate assays. Stained cells (at least two hundred cells per treatment) were scored in a microscope equipped with epifluorescence (Nikon Labophot, Tokyo, Japan). The presence of a bright staining over the acrosome indicated intact spermatozoa; those presenting staining restricted to the equatorial segment or lack of staining were considered acrosome-reacted.

Electrophoresis and Western Immunoblotting

Proteins from spermatozoa incubated under different conditions were analyzed by SDS-PAGE and Western immunoblotting. Spermatozoa were washed twice with PBS and resuspended in Laemmli sample buffer (0.025 M Tris, 0.5% SDS, 5% glycerol, pH 6.8) containing 5 mg/ml deoxyribonuclease (DNase). After a 15-min incubation at room temperature, samples were centrifuged at 6000 x g for 5 min. The supernatants were removed and heated at 100°C in the presence of 70 mM 2-ß-mercaptoethanol for 5 min. After centrifugation, the supernatants were stored at -20°C until use. Solubilized proteins (corresponding to 2 x 106 spermatozoa per lane) were separated on 7% SDS-polyacrylamide gels [33]. Prestained molecular weight standards (GIBCO BRL, Life Technologies, Inc., Gaithersburg, MD) were run in parallel. Electrophoretic transfer of proteins to nitrocellulose (Sigma Chemical Co.) was carried out according to the method of Towbin et al. [34], at 35 V, 4°C for 18 h. To block nonspecific binding sites, the membrane was first incubated with 2% dry skimmed milk in PBS-0.1% Tween 20 (blocking solution). Afterwards, nitrocellulose was incubated for 1 h with a monoclonal antiphosphotyrosine antibody (Upstate Biotechnology Inc., Lake Placid, NY; cat. #05-321) diluted 1:10 000 in blocking solution. After 4 washings with PBS-0.1% Tween 20, antimouse peroxidase-conjugated IgG (Jackson ImmunoResearch Laboratories Inc., West Grove, PA; cat, #115-035-003) 1:5000 in blocking solution was added. After 1 h, the membrane was extensively washed, and reactive bands were detected by enhanced chemiluminescence using the ECL kit (Amersham Life Science Inc., Oakville, ON, Canada) according to the manufacturer's instructions. All incubations were performed at room temperature. In some experiments, the antiphosphotyrosine antibody was blocked with 40 mM O-phosphotyrosine (Sigma Chemical Co.) for 1 h with constant rotation, before use for immunoblotting.

Computer-Assisted Sperm Analysis

Aliquots of 5 µl of spermatozoa incubated 4 h in HSM Ca2+ or HSM Sr2+ at a concentration of 20 x 106 cells/ml were placed in a Makler chamber (Sefi-Medical Instruments, Rehovot, Israel). Sperm curvilinear velocity (VCL), straight line velocity (VSL), and percentage of linearity (% LIN) were measured using a CellSoft Automatic Semen Analyzer (Series 3000; Cryo Resources Ltd., Montgomery, NY) coupled to a warmed (37°C) phase contrast microscope (Olympus Optical Co., Tokyo, Japan). The image was fed into a television monitor through a video camera (AG-6720A; Panasonic, Tokyo, Japan) mounted on the microscope. The settings used during the analysis were as follows: number of frames analyzed per second = 20, frequency = 30 Hz, threshold velocity = 10 µm/sec, minimum sampling for motility = 1 frame, minimum sampling for velocity = 7 frames. A minimum of 10 fields were videotaped, and at least 100 motile spermatozoa were analyzed per sample. Individual cells with VCL > 100 µm/sec, VSL < 35 µm/sec, and % LIN < 35 were considered hyperactivated, and spermatozoa with VCL > 100 µm/sec, VSL < 60 µm/sec, and % LIN < 60 were classified as transitional (adapted from Mortimer and Mortimer [35]).

Hemizona (HZ) Assay (HZA)

Human oocytes were obtained during egg retrieval from women superovulated as previously described [30]. Noninseminated, surplus oocytes were used with the patients' consent. Oocytes were incubated 6–8 h in HTF medium (Irvine Scientific, Santa Ana, CA) for maturation. Cumulus cells were removed by treatment with 80 IU hyaluronidase (Sigma Chemical Co.) for 20 sec and then washed with PBS. Oocytes in metaphase II were placed in a 0.1 M Tris, 1.5 M (NH4)2 SO4, 0.5% dextran, pH 7 solution at 4°C until use [36].

The day before the HZA, oocytes were washed 4 times by pipetting on drops of PBS, and placed in a Petri dish (BWR Scientific Products, Bridge Port, NJ) at 37°C for 12 h. Oocytes were immobilized with holding pipettes (Cook, Queensland, Australia) attached to a micromanipulator (Narishigue, Tokyo, Japan) coupled to an inverted microscope (Nikon, Diaphot, Tokyo, Japan) and bisected into two equal halves with a microscalpel. HZ were placed in a 100-µl drop of HSM Ca2+ or HSM Sr2+ supplemented with 3.5% human serum albumin (Sigma Chemical Co.), and 3 x 104 motile spermatozoa preincubated 6 h in the same medium were added to each drop. After a 4-h incubation, HZ were washed by repeated vigorous pipetting in drops of the corresponding medium. The number of spermatozoa tightly bound to the outer surface was counted under a x400 magnification using Hoffman interference optics (Modulation Optics Inc., Greenvale, NY) [37].

In order to analyze the importance of traces of Ca2+ present in HSM Sr2+ on sperm-ZP interaction, spermatozoa were also incubated for 4 h in HSM Sr2+ with or without 0.1 mM EGTA, and the HZA were performed in the same media.

Statistical Analysis

Data were expressed as mean ± SEM. To assume normal distribution, percentages were converted to ratios and subjected to the arc sine square root transformation. Acrosome reaction results were expressed as percentage of acrosome-reacted spermatozoa or AR inducibility (% hFF-induced AR minus % spontaneous AR), and were compared by one-way ANOVA and Student-Neuman-Keuls multiple-comparison test. The percentages of transitional and hyperactivated cells incubated in HSM Ca2+ or HSM Sr2+ as well as the numbers of spermatozoa bound in the HZA were compared by Student's t-test. Percentages of motility and vitality of spermatozoa incubated in different media for several periods of time were analyzed by two-way ANOVA and the Student-Neuman-Keuls multiple comparison test. Statistical analyses were done with an IBM-compatible computer using the GraphPad InStat program (GraphPad Software, San Diego, CA).


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Sr 2+ and hFF-Induced AR

AR inducibility by progesterone or hFF has been described as an indicator of the capacitation status of a sperm population [1, 3, 38]. The effectiveness of Sr2+ in supporting capacitation was determined by comparing sperm ability to undergo hFF-induced AR in Ca2+- and Sr2+-containing media. In spermatozoa incubated for 18 h in HSM Ca2+ and exposed to hFF in the same medium, a significant increase in the percentage of AR was observed over control values (14 ± 1% spontaneous AR vs. 36 ± 2% hFF-induced AR, p < 0.001; Fig. 1, left panel). In contrast, when spermatozoa were incubated and induced in HSM Sr2+, there was no difference between spontaneous (15 ± 1%) and hFF-induced AR (18 ± 2%) (Fig. 1, right panel). The percentages of spontaneous AR were similar in spermatozoa incubated overnight in HSM Ca2+ and in HSM Sr2+.



View larger version (13K):
[in this window]
[in a new window]
  		FIG. 1. AR in spermatozoa incubated in Ca2+- or Sr2+-containing medium. After 18 h in HSM Ca2+ (left panel) or HSM Sr2+ (right panel), spermatozoa were incubated with 10% hFF or buffer (Spt, spontaneous AR) as control. The percentage of acrosome-reacted sperm was determined by Pisum sativum staining. Results are expressed as mean ± SEM, n = 14. *p < 0.001 vs. Ca2+ Spt, Sr2+ Spt, and Sr2+ hFF values.

In order to determine whether the replacement of Ca2+ for Sr2+ affected capacitation and/or AR, spermatozoa were incubated 18 h in HSM Ca2+ or HSM Sr2+ and resuspended in each medium before AR induction. Figure 2 shows AR inducibility (% hFF-induced AR minus % spontaneous AR) under every condition. In agreement with the results shown in Figure 1, AR inducibility of spermatozoa incubated and exposed to hFF in the presence of Sr2+ (Sr2+/Sr2+) was significantly reduced in comparison with those of the Ca2+/Ca2+ group (5 ± 1% vs. 28 ± 4%, p < 0.01). Spermatozoa incubated overnight in HSM Sr2+ and exposed to hFF in Ca2+ medium (Sr2+/Ca2+) had the same AR inducibility as that found under the Ca2+/Ca2+ condition (26 ± 6% vs. 28 ± 4%). However, when spermatozoa were capacitated in HSM Ca2+ and exposed to hFF in HSM Sr2+ (Ca2+/Sr2+), a significant reduction in AR inducibility was observed in comparison to Ca2+/Ca2+ treatment (11 ± 3% vs. 28 ± 4%, p < 0.01).



View larger version (12K):
[in this window]
[in a new window]
  		FIG. 2. AR inducibility in Ca2+- vs. Sr2+-containing medium. Motile spermatozoa were incubated for 18 h in HSM Ca2+ or HSM Sr2+, centrifuged, and resuspended in Ca2+- or Sr2+-containing medium. Four experimental conditions were then obtained as indicated beneath the bars. AR was induced as described in Materials and Methods. AR inducibility was calculated as % hFF-induced AR minus % spontaneous AR. Results are expressed as mean ± SEM, n = 6. *p < 0.01 vs. Ca2+/Ca2+ and Sr2+/Ca2+. **p < 0.05 vs Ca2+/Sr2+.

Effectiveness of Sr 2+ in Supporting Sperm Protein Tyrosine Phosphorylation

Capacitation has been associated with an increase in protein tyrosine phosphorylation in spermatozoa from several species, including human [39–45]. To determine whether Sr2+ can replace Ca2+ in supporting capacitation, protein tyrosine phosphorylation in spermatozoa incubated in both media was compared. Similar low levels of tyrosine phosphorylation were observed in sperm proteins at Time 0 of incubation either in HSM Ca2+ or HSM Sr2+ (Fig. 3, lanes A and B). After 18 h under capacitating conditions, there was an enhancement in protein tyrosine phosphorylation. However, no differences were found between patterns from spermatozoa incubated in the presence of Ca2+ or Sr2+ (Fig. 3, lanes C and D).



View larger version (77K):
[in this window]
[in a new window]
  		FIG. 3. Protein tyrosine phosphorylation pattern in spermatozoa incubated in Ca2+- vs. Sr2+-containing medium. Motile spermatozoa were resuspended in HSM Ca2+ (lanes A and C) or Sr2+ (lanes B and D) and incubated for 0 (lanes A and B) and 18 h (lanes C and D). Sperm proteins patterns were analyzed by Western immunoblotting using a monoclonal antiphosphotyrosine antibody. Molecular weight standards (Mr x 10-3) are indicated on the left. A typical experiment is shown. This experiment was performed four times with similar results.

In order to study the kinetics of protein tyrosine phosphorylation in HSM Ca2+ and HSM Sr2+, spermatozoa were incubated for 1, 2, 4, and 6 h under both conditions, and the corresponding patterns of phosphotyrosine-positive bands were compared. Sperm protein tyrosine phosphorylation was similar in both media at any time studied (data not shown).

Phosphorylation was specific for tyrosine residues, since the immunoreactivity was completely abolished when the antibody was previously blocked with O-phosphotyrosine.

Sr 2+ and Sperm Motility

When spermatozoa were incubated for 2, 4, or 18 h in HSM Sr2+ or HSM Ca2+, similar percentages of motile cells were found (Table 1). However, after 18-h incubation, this parameter was significantly reduced (p < 0.05) both in Ca2+- and Sr2+-containing media.


View this table:
[in this window]
[in a new window]
  		TABLE 1. Percentage of motile spermatozoa incubated for different periods of time in HSM Ca2+ or HSM Sr2+ (mean ± SEM; n = 3).

Under capacitating conditions, spermatozoa develop a distinctive type of motility, known as hyperactivation [3]. It is characterized by an increased velocity, a decreased linearity, a wide lateral head displacement, and a whiplash movement of the flagellum. In the present study, the ability of Sr2+ to sustain the development of hyperactivity was determined. Using a computer-assisted analysis, several motion parameters of spermatozoa incubated in HSM Ca2+ or HSM Sr2+ were measured, and the proportions of cells showing nonprogressive (hyperactivated) or transitional trajectories were compared. Under both conditions, similar percentages of transitional and hyperactivated spermatozoa were obtained (Table 2).


View this table:
[in this window]
[in a new window]
  		TABLE 2. Percentage of transitional and hyperactivated spermatozoa after 4-h incubation in HSM Ca2+ or HSM Sr2+ (mean ± SEM; n = 5).a

Sr 2+ and Sperm-ZP Interaction

To examine whether human sperm binding to homologous ZP depends on Ca2+ or can also be achieved in the presence of Sr2+, spermatozoa were preincubated in HSM Ca2+ or HSM Sr2+, and HZA in both media were performed. The number of spermatozoa bound per HZ was not significantly different between these conditions (25 ± 6 in HSM Ca2+ vs. 33 ± 6 in HSM Sr2+; Fig. 4).



View larger version (14K):
[in this window]
[in a new window]
  		FIG. 4. Effectiveness of Sr2+ in supporting sperm-ZP interaction. After 6-h incubation in HSM Ca2+ or HSM Sr2+, spermatozoa were incubated 4 h with HZ placed in the corresponding medium. Spermatozoa tightly bound to the outer face of each HZ were counted. Results are expressed as mean ± SEM (n = 5 HZ). Differences were not significant.

Capacitation in Ca2+-Deficient Media

From the results presented above, it can be suggested that either Sr2+ is able to replace Ca2+ in maintaining capacitation-related events or Ca2+ is not necessary for capacitation. To explore the latter possibility, spermatozoa were incubated in medium with or without the addition of Ca2+, and several parameters were compared. Protein tyrosine phosphorylation at 1, 2, 4, and 6 h was enhanced in spermatozoa incubated in HSM (-) (not supplemented with Ca2+ or Sr2+) in comparison with those incubated in HSM Ca2+ (data not shown). When spermatozoa were incubated for more than 4 h in HSM (-), their motility was significantly reduced (p < 0.01, Table 3). Regarding sperm vitality, similar percentages were obtained in HSM (-) or HSM Ca2+, although a significant reduction in the proportion of live cells (p < 0.01) was seen after 6-h incubation under both conditions (Table 3).


View this table:
[in this window]
[in a new window]
  		TABLE 3. Percentage of motility (% Mot.) and vitality (% Vital.) of spermatozoa incubated for different periods of time in HSM Ca2+ or HSM (-) (mean ± SEM; n = 3).

Although no Ca2+ was added to HSM (-) or HSM Sr2+, the contamination of other components of the media with this cation cannot be ruled out. The actual Ca2+ concentration in HSM (-) and HSM Sr2+ supplemented with 2.6% BSA was found to be around 60 µM when assessed by atomic absorption. To determine whether these traces of Ca2+ were responsible for the maintenance of human sperm function in HSM Sr2+, spermatozoa were incubated overnight in this medium with or without the addition of 0.1 mM EGTA. A similar percentage of motile spermatozoa and the same pattern of protein tyrosine phosphorylation was obtained after 18-h incubation in the presence or absence of the Ca2+ chelator (data not shown). In addition, spermatozoa incubated under both conditions showed a similar binding capacity in the HZA (27 ± 5 vs. 31 ± 6 spermatozoa bound per HZ in HSM Sr2+ and in HSM Sr2+ EGTA, respectively; Fig. 5) and AR inducibility when exposed to hFF in the presence of Ca2+ (10 ± 3% in Sr2+/Ca2+ vs. 9 ± 3% in Sr2+ EGTA/Ca2+; Fig. 6).



View larger version (15K):
[in this window]
[in a new window]
  		FIG. 5. Sperm-ZP interaction in HSM Sr2+ with or without the addition of EGTA. After 6-h incubation in HSM Sr2+ or in HSM Sr2+ supplemented with EGTA, spermatozoa were incubated for 4 h with HZ placed in the corresponding medium. Spermatozoa tightly bound to the outer face of each HZ were counted. Results are expressed as mean ± SEM (n = 7 HZ). Differences were not significant.



View larger version (13K):
[in this window]
[in a new window]
  		FIG. 6. AR inducibility after sperm incubation in Sr2+ vs. Sr2+-plus-EGTA medium. Motile spermatozoa were incubated for 18 h in HSM Ca2+, HSM Sr2+, or HSM Sr2+ with the addition of EGTA, centrifuged, and resuspended in medium containing Ca2+, Sr2+, or Sr2+EGTA. Five experimental conditions were then obtained as indicated beneath the bars. AR inducibility was calculated as % hFF-induced AR minus % spontaneous AR. Data represent mean ± SEM, n = 5. *p < 0.001 vs. Ca2+/Ca2+.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
The completion of capacitation has been defined as the stage at which spermatozoa are able to acrosome-react in response to a physiological stimulus. In humans, follicular fluid is one of the identified inducers, and its ability to elicit the AR is displayed only on spermatozoa that have undergone changes associated with capacitation [38]. Additionally, it has been demonstrated that sperm capacity to undergo the AR when exposed to hFF is indicative of its fertilizing competence [46].

The ability of Sr2+ to replace Ca2+ in supporting human sperm function has been previously analyzed, although it has yet not been completely determined. In the present study, AR inducibility in response to follicular fluid was used to compare the effectiveness of both divalent cations in maintaining capacitation and AR.

Our results show that spermatozoa that had been incubated for 18 h in HSM Sr2+ were unable to increase the AR rate when exposed to hFF in Sr2+-containing medium. From this evidence, it could be inferred that Sr2+ could not support capacitation and/or AR. To distinguish between these possibilities, cells were incubated in HSM Sr2+ overnight and then in HSM Ca2+ at the time of AR induction. In this case, spermatozoa responded to hFF, suggesting that after overnight incubation in HSM Sr2+, they were capacitated since they were able to undergo the AR if Ca2+ was added during the induction process. It was also possible that traces of Ca2+ contained by HSM Sr2+ were responsible for sperm capacitation. In order to study this point, spermatozoa were incubated overnight in HSM Sr2+ with the addition of EGTA, and induced in the presence of Ca2+. Acrosome reaction inducibility was similar in cells incubated in medium with or without EGTA supplementation. This indicates that Sr2+, and not the traces of Ca2+ present in HSM Sr2+, maintained sperm capacitation in this medium. However, it cannot be ruled out that Sr2+ might have initiated the capacitation process, which could later be completed by Ca2+.

To study the ability of Sr2+ to support induced AR, spermatozoa that were capacitated in HSM Ca2+ were stimulated with hFF in Sr2+-containing medium. Under this condition, spermatozoa did not show significant levels of AR, indicating that Sr2+ was unable to maintain the events leading to acrosome exocytosis in response to hFF. Nevertheless, these spermatozoa had a higher AR inducibility compared with those incubated and induced in the presence of Sr2+. This could be attributed to the accumulation of intracellular Ca2+ during overnight incubation in HSM Ca2+.

Regarding spontaneous AR, similar percentages of acrosomal loss were found after 18-h incubation in HSM Sr2+ or Ca2+. These results are in agreement with previous reports in guinea pigs [19], mice [20–22], and humans [25]. However, an ultrastructural analysis revealed that human spermatozoa incubated in Sr2+ medium showed earlier AR stages than those incubated in the presence of Ca2+, suggesting that Sr2+ is less efficient in promoting complete exocytosis of the acrosomal content [12].

Although the molecular mechanisms underlying sperm capacitation have not yet been completely elucidated, it has been established that an increase in protein tyrosine phosphorylation accompanies this process. In mouse spermatozoa, this event has been reported to be Ca2+-dependent [40]. In contrast, it has been demonstrated that in humans, Ca2+ negatively modulates not only sperm protein tyrosine phosphorylation but also tyrosine kinase activity [47, 48]. In our study, sperm incubation in HSM Ca2+ and HSM Sr2+ (either in the presence or in the absence of EGTA) produced the same protein tyrosine phosphorylation pattern at any time point analyzed. When spermatozoa were incubated in medium deficient in both cations, an enhanced pattern of tyrosine phosphorylation was obtained, in agreement with previous evidence [41, 47, 48].

It is worth noting that although spermatozoa incubated for 18 h in HSM Ca2+ and HSM Sr2+ showed a similar pattern of protein tyrosine phosphorylation, the induction of AR in response to hFF was different under these conditions. This indicates differences in ionic requirements between these processes and suggests that Ca2+-mediated events necessary for hFF-induced AR are downstream of protein tyrosine phosphorylation observed during capacitation.

In the present study, after incubation in HSM Sr2+ (with or without EGTA) and HSM Ca2+, spermatozoa showed similar motility, in accordance with previous reports [26, 27]. Moreover, the analysis of motility parameters revealed the same proportions of transitional and hyperactivated spermatozoa in Ca2+- and Sr2+-containing media; similar results were reported for the guinea pig [19] and mouse [20–22]. Nevertheless, under the conditions used in this study, when spermatozoa were incubated for more than 4 h in HSM (-) not supplemented with any of these divalent cations, a severe decline in motility was observed. For this reason, experiments involving overnight incubation in HSM (-) could not be carried out. Taken together, these results and those of sperm protein tyrosine phosphorylation lead to the conclusion that Sr2+ effectively replaces Ca2+ in the intracellular mechanisms involved in these events. It is worth mentioning that other divalent cations, such as Ba2+ and Mg2+, were unable to maintain sperm function [12, 19, 22, 23, 49], results that stress the specificity of Sr2+ in replacing Ca2+.

Several studies have dealt with the extracellular Ca2+ requirement for capacitation in mouse and human spermatozoa [11–13]. It has been reported that Ca2+ exerts its action by stimulating the adenylate cyclase, with a subsequent increase in cAMP concentrations [50–53]. Elevated cAMP would, in turn, modulate protein kinase A (PKA) activity, which has been reported to be involved in sperm motility [54–56] and protein tyrosine phosphorylation in a variety of species [42, 44, 57, 58]. Given that Sr2+ supports human sperm motility and tyrosine phosphorylation in a manner similar to that of Ca2+, it could be inferred that Sr2+ would be able to activate adenylate cyclase or PKA, but this possibility remains to be proven.

Concerning human sperm-oocyte interaction, a similar number of spermatozoa bound to homologous ZP when the assay was carried out in media containing Ca2+, Sr2+, or Sr2+ plus EGTA. Although this observation seems to contradict a previous report [25], some methodological differences should be stressed. Mortimer et al. [25] used whole oocytes and noncapacitated spermatozoa for binding assays. In the present study, HZA were carried out, providing an accurate control in the equivalent half of each HZ. The other possible source of discrepancy is that we incubated spermatozoa for 6 h before the assays. However, this possibility was excluded because similar binding results were obtained using either capacitated or noncapacitated spermatozoa (data not shown). From these data, it could be inferred that Sr2+ can support all sperm surface changes necessary to ZP recognition and the tight binding of gametes. Whether Sr2+ allows ZP-induced AR and sperm-oocyte fusion in humans remains unknown. However, evidence in the mouse indicate that this cation supports sperm-ZP penetration and fertilization [22].

In summary, the results presented in this study lead us to conclude that Sr2+ can effectively replace Ca2+ in maintaining human sperm capacitation-related events, such as protein tyrosine phosphorylation and hyperactivation, and also sperm-ZP interaction. This effect seems to be independent of traces of Ca2+ present in HSM Sr2+, since with the addition of EGTA to this medium, similar results were obtained. On the other hand, Sr2+ was unable to support hFF-induced acrosome reaction. The exact point of the AR cascade at which Sr2+ cannot replace Ca2+ has not been determined and is currently under study in our laboratory.

The incubation of spermatozoa in Sr2+-containing media constitutes an interesting experimental model for the study of molecular events related to capacitation and a useful model for analyzing this process while avoiding the consequent induction of the AR.


   ACKNOWLEDGMENTS
 
The authors especially thank Dr. Juan C. Calamera from LER, Laboratorio de Estudios en Reproducción, for his generous and invaluable help in CASA analysis. We would also like to thank Patricia Delcourt for her technical assistance.


   FOOTNOTES
 
1 This work was supported by the Rockefeller Foundation (grant #9439), the World Health Organization (Career Development Fellowship to P.V.M. and project #97175), the Programa Latimoamericano de Capacitación e Investigación en Reproducción Humana (PLACIRH, Re-Entry Grant PRE-014/97), and the Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET) of Argentina. Back

2 Correspondence: Clara I. Marín-Briggiler, Instituto de Biología y Medicina Experimental, Vuelta de Obligado 2490, (1428) Buenos Aires, Argentina. FAX: 54 11 4786 2564; cmarin{at}dna.uba.ar Back

Accepted: April 22, 1999.

Received: December 15, 1998.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

  1. Florman HM, Babcock DF. Progress towards understanding the molecular basis of capacitation. In: Wassarman PM (ed.), Elements of Mammalian Fertilization, vol. 1, Basic Concepts. Boca Raton, FL: CRC Press; 1991: 105–132.
  2. Cohen-Dayag A, Eisenbach M. Potential assays for sperm capacitation in mammals. Am J Physiol Cell Physiol 1994; 267:C1167-C1176.
  3. Yanagimachi R. Mammalian fertilization. In: Knobil E, Neill JD (eds.), The Physiology of Reproduction, 2nd ed. New York: Raven Press; 1994: 189–317.
  4. Visconti PE, Galantino-Homer HL, Moore GD, Bailey JL, Ning X, Fornes M, Kopf GS. The molecular basis of sperm capacitation. J Androl 1998; 19:242–248.[Free Full Text]
  5. Thomas P, Meizel S. An influx of extracellular calcium is required for initiation of the human sperm acrosome reaction induced by human follicular fluid. Gamete Res 1988; 20:397–413.[CrossRef][Medline]
  6. Florman HM, Tombes RM, First NL, Babcock DF. An adhesion-associated agonist from the zona pellucida activates G protein-promoted elevations of internal Ca2+ and pH that mediate mammalian sperm acrosomal exocytosis. Dev Biol 1989; 135:133–146.[CrossRef][Medline]
  7. Blackmore PF, Beebe SJ, Danforth DR, Alexander N. Progesterone and 17{alpha}-hydroxyprogesterone. Novel stimulators of calcium influx in human sperm. J Biol Chem 1990; 265:1376–1380.[Abstract/Free Full Text]
  8. Storey BT, Hourani CL, Kim JB. A transient rise in intracellular Ca2+ is a precursor reaction to the zona pellucida-induced acrosome reaction in mouse sperm and is blocked by the induced acrosome reaction inhibitor 3-quinuclidinyl benzilate. Mol Reprod Dev 1992; 32:41–50.[CrossRef][Medline]
  9. Brewis IA, Clayton R, Barratt CLR, Hornby DPJ, Moore HD. Recombinant human zona pellucida glycoprotein 3 induces calcium influx and acrosome reaction in human spermatozoa. Mol Hum Reprod 1996; 2:583–589.[Abstract/Free Full Text]
  10. Meizel S, Turner KO, Nuccitelli R. Progesterone triggers a wave of increased free calcium during the human sperm acrosome reaction. Dev Biol 1997; 182:67–75.[CrossRef][Medline]
  11. Fraser LR. Minimum and maximum extracellular Ca2+ requirements during mouse sperm capacitation and fertilization in vitro. J Reprod Fertil 1987; 81:77–89.[Abstract]
  12. Stock CE, Fraser LR. Divalent cations, capacitation and the acrosome reaction in human spermatozoa. J Reprod Fertil 1989; 87:463–478.[Abstract]
  13. DasGupta S, Mills CL, Fraser LR. Ca2+-Related changes in the capacitation state of human spermatozoa assessed by a chlortetracycline fluorescence assay. J Reprod Fertil 1993; 99:135–143.[Abstract]
  14. Coronel CE, Lardy HA. Characterization of Ca2+ uptake by guinea pig epididymal spermatozoa. Biol Reprod 1987; 37:1097–1107.[Abstract]
  15. White DR, Aitken RJ. Relationship between calcium, cyclic AMP, ATP, and intracellular pH and the hamster spermatozoa to express hyperactivated motility. Gamete Res 1989; 22:163–177.[CrossRef][Medline]
  16. Zhou R, Shi B, Chou KCK, Oswalt MD, Haug A. Changes in intracellular calcium of porcine sperm during in vitro incubation with seminal plasma and a capacitating medium. Biochem Biophys Res Commun 1990; 172:47–53.[CrossRef][Medline]
  17. Baldi E, Casano R, Falsetti C, Krausz C, Maggi M, Forti G. Intracellular calcium accumulation and responsiveness to progesterone in capacitating human spermatozoa. J Androl 1991; 12:323–330.[Abstract/Free Full Text]
  18. Okamura N, Tanba M, Fukuda A, Sugita Y, Nagai T. Forskolin stimulates porcine sperm capacitation by increasing calcium uptake. FEBS Lett 1993; 316:283–286.[CrossRef][Medline]
  19. Yanagimachi R, Usui N. Calcium dependence of the acrosome reaction and activation of guinea pig spermatozoa. Exp Cell Res 1974; 89:161–174.[CrossRef][Medline]
  20. Heffner LJ, Saling PM, Storey BT. Separation of calcium effects on motility and zona binding ability in mouse spermatozoa. J Exp Zool 1980; 212:53–60.[CrossRef][Medline]
  21. Cooper TG. The onset and maintenance of hyperactivated motility of spermatozoa from the mouse. Gamete Res 1984; 9:55–74.
  22. Fraser LR. Strontium supports capacitation and the acrosome reaction in mouse sperm and rapidly activates mouse eggs. Gamete Res 1987; 18:363–374.[CrossRef][Medline]
  23. Roldan ERS, Harrison RAP. Polyphosphoinositide breakdown and subsequent exocytosis in the Ca2+/ionophore-induced acrosome reaction of mammalian spermatozoa. Biochem J 1989; 259:397–406.[Medline]
  24. Yanagimachi R. Calcium requirement for sperm-egg fusion in mammals. Biol Reprod 1978; 19:949–958.[Abstract]
  25. Mortimer D, Chorney MJ, Curtis EF, Trounson AO. Calcium dependence of human sperm fertilizing ability. J Exp Zool 1988; 246:194–201.[CrossRef][Medline]
  26. Mortimer D, Curtis EF, Dravland JE. The use of strontium-substituted media for capacitating human spermatozoa: an improved sperm preparation method for the zona-free hamster egg penetration test. Fertil Steril 1986; 46:97–103.[Medline]
  27. Mortimer D. Comparison of the fertilizing ability of human spermatozoa incubated in calcium- and strontium-containing media. J Exp Zool 1986; 237:21–24.[CrossRef][Medline]
  28. Suarez SS, Wolf DP, Meizel S. Induction of the acrosome reaction in human spermatozoa by a fraction of human follicular fluid. Gamete Res 1986; 14:107–121.
  29. World Health Organization: WHO Laboratory Manual for the Examination of Human Semen and Sperm-Cervical Mucus Interaction, 3rd ed. New York: Cambridge University Press; 1992.
  30. Meldrum DR, Wisot A, Hamilton F, Gutlay AL, Huynh D, Kempton W. Timing of initiation and dose schedule of leuprolide influence the time course of ovarian suppression. Fertil Steril 1988; 50:400–402.[Medline]
  31. Mendoza C, Carreras A, Moos J, Tesarik J. Distinction between true acrosome reaction and degenerative acrosome loss by a one-step staining method using Pisum sativum agglutinin. J Reprod Fertil 1992; 95:755–763.[Abstract]
  32. Cross NL, Morales P, Overstreet JW, Hanson FW. Two simple methods for detecting acrosome-reacted human sperm. Gamete Res 1986; 15:213–226.[CrossRef]
  33. Laemmli UK. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 1970; 227:680–685.[CrossRef][Medline]
  34. Towbin H, Staehelin T, Gordon J. Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Proc Natl Acad Sci USA 1979; 76:4350–4354.[Abstract/Free Full Text]
  35. Mortimer ST, Mortimer D. Kinematics of human spermatozoa incubated under capacitating conditions. J Androl 1990; 11:195–203.[Abstract/Free Full Text]
  36. Fayrer-Hosken RA, Brackett BG. Use of salt-stored zonae pellucidae for assessing rabbit sperm capacitation for in vitro fertilization. Gamete Res 1987; 17:191–201.[CrossRef][Medline]
  37. Burkman LJ, Coddington CC, Franken DR, Kruger TF, Rosenwaks Z, Hodgen GD. The hemizona assay (HZA): development of a diagnostic test for the binding of human spermatozoa to the human hemizona pellucida to predict fertilization potential. Fertil Steril 1988; 49:688–697.[Medline]
  38. Calvo L, Vantman D, Banks SM, Tezon JG, Koukoulis G, Dennison L, Sherins RJ. Follicular fluid-induced acrosome reaction distinguishes a subgroup of men with unexplained infertility not identified by semen analysis. Fertil Steril 1989; 52:1048–1054.[Medline]
  39. Naz RK, Ahmad K, Kumar R. Role of membrane phosphotyrosine proteins in human spermatozoal function. J Cell Sci 1991; 99:157–165.[Abstract/Free Full Text]
  40. Visconti PE, Bailey JL, Moore GD, Pan D, Olds-Clarke P, Kopf GS. Capacitation of mouse spermatozoa. I. Correlation between the capacitation state and protein tyrosine phosphorylation. Development 1995; 121:1129–1137.[Abstract]
  41. Emiliozzi C, Fenichel P. Protein tyrosine phosphorylation is associated with capacitation of human sperm in vitro but is not sufficient for its completion. Biol Reprod 1997; 56:674–679.[Abstract]
  42. Leclerc P, De Lamirande E, Gagnon C. Cyclic adenosine 3',5'-monophosphate-dependent regulation of protein tyrosine phosphorylation in relation to human sperm capacitation and motility. Biol Reprod 1996; 55:684–692.[Abstract]
  43. Pukazhenthi BS, Wildt DE, Ottinger MA, Howard J. Compromised sperm protein phosphorylation after capacitation, swim-up, and zona pellucida exposure in teratospermic domestic cats. J Androl 1996; 17:409–419.[Abstract/Free Full Text]
  44. Galantino-Homer HL, Visconti PE, Kopf GS. Regulation of protein tyrosine phosphorylation during bovine sperm capacitation by a cyclic adenosine 3',5'-monophosphate-dependent pathway. Biol Reprod 1997; 56:707–719.[Abstract]
  45. Tomes CN, Carballada R, Moses DF, Katz DF, Saling PM. Treatment of human spermatozoa with seminal plasma inhibits protein tyrosine phosphorylation. Mol Hum Reprod 1998; 4:17–25.[Abstract/Free Full Text]
  46. Calvo L, Dennison-Lagos L, Banks SM, Dorfmann A, Thorsell LP, Bustillo M, Schulman JD, Sherins RJ. Acrosome reaction inducibility predicts fertilization success at in-vitro fertilization. Hum Reprod 1994; 9:1880–1886.[Abstract/Free Full Text]
  47. Carrera A, Moos J, Ning XP, Gerton GL, Tesarik J, Kopf GS, Moss SB. Regulation of protein tyrosine phosphorylation in human sperm by a calcium/calmodulin-dependent mechanism: identification of a kinase anchor proteins as major substrates for tyrosine phosphorylation. Dev Biol 1996; 180:284–296.[CrossRef][Medline]
  48. Luconi M, Krausz C, Forti G, Baldi E. Extracellular calcium negatively modulates tyrosine phosphorylation and tyrosine kinase activity during capacitation of human spermatozoa. Biol Reprod 1996; 55:207–216.[Abstract]
  49. Roldan ERS, Fragio C. Phospholipase A2 activity and exocytosis of the ram sperm acrosome: regulation by divalent cations. Biochim Biophys Acta 1993; 1168:108–114.[Medline]
  50. Hyne RV, Garbers DL. Calcium dependent elevation of adenosine 3',5'-monophosphate and induction of the acrosome reaction in guinea pig spermatozoa. Proc Natl Acad Sci USA 1979; 76:5699–5703.[Abstract/Free Full Text]
  51. Hyne RV, Garbers DL. Regulation of guinea pig sperm adenylate cyclase by calcium. Biol Reprod 1979; 21:1135–1142.[Abstract]
  52. Gross MK, Toscano DG, Toscano WA. Calmodulin-mediated adenylate cyclase from mammalian sperm. J Biol Chem 1987; 262:8672–8676.[Abstract/Free Full Text]
  53. Parinaud J, Milhet P. Progesterone induces Ca++-dependent 3',5'-cyclic adenosine monophosphate increase in human sperm. J Clin Endocrinol Metab 1996; 81:1357–1360.[Abstract]
  54. Tash JS, Means AR. Cyclic adenosine 3',5' monophosphate, calcium and protein phosphorylation in flagellar motility. Biol Reprod 1983; 28:75–104.[Abstract]
  55. Tash JS. Role of cAMP, calcium and protein tyrosine phosphorylation in sperm motility. In: Gagnon C (ed.), Controls of Sperm Motility: Biological and Clinical Aspects. Boca Raton, FL: CRC Press; 1990: 229–241.
  56. San Agustin JT, Witman GB. Role of cAMP in the reactivation of demembranated ram spermatozoa. Cell Motil Cytoskeleton 1994; 27:206–218.[CrossRef][Medline]
  57. Visconti PE, Moore GD, Bailey JL, Leclerc P, Connors SA, Pan D, Olds-Clarke P, Kopf GS. Capacitation of mouse spermatozoa. II. Protein tyrosine phosphorylation and capacitation are regulated by a cAMP-dependent pathway. Development 1995; 121:1139–1150.[Abstract]
  58. Kaláb P, Peknicová J, Geussová G, Moos J. Regulation of protein tyrosine phosphorylation in boar sperm through a cAMP-dependent pathway. Mol Reprod Dev 1998; 51:304–314.[CrossRef][Medline]



This article has been cited by other articles:


Home page
 Mol Hum ReprodHome page
K. Zitta, E. V. Wertheimer, and P. V. Miranda
Sperm N-acetylglucosaminidase is involved in primary binding to the zona pellucida
Mol. Hum. Reprod., September 1, 2006; 12(9): 557 - 563.
[Abstract] [Full Text] [PDF]

Home page
 J. Cell Sci.Home page
C. I. Marin-Briggiler, K. N. Jha, O. Chertihin, M. G. Buffone, J. C. Herr, M. H. Vazquez-Levin, and P. E. Visconti
Evidence of the presence of calcium/calmodulin-dependent protein kinase IV in human sperm and its involvement in motility regulation
J. Cell Sci., May 1, 2005; 118(9): 2013 - 2022.
[Abstract] [Full Text] [PDF]

Home page
 Mol Hum ReprodHome page
K. Zitta, E. Wertheimer, and P. V. Miranda
Analysis of the participation of N-acetylglucosamine in the different steps of sperm-zona pellucida interaction in hamster
Mol. Hum. Reprod., December 1, 2004; 10(12): 925 - 933.
[Abstract] [Full Text] [PDF]

Home page
 J. Cell Sci.Home page
K. L. Asquith, R. M. Baleato, E. A. McLaughlin, B. Nixon, and R. J. Aitken
Tyrosine phosphorylation activates surface chaperones facilitating sperm-zona recognition
J. Cell Sci., July 15, 2004; 117(16): 3645 - 3657.
[Abstract] [Full Text] [PDF]

Home page
 Mol Hum ReprodHome page
A. A. Schuffner, H. S. Bastiaan, H. E. Duran, Z.-Y. Lin, M. Morshedi, D. R. Franken, and S. Oehninger
Zona pellucida-induced acrosome reaction in human sperm: dependency on activation of pertussis toxin-sensitive Gi protein and extracellular calcium, and priming effect of progesterone and follicular fluid
Mol. Hum. Reprod., August 1, 2002; 8(8): 722 - 727.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Marín-Briggiler, C. I.
Right arrow Articles by Miranda, P. V.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Marín-Briggiler, C. I.
Right arrow Articles by Miranda, P. V.
Agricola
Right arrow Articles by Marín-Briggiler, C. I.
Right arrow Articles by Miranda, P. V.