Progesterone-Induced Acrosome Reaction in Stallion Spermatozoa Is Mediated by a Plasma Membrane Progesterone Receptor

^a Department of Herd Health & Reproduction, ^b Laboratory of Veterinary Biochemistry ^c and Department of Functional Morphology, Graduate School of Animal Sciences, Faculty of Veterinary Medicine, Utrecht University, 3584 CL Utrecht, The Netherlands

	ABSTRACT

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
The aim of the present study was to investigate whether the induction of stallion sperm acrosome reaction (AR) by progesterone is mediated by binding of progesterone to a receptor on the sperm plasma membrane or to an intracellular progesterone receptor. Progesterone-BSA conjugate labeled with fluorescein isothiocyanate (P-BSA-FITC) in combination with a vital stain, ethidium homodimer, was applied to visualize the presence of the progesterone receptor on living spermatozoa. Alternatively, an indirect immunofluorescence technique employing a monoclonal antibody (C-262) against human intracellular progesterone receptor was conducted to validate the presence of the progesterone receptor. Immunogold labeling techniques enabled ultrastructural localization of P-BSA-FITC or C-262 with transmission electron microscopy. The dynamic changes in labeling patterns were monitored for sperm cells, using fluorescence microscopy and flow cytometry during a 5-h capacitation period. An increasing number of viable cells showed affinity for P-BSA-FITC or C-262 at the acrosomal plasma membrane region of the sperm head, while a decreasing number of viable cells were not labeled. In contrast, almost all deteriorated cells were labeled in the cytosol of the postequatorial region of the sperm head. Incubation with P-BSA-FITC resulted in the induction of AR but to a lesser extent than that for sperm incubated with free progesterone. Therefore, coupling of progesterone to its receptor on the sperm plasma membrane appears to be an important step in the induction of the AR.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
The sperm acrosome reaction (AR) is a calcium dependent, exocytotic event required for mammalian fertilization. The AR facilitates the penetration of the zona pellucida by spermatozoa and the subsequent fusion of the sperm plasma membrane with the oocyte's oolemma. Ejaculated spermatozoa require a series of preparatory changes in order to undergo the AR. These physiological changes are collectively termed capacitation and involve primary membrane modifications [1]. Capacitation can occur either in vivo, during the passage of spermatozoa through the female genital tract, or in vitro, during incubation of washed spermatozoa under proper conditions. In vitro studies have shown that the AR can be initiated in preincubated spermatozoa either spontaneously or by various physiological (zona pellucida glycoproteins and follicular fluid) and nonphysiological (calcium ionophore) inducers [2, 3]. However, a biologically effective AR will depend on the presence and the activity of physiological inducers as well as the capacity of spermatozoa to respond to these inducers.

According to the current dogma, capacitated and acrosome-intact spermatozoa initiate binding to the zona pellucida [2]. Although the zona pellucida is considered the prime physiological inducer of AR, previous studies have shown a low incidence of AR in zona pellucida-bound stallion spermatozoa after 1-h in vitro binding [4–6]. This low incidence suggests that, besides the zona pellucida glycoproteins, another major factor might be responsible for AR. In addition to the zona pellucida, protein-bound progesterone, present in human follicular fluid, has been demonstrated to induce AR in spermatozoa [7]. It has been shown that free progesterone is capable of inducing AR in human [8], pig [9], mouse [10], and stallion spermatozoa [11, 12].

How progesterone induces AR has been the subject of a number of studies. Originally progesterone was thought to act through the classical mechanism of steroid action: progesterone enters the cell and binds to a cytoplasmic receptor, and this complex then binds to DNA and acts as a transcription factor [13]. In mature sperm cells, such a scenario is unlikely because they have completely shut down DNA replication and transcription [2]. Interestingly, Meizel and Turner [8] showed that progesterone probably exerts its effects on human spermatozoa by binding to a plasma membrane receptor. In addition, Castilla et al. [14] were unable to detect the genomic progesterone receptor (mediating the classical mechanism) in human spermatozoa. Recently, plasma membrane receptors for progesterone have been reported in human spermatozoa [15] and in rat brain nerve cells [16]. Indeed, these progesterone receptors mediate sperm functions by means other than cytosolic/nuclear steroid receptors in somatic cells as reviewed by Meizel [17]. The presence and action of nongenomic steroid receptors have also been described for other steroids [16, 18].

The aim of the present study was to investigate whether the induction of stallion sperm AR by progesterone is mediated by binding to a plasma membrane receptor or an intracellular progesterone receptor. Therefore, the effect of progesterone on stallion spermatozoa was studied with fluorescein isothiocyanate-conjugated progesterone-BSA (P-BSA-FITC), which because of its large size and hydrophilic nature cannot penetrate the sperm plasma membrane. In addition, the (ultra)localization of the progesterone receptor on stallion spermatozoa was studied by using P-BSA-FITC or, alternatively, a monoclonal antibody against the progesterone receptor and secondary FITC-conjugated antibody. Finally, the binding dynamics of P-BSA-FITC to subpopulations of sperm cells was evaluated during capacitation in vitro using fluorescence microscopy and flow cytometry analysis.

	MATERIALS AND METHODS

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Treatment and Preincubation of Stallion Spermatozoa

After collecting and filtering the ejaculate, a semen sample (5 ml) was mixed with an equal volume of egg yolk extender (D11, defined in [19]) and centrifuged (10 min at 900 x g) at room temperature. After the supernatant was decanted, the sperm pellet was resuspended in 10 ml of egg yolk extender. Subsequently, the sample was centrifuged (10 min at 50 x g) to sediment any remaining gel fraction, seminal debris, clumped spermatozoa, and large egg yolk particles. One milliliter of the supernatant was transferred to another tube and then gently overlayered with 2 ml of prewarmed (37°C) modified Tyrode's medium containing 2.0 mM CaCl₂, 3.1 mM KCl, 0.4 mM MgCl₂, 100.0 mM NaCl, 25.0 mM NaHCO₃, 0.3 mM NaH₂PO₄, 1.0 mM sodium pyruvate, 21.6 mM sodium lactate, 10.0 mM Hepes, 10 mg/ml BSA, and 1 mg/ml polyvinyl alcohol at pH 7.2 (SP-TALP) [20]. Tubes were placed in a slant rack to position them at an angle 20° from upright in an incubator at 37°C, in a humidified atmosphere saturated with 5% CO₂. After 1 h, the top 1 ml medium was collected, and the combined swim-up fraction (sperm concentration: 10–15 x 10⁶/ml) was maintained at 37°C in a humidified atmosphere saturated with 5% CO₂ until use.

Testes and epididymides were obtained from stallions after routine castration in the clinic. The caudae epididymides were dissected and flushed with 2 ml SP-TALP. Subsequently they were cut 10–20 times with a razor blade, and the SP-TALP and sperm (approximately 200 million per cauda) were collected and diluted to a concentration of approximately 70 million sperm cells/ml SP-TALP.

Assessment of Sperm Viability

Calcein-AM in combination with ethidium homodimer (EthD-1) (Live/Dead kit; Molecular Probes Inc., Eugene, OR) was used as described by Borg et al. [21] to assess whether sperm were viable (alive). For quantitative assessment of sperm viability, 200 spermatozoa from a stained sample were assessed in randomly selected fields under an epifluorescence microscope (x500) equipped with a dichroic mirror unit set of filters (BH2-RFCA; Olympus, Tokyo, Japan). Spermatozoa with accumulated green calcein in all cellular compartments were classified as viable, whereas spermatozoa displaying red EthD-1 over the head and spermatozoa with accumulated green calcein only in the acrosome and displaying red EthD-1 in the postacrosomal region were classified as nonviable (dead).

Assessment of Acrosomal Status

For the assessment of acrosomal status, 400 µl of sperm suspension was mixed with 45 µl of 20 µM EthD-1 in SP-TALP and incubated for 3 min at 37°C. Then, 20 µl of a solution containing 1 mg/ml DNA (Sigma Chemical Co., St. Louis, MO) in PBS was added [22] to bind the excess EthD-1 for 2 min. Immediately, 465 µl of 2% paraformaldehyde in PBS was added and mixed. The sperm suspension was then centrifuged at 600 x g for 3 min. After removal of supernatant, the fixed sperm cells were washed with 1 ml PBS and centrifuged at 600 x g for 3 min to remove remaining paraformaldehyde. After washing, the sperm pellet was resuspended in 100 µl of PBS. Subsequently, 100 µl of 0.1% Nonidet-P40 (Fluka, Buchs, Germany) was added and mixed to permeabilize sperm membranes. After 5 min, 1 ml of PBS was added and the sperm suspension was centrifuged at 600 x g for 3 min to remove remaining Nonidet-P40. The washing procedure was repeated once after supernatant removal. The sperm pellet was resuspended in 100 µl of PBS, and 100 µl of a solution containing 100 µg/ml FITC-labeled peanut agglutinin (PNA-FITC; E-Y. Lab. Inc., San Mateo, CA) in PBS was added at 37°C for 30 min to stain the sperm acrosome. Subsequently, 1 ml of PBS was added, and the sperm suspension was centrifuged at 600 x g for 3 min to remove the excess PNA-FITC. The washing procedure was repeated once after removal of supernatant. Finally, the sperm pellet was resuspended in 200 µl PBS, and 10 µl of this suspension was transferred to a glass slide, mounted with 5 µl antifade solution as described by Johnson and Gloria [23], covered with a coverslip, and sealed with nail polish. Slides were then kept in the dark until examined.

To estimate the proportion of AR in spermatozoa, 200 spermatozoa were assessed in randomly selected fields under an epifluorescence microscope as described above. Spermatozoa with an EthD-1-stained nucleus were classified as nonviable, and those with a nonstained nucleus were classified as alive. Spermatozoa were considered to have an intact acrosome if the PNA-FITC staining was distributed over the entire acrosome. Patchy-like, disrupted PNA-FITC fluorescence of acrosome indicated that the AR was in process. Spermatozoa with PNA-FITC staining on the equatorial segment had completed the AR and had shed the outer acrosomal membrane [5].

Treatment of Spermatozoa with Progesterone-BSA-FITC Conjugate

Progesterone 3-(o-carboxymethyl)oxime:BSA-FITC conjugate (Sigma Chemical Co.) was dissolved in PBS at a concentration of 1 mg/ml. Nonconjugated progesterone was removed from the probe by dextran-charcoal treatment as described by Meizel and Turner [8].

After 4-h preincubation in SP-TALP, sperm samples were treated as follows:

Four hundred microliters sperm suspension was added to 50 µl of P-BSA-FITC solution and 50 µl of EthD-1 (20 µM) in PBS, resulting in a final concentration of 100 µg conjugate/ml and 2 µM EthD-1. This conjugate concentration corresponded to 10 µM progesterone concentration. After 5-min incubation at 37°C, 25 µl of 1 mg/ml DNA in PBS was added (final concentration: 25 µg/ml) for 2 min to compete for the unbound EthD-1. To fix spermatozoa, 525 µl of 4% paraformaldehyde and 1% glutaraldehyde in PBS was added. The sperm suspension was centrifuged at 600 x g for 3 min. After supernatant removal, the fixed sperm cells were washed with 1 ml PBS and centrifuged at 600 x g for 3 min to remove remaining aldehyde residues. This washing procedure was repeated twice, and the sperm pellet was resuspended in 200 µl PBS. Ten microliters sperm suspension was transferred to a glass slide, mounted with 5 µl antifade solution, covered with a coverslip, and sealed with nail polish. Slides were kept in the dark until examined under an epifluorescence microscope. Spermatozoa not stained with EthD-1 were considered alive. Another 400 µl of sperm suspension was processed to assess acrosomal status.

The potential of free progesterone to compete for receptor binding with P-BSA-FITC on stallion sperm was tested. A solution of EthD-1, progesterone, and dimethyl sulfoxide (DMSO) was added to a 400-µl sperm suspension (final concentrations of 32 µM progesterone and 0.1% DMSO [v:v] and 2 µM EthD-1) for 5 min. Subsequently, this sperm suspension was stained with P-BSA-FITC (final concentration of 100 µg/ml) as described above with the exception that during the last 2 min of P-BSA-FITC staining, DNA (25 µg/ml) was added (reduction of progesterone-induced ARs).

Four hundred microliters of sperm suspension was added to 50 µl BSA-FITC or ß-estradiol 6-(O-carboxy-methyl)oxime:BSA-FITC (E-BSA-FITC), both in combination with 50 µl EthD-1 (20 µM), and then processed as described for P-BSA-FITC staining. Another 400 µl was processed to assess acrosomal status.

For all experiments, ejaculates with more than 70% progressive sperm motility, from 3 known-fertile Dutch warmblood stallions, were used. Sperm motility was assessed as described by Parlevliet et al. [24].

Freshly ejaculated sperm samples washed through a discontinuous Percoll gradient (using 35% and 70% isotonic SP-TALP-based Percoll layers, centrifugation at 300 x g for 10 min and at 750 x g for 15 min) to remove seminal plasma and plasma membrane coating material. The Percoll-washed sperm cells were stained with P-BSA-FITC and EthD-1 as described above.

A portion of 400 µl SP-TALP-diluted epididymal stallion sperm (approximately 10 million cells/ml) was stained with 50 µl (1 mg/ml) P-BSA-FITC and 50 µl EthD-1 (20 µM).

Control labeling experiments were carried out on 400-µl samples of ejaculated, swim-up sperm with 50 µl (1 mg/ml) BSA-FITC or with 50 µl (1 mg/ml) E-BSA-FITC in combination with 50 µl (20 µM) EthD-1.

Treatment of Spermatozoa with Monoclonal Antibody against Progesterone Receptor

Localization of the progesterone receptor in stallion spermatozoa was carried out by indirect immunofluorescence using C-262 and rabbit anti-mouse IgG conjugated with FITC (RAM-FITC; Zymed, San Francisco, CA). The mouse monoclonal antibody C-262 (code SRA-1110; purchased from Stress Gen, Victoria, BC, Canada) was raised against synthetic peptides corresponding to the C-terminal tail of the human intracellular progesterone receptor representing the steroid-binding site [25].

After 4-h preincubation in SP-TALP, 500 µl sperm suspension was mixed with 500 µl 2% paraformaldehyde in PBS for 10 min. The sperm suspension was then centrifuged at 600 x g for 3 min. After supernatant removal, fixed sperm cells were washed with 1 ml PBS and centrifuged at 600 x g for 3 min to remove remaining aldehyde residues. The washing procedure was repeated twice. After supernatant removal, the sperm pellet was resuspended in 180 µl PBS supplemented with 3 mg/ml BSA and incubated with 20 µl of a solution of 1.9 mg/ml C-262 in PBS supplemented with 3 mg/ml BSA for 1 h at 37°C. The sperm suspension was then centrifuged at 600 x g for 3 min. After removal of the supernatant, the spermatozoa were washed twice as described above to remove unbound primary antibody. The sperm pellet was resuspended in 245 µl PBS supplemented with 3 mg/ml BSA and incubated with 5 µl of a solution of 0.75 mg/ml RAM-FITC in PBS supplemented with 3 mg/ml BSA for 1 h at 37°C. The sperm suspension was then centrifuged at 300 x g for 3 min. After supernatant removal, the spermatozoa were washed twice as described above to remove unbound secondary antibody. As a control, a duplicate sperm sample was processed and incubated with RAM-FITC only. Spermatozoa were resuspended in 200 µl of PBS and counterstained with 5 µl of 0.5% Evans blue in PBS as described by Farlin et al. [26]. Ten microliters of the suspension was transferred to a glass slide, mounted with 5 µl antifade solution, covered with a coverslip, and sealed with nail polish. Slides were kept in the dark until examined under an epifluorescence microscope as described above.

The affinity of C-262 for proteins isolated from stallion sperm and human sperm (the latter from healthy and proven fertile donors, Academic Hospital Utrecht) was assessed. Proteins were prepared for SDS-PAGE and Western blotting as described by Sabeur et al. [15]. In addition, ELISA and spot-blot assays were carried out with native and reduced proteins samples from stallion and human sperm (immunoincubation steps as for Western blotting [15]).

Ultrastructural Localization of the Progesterone Receptor

Sperm samples (10 x 10⁶ spermatozoa) were treated either with C-262/RAM-FITC (as described above) or with P-BSA-FITC and fixed in 2% paraformaldehyde/0.5% glutaraldehyde and stored overnight at 4°C. Subsequently the sample was centrifuged (6000 x g, 10 min), and the pellet was recovered in PBS containing 50 mM glycine (pH 7.4) to block free aldehyde groups. After 10 min the procedure was repeated and the recovered sample was left for 1 h in PBS-glycine. The spermatozoa were centrifuged for 10 min (6000 x g), and the pellet was resuspended in 10% (w:v) gelatine in 0.1 M sodium phosphate (pH 7.4) at 37°C. Subsequently the sample was centrifuged for 2 min at 9500 x g. After cooling down to 4°C, the sample was cut out of the tube and placed in fixative. After infiltration with 2.3 M sucrose and freezing in liquid nitrogen, ultrathin cryosections (50–100 nm) were cut at -120°C on a Reichert UltracutS/FCS (Leika Aktiengesellschaft, Wien, Austria) using a cryo diamond knife (Drukker International, Cuyk, The Netherlands) and were transferred to Formvar carbon-coated nickel grids. Thawed ultrathin cryosections were subsequently incubated in the following solutions: a) PBS-glycine for 15 min; b) block buffer (5% w:v BSA, 0.1% cold water fish skin gelatin [Aurion, Wageningen, The Netherlands] in PBS, pH 7.4) for 30 min; c) incubation buffer (0.1% w:v BSA-C [Aurion] in PBS, pH 7.4) for 5 min; d) rabbit anti-FITC (Molecular Probes) in incubation buffer (1 µg/ml) for 1 h; e) incubation buffer (three times, 10 min each); f) 10 nm protein A-gold particle solution in incubation buffer for 1 h; g) incubation buffer (three times, 10 min each); h) PBS (two times, 5 min each); i) distilled water (four times, 5 min each); j) 2% w:v uranyl acetate-oxalate (pH 7) for 10 min. The stained cryosections were embedded in 1.8% w:v methylcellulose containing 0.3% w:v uranyl acetate. Immunogold-labeled cryosections were studied and photographed in a Philips CM10 electron microscope (Philips, Eindhoven, The Netherlands) at 80 kV.

Induction of the AR with Progesterone and P-BSA-FITC

After 4-h preincubation in SP-TALP, a 500-µl sperm suspension was incubated for 30 min with either 10 µM progesterone or 100 µg/ml P-BSA-FITC (equal to 10 µM progesterone) or 0.1% (v:v) DMSO (vehicle only) at 37°C in humidified air saturated with 5% CO₂. Subsequently, the samples were processed for assessment of acrosomal status and viability.

Effect of Incubation Time on P-BSA-FITC Staining Patterns

After swim-up, spermatozoa were incubated in SP-TALP for 7 h. At onset of incubation and subsequently every hour from 1 up to 7 h, aliquots were taken and treated with P-BSA-FITC as described above. Two hundred viable spermatozoa were classified according to their staining patterns. Simultaneously, sperm aliquots were examined to estimate motility and viability with calcein-AM and EthD-1.

For flow cytometry analysis, sperm samples were collected after 0-, 1-, 3-, and 5-h incubation in SP-TALP and processed to analyze P-BSA-FITC staining in combination with EthD-1. Samples were divided for flow cytometry analysis and fluorescence microscopy.

Flow Cytometry Analysis of P-BSA-FITC Staining

Sperm suspensions were mixed with 10 µg/ml P-BSA-FITC and 2 µM EthD-1. The suspensions were immediately analyzed in a FACS-Scan flow cytometer with 100 mW argon laser (Becton Dickinson, San Jose, CA). Cells were excited at 488 nm, and the fluorescence data were collected in a logarithmic scale, while forward and sideward light-scatter data were collected in a linear scale. At the right light-scatter settings, specific sperm events were recognizable as a typical L-shaped scattering profile. The nonspermatic events (mostly small particles, 3% of total events) were gated out of further analyses. P-BSA-FITC-stained spermatozoa were detected by the FL-1 detector using 530/30-nm band-pass filter, while the FL-3 detector was used to detect EthD-1 fluorescence using 620-nm long-pass filter. For each sample, data were collected for 10 000 spermatic events at a rate of 500–1000/sec. The level of P-BSA-FITC was chosen to be 10 µg/ml so that positive cells gave a signal around 10² logarithmic scale arbitrary units of fluorescence intensity on the FL-1 detector with a sensitivity setting of approximately 600 V, while negative cells gave a signal around 10¹. The combination of P-BSA-FITC and EthD-1 allowed compensations to be kept below 10%. EthD-1 staining of nonviable spermatozoa was accomplished within 30 sec. The proportion of EthD-1-stained cells corresponded well to the proportion of cells stained with propidium iodide (a well-established supravital dye used in flow cytometry; Molecular Probes Inc., Leiden, The Netherlands). The viable sperm population remained negative to EthD-1 for at least 1 h. P-BSA-FITC staining was slower but essentially complete for positive spermatozoa within 2 min. In control experiments, spermatozoa were stained with BSA-FITC or with E-BSA-FITC in combination with EthD-1.

Flow cytometry data were stored and analyzed in WinMDI (Becton Dickinson). Two-dimensional dot plots of either FSC/SSC or FL-1/FL-3 were analyzed by drawing regions to delineate cell population categories. The percentage of cells and the average amount of cell-associated FITC were calculated for each cell population.

Statistical Analysis

The difference in proportion of spermatozoa classified according to the acrosomal status and viability was analyzed by nonparametric paired (Wilcoxon) test using SPSS statistics (Statistical Program for Social Sciences, Chicago, IL). The level of significance was p < 0.05.

	RESULTS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
After treatment of stallion spermatozoa with P-BSA-FITC, three main staining patterns could be distinguished on viable cells (excluding the supravital stain EthD-1): 1) Cells with uniform P-BSA-FITC staining on the whole acrosomal cap of the sperm head (Fig. 1A); 2) cells that were not stained with P-BSA-FITC at all (not shown); and 3) cells with various degrees of patchy P-BSA-FITC labeling (for instance see Fig. 1B). Most nonviable spermatozoa (EthD-1 positive) displayed postacrosomal P-BSA-FITC staining (Fig. 1C), while some displayed no staining at all. Only few deteriorated cells (i.e., with EthD-1 and postacrosomal P-BSA-FITC staining) were also stained at the apical head region with P-BSA-FITC (< 3% of the deteriorated sperm cell population). Postacrosomal P-BSA-FITC staining was not observed in living sperm cells (i.e., EthD-1-negative cells). When BSA-FITC or E-BSA-FITC was applied instead of P-BSA-FITC, no staining of the acrosomal cap was observed, demonstrating the involvement of the progesterone moiety in the binding of P-BSA-FITC to stallion sperm.

View larger version (14K): [in this window] [in a new window]   FIG. 1. Fluorescence pattern of stallion spermatozoa preincubated in SP-TALP for 4 h and stained for progesterone receptor localization. A) Binding of P-BSA-FITC to a viable spermatozoon displaying fluorescence over the acrosomal region of the head. B) Same, but patchy apical P-BSA-FITC binding. C) Binding of P-BSA-FITC to a nonviable spermatozoon (EthD-1 positive) displaying postacrosomal staining. D) Localization of the progesterone receptor on a spermatozoon by indirect immunofluorescence with monoclonal antibody against progesterone receptor. x2500.

Thirty-eight percent of the viable spermatozoa displayed P-BSA-FITC staining on the acrosomal cap. In total, 88% of the viable spermatozoa, including almost all apical P-BSA-FITC-stained spermatozoa, had an intact acrosome. Pretreatment with 32 µM progesterone reduced the proportion of spermatozoa displaying P-BSA-FITC staining on the acrosomal cap from 38% to 7% (Fig. 2A), demonstrating competition in receptor binding.

View larger version (20K): [in this window] [in a new window]   FIG. 2. P-BSA-FITC and BSA-FITC binding to stallion spermatozoa. A) Conventionally washed stallion semen preincubated in SP-TALP for 4 h. B) Percoll-washed sperm cells. Black bars represent percentage of viable spermatozoa displaying P-BSA-FITC binding to the acrosomal region without any pretreatment and after a 5-min pretreatment with 32 µM progesterone. Open bars represent the percentage of live acrosome-intact spermatozoa (n = 200 viable spermatozoa). Means ± SD from ejaculates from 3 fertile stallions are shown.

The localization of the progesterone receptor in preincubated stallion spermatozoa was also determined by indirect immunofluorescence using C-262 against the progesterone receptor. After fixation, spermatozoa with immunoreactivity against C-262 exhibited a staining pattern on the acrosomal cap (Fig. 1D) that was similar to P-BSA-FITC staining. Treatment of a sperm sample with C-262 resulted in 22% of spermatozoa displaying acrosomal cap staining (n = 200), whereas a parallel sample treated with P-BSA-FITC resulted in 24% of spermatozoa displaying acrosomal cap staining. No staining of acrosomal cap was observed when spermatozoa were incubated with RAM-FITC without prior treatment with C-262. Some spermatozoa (15%, corresponding to 19% of P-BSA-FITC staining) displayed a postacrosomal staining pattern (as seen in Fig. 1C), characteristic for nonviable spermatozoa.

To determine the C-262 affinity for sperm progesterone receptor, Western blots of SDS-PAGE were used to separate human and stallion sperm protein samples, according to the procedure of Sabeur et al. [15], and positive protein bands were obtained that were similar to those described previously ([15], data not shown). In order to detect affinity of C-262 on the sperm protein samples, we carried out ELISA and spot-blot assays with native and with reduced proteins (the latter were also used for SDS-PAGE). Importantly, we detected that the affinity of C-262 for the reduced progesterone receptor decreased by a factor of more than 70 in comparison to its affinity for the native receptor. Similarly, P-BSA-FITC did not have any affinity for denatured sperm proteins but had high affinity for native stallion and human sperm proteins.

Ultrathin cryosections were made from sperm samples prelabeled with P-BSA-FITC, BSA-FITC, E-BSA-FITC, RAM-FITC, or C-262/RAM-FITC. FITC bound to sperm was visualized for electron microscopy using an anti-FITC antibody in combination with protein A-gold. The labeling patterns confirmed the observations made by fluorescence microscopy. Three types of immunogold labeling patterns were seen on sperm cells prelabeled with P-BSA-FITC: 1) a subpopulation of intact cells not labeled with gold particles (not shown); 2) a subpopulation of sperm cells that was exclusively labeled at the apical plasma membrane (viable cells, Fig. 3A); and 3) a subpopulation of sperm cells labeled in the cytosol of the postequatorial region of the sperm head (deteriorated cells, Fig. 3B). The cell depicted in Figure 3B is considered to be deteriorated since postacrosomal P-BSA-FITC labeling was observed only in EthD-1-positive (i.e., membrane damaged) cells (observation made by fluorescence microscopy; for pattern see Fig. 1C). Probably deterioration of the cell depicted in Figure 3B occurred after the initiation of the AR, since the cell has lost the apical plasma membrane while mixed vesicles (between plasma membrane and the outer acrosomal membrane) have emerged at the equatorial segment. The apical P-BSA-FITC binding sites on the cell surface disappear after the loss of the apical plasma membrane of stallion sperm cells. In fact, many free P-BSA-FITC-positive acrosomal caps could be detected in our microscopical preparations. Similar immunogold labeling patterns were observed for sperm cells prelabeled with C-262/RAM-FITC. In control labeling experiments, no label was detected on sperm cells incubated with RAM-FITC (omission of C-262), BSA-FITC, or E-BSA-FITC (data not shown).

View larger version (114K): [in this window] [in a new window]   FIG. 3. Immunogold labeling of ultrathin cryosections of spermatozoa previously labeled with P-BSA-FITC using rabbit anti-FITC followed by protein A-gold (10 nm). A) The exclusive localization of the receptor on the apical region of the plasma membrane (arrow) is shown. B) Postequatorial labeling in the cytosol on deteriorated sperm cell. Note the absence of the apical head plasma membrane and the presence of membrane vesicles at the equatorial head region. Bar = 0.5 µm.

Incubation of preincubated spermatozoa in SP-TALP supplemented with either P-BSA-FITC or progesterone showed an increase in the percentage of viable acrosome-reacted spermatozoa in comparison to incubation of spermatozoa in nonsupplemented SP-TALP (p < 0.05). The percentage of viable AR spermatozoa was higher after incubation with progesterone than the percentages after incubation with P-BSA-FITC (p < 0.05). Sperm incubation with either progesterone or P-BSA-FITC only marginally affected the viability of sperm cells within a period of 5 min, as determined by the proportion of spermatozoa negative for EthD-1 (Fig. 4).

View larger version (18K): [in this window] [in a new window]   FIG. 4. Effect of P-BSA-FITC and progesterone on stallion spermatozoa preincubated for 4 h in SP-TALP. Spermatozoa were incubated in SP-TALP containing 100 µg/ml P-BSA-FITC or 10 µM progesterone for 30 min. Means ± SD of ejaculates from 3 fertile stallions are shown.

The effect of a prolonged incubation in SP-TALP on the P-BSA-FITC binding is presented in Figure 5. Initially, the proportion of live spermatozoa (n = 200) that displayed staining with P-BSA-FITC on the whole acrosomal cap increased with incubation time. After 5-h incubation, this proportion did not increase further, and eventually only 42% of viable spermatozoa displayed staining on the acrosomal cap. Concurrently, both sperm viability and motility decreased with incubation time from 88% and 83% to 57% and 43%, respectively, after 7-h incubation.

View larger version (17K): [in this window] [in a new window]   FIG. 5. Effect of prolonged incubation in SP-TALP on the P-BSA-FITC binding to stallion spermatozoa. Percentage of viable spermatozoa (n = 200) demonstrating P-BSA-FITC binding to the acrosomal region is shown; means ± SD of ejaculates from 3 fertile stallions.

Interestingly, when sperm cells were washed to a discontinuous Percoll gradient, more than 95% of the living sperm cells showed apical P-BSA-FITC labeling (Fig. 2B). Similar to findings for SP-TALP-washed sperm cells (Fig. 2A), P-BSA-FITC binding was efficiently blocked by a preincubation of 5 min with 32 µM progesterone (apical labeling observed for only 15% of the cells; Fig. 2B).

When spermatozoa collected from the swim-up fraction were treated with P-BSA-FITC and EthD-1 and analyzed with a flow cytometer (n = 10 000), only a small proportion of spermatozoa were nonviable (< 10%), and essentially all of these nonviable cells were P-BSA-FITC positive. Nonviable spermatozoa had a characteristic P-BSA-FITC staining pattern as illustrated in Figure 1C. Fourteen percent of the viable spermatozoa were P-BSA-FITC positive (Fig. 6A), similar to the data (11%) obtained from fluorescence microscopy observations. After further incubation, the percentage of P-BSA-FITC-positive viable spermatozoa increased to 25%, 37%, and 52% after 1-, 3-, and 5-h incubation, respectively (Fig. 6, B–D). The corresponding data obtained from fluorescence microscopy were 22%, 33%, and 46%. Sperm viability decreased in time, and the percentages of dead spermatozoa as analyzed by flow cytometry were 15%, 26%, and 34% after 1-, 3-, and 5-h incubation, respectively (Fig. 6, B–D). The corresponding data obtained from fluorescence microscopy were 19%, 25%, and 37%.

View larger version (81K): [in this window] [in a new window]   FIG. 6. Flow cytometry detection of P-BSA-FITC binding to stallion spermatozoa. Sperm cells were preincubated in SP-TALP for 0 h (A), 1 h (B), 3 h (C), and 5 h (D). Spermatozoa were stained with P-BSA-FITC and EthD-1. The amount of P-BSA-FITC binding as detected by the FL-1 detector in arbitrary units (A.U.) is indicated on the x-axis. The amount of EthD-1 binding per sperm cell as detected by the FL-3 detector in arbitrary units is indicated on the y-axis. All sperm cell events with a greater amount of EthD-1 binding than indicated by the horizontal line were gated as nonviable cells (indicated as red dots). The viable sperm cells with greater amount of P-BSA-FITC binding than indicated by the vertical line were gated as spermatozoa demonstrating the progesterone receptor (indicated as green dots). The black dots represent sperm cells that were not stained by P-BSA-FITC and EthD-1. Each panel depicts 10 000 specific sperm events.

The amount of autofluorescence and binding of P-BSA-FITC, E-BSA-FITC, or BSA-FITC to spermatozoa was analyzed by flow cytometry in the FL-1 detector. Spermatozoa not incubated with P-BSA-FITC had an FL-1 signal identical to that of spermatozoa classified as negative after P-BSA-FITC treatment (autofluorescence). Spermatozoa scored as P-BSA-FITC positive showed a 10-fold increase of FL-1 signal. Spermatozoa stained with BSA-FITC or E-BSA-FITC gave only a weak FL-1 signal that was not distinguishable from the autofluorescent signal.

	DISCUSSION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
The existence of binding sites for P-BSA-FITC conjugate on the plasma membrane of stallion spermatozoa is supported by the staining of acrosome-intact spermatozoa and the nature of this membrane-impermeable conjugate. The decreasing number of spermatozoa displaying this binding after pretreatment with progesterone, and the observation that BSA-FITC as well as E-BSA-FITC did not bind to viable spermatozoa, support the conclusion of the existence of the progesterone receptor. Further evidence for the presence and localization of the progesterone receptor is provided by the observed staining patterns of spermatozoa with C-262 (a monoclonal antibody against the progesterone receptor) and RAM-FITC, which corresponded to those observed with P-BSA-FITC. C-262 recognizes the progesterone-binding site in the carboxyl-terminus of cytosolic/nuclear progesterone receptor in somatic cells [25] and also the progesterone-binding site of the nongenomic progesterone receptor in sperm cells. C-262 also blocks the progesterone effect on human sperm AR by inhibiting the Ca²⁺ and Cl^- fluxes [15]. Thus, C-262 can be considered as a kind of antagonist that binds the progesterone receptor. Immunogold labeling of P-BSA-FITC showed the exclusive localization of the gold particles on the apical plasma membrane of viable cells (Fig. 3A) and in the cytosol of deteriorated cells (Fig. 3B). Identical results were obtained by immunogold labeling of C-262/RAM-FITC. The presence of progesterone receptors in the apical plasma membrane of spermatozoa has previously been reported for humans [27, 28]. In contrast, Sabeur et al. [15] demonstrated progesterone binding to the equatorial region on the human sperm head. Since Sabeur et al. [15] subjected sperm cells to ethanol permeabilization prior to the immunolabeling procedure, this postequatorial staining most likely reflects intracellular labeling of progesterone receptors (as is depicted for deteriorated stallion sperm cells in Figs. 1B and 3B).

The data presented in Figure 5 showed that at onset of incubation the progesterone receptor was available in a rather low percentage of spermatozoa (< 10%). This percentage gradually increased during incubation, reaching a maximum level after 5-h incubation. The results of flow cytometry analysis supported these dynamic changes in the sperm population demonstrating the presence of the progesterone receptor. Thus, only a limited proportion of the sperm population in an ejaculate appears to show the progesterone receptor after prolonged incubation. Tesarik et al. [28] also reported the presence of a selective sperm population demonstrating the progesterone receptor in humans. Protein synthesis is completely shut down during spermatogenesis [29], and vesicle-mediated protein transport to the plasma membrane has not been demonstrated in mature sperm cells [30]. Therefore, the progesterone receptor is most likely present on spermatozoa before ejaculation but coated by extracellular material. Although (glyco)proteins from seminal plasma are reported to form such a protective coat covering the sperm plasma membrane [2, 31, 32], preliminary experiments demonstrated that cauda epididymal sperm cells did not show any affinity for C-262/RAM-FITC or for P-BSA-FITC. Therefore, the progesterone receptor is probably already coated by proteins that originate from higher parts of the epididymis or the testis before mixing with accessory sex gland secretions during ejaculation. The temporary coat may sterically hinder the binding of progesterone to its receptor on the apical plasma membrane and thus prevent subsequent intracellular signal transduction evoked by progesterone binding [10, 15, 33–36]. During capacitation, this protection is then gradually removed from the plasma membrane, especially in the acrosomal region [2, 31, 32], resulting in the exposure of the progesterone receptor. In this scenario the sperm uncoating process of P-BSA-FITC-negative sperm cells was still not effective. An alternative explanation for the sperm cell population that remained negative for P-BSA-FITC or for C-262/RAM-FITC staining (after 5-h capacitation period; Figs. 5 and 6) is the possibility that they lack the progesterone receptor. However, this is unlikely, since after Percoll washing, most cells (> 95%) were positively stained for the progesterone receptor at the apical plasma membrane (Fig. 2B). In fact, those cells were more sensitive to progesterone treatments than conventional washed cells (data not shown). The observed low percentage of sperm cells that stained positively for the progesterone receptor prior to incubation explains why progesterone cannot effectively induce the AR in freshly ejaculated stallion spermatozoa [11]. Interestingly, it has been shown that the inability of spermatozoa to respond to progesterone in vitro might be related to male infertility [11, 33, 37]. During the AR, sperm cells have a patchy apical P-BSA-FITC or C-262/RAM-FITC labeling (Fig. 1C). Deteriorated cells were labeled at the postacrosomal region (Figs. 1C and 3B). The damaged plasma membrane enables labeling of a cytosolic progesterone receptor at the postequatorial region of the sperm head (Figs. 1C and 3B). Only a few deteriorated cells could be detected with a combination of patchy apical labeling together with the postequatorial labeling (probably cells that deteriorated during the AR). The majority of deteriorated cells, however, showed only postacrosomal intracellular P-BSA-FITC binding sites. This could be the case because such cells have shed the apical plasma membrane (with its P-BSA-FITC binding sites) during the AR, prior to deterioration, as is depicted for one sperm cell in Figure 3B. The fact that we often observed P-BSA-FITC-positive free acrosome caps in our microscopical preparations supports this idea. An alternative explanation is that the deteriorated cells did not expose the progesterone receptor prior to deterioration.

Flow cytometry analysis of P-BSA-FITC conjugate binding to spermatozoa demonstrates three distinct subpopulations during incubation time. One population corresponds to viable spermatozoa that are not stained with P-BSA-FITC. One population corresponds to viable spermatozoa that are positively stained with P-BSA-FITC and a population of nonviable spermatozoa (see Fig. 6). Similar to the observation with fluorescence microscopy, the flow cytometry analyses showed a clear exposure of the progesterone receptor on the sperm surface during the 5-h incubation period. Simultaneously, a subpopulation of spermatozoa demonstrating the progesterone receptor deteriorated and became nonviable during the incubation. These nonviable spermatozoa remained P-BSA-FITC positive, probably due to postacrosomal cytosolic staining (see Figs. 1C and 3B). Although a slightly higher proportion of P-BSA-FITC spermatozoa was determined with the flow cytometer in comparison with the fluorescence microscopy observation, the differences were smaller than 3–6%. It should be mentioned that P-BSA-FITC-stained sperm samples were analyzed as fixed samples for the fluorescence microscopy but were not fixed for analysis with the flow cytometer [38]. This may explain the small differences observed. Furthermore, the flow cytometer cannot distinguish between different localization patterns of P-BSA-FITC but only discriminates between the intensity of P-BSA-FITC bound to individual sperm cells. The difficulty in distinguishing the actual staining patterns was probably due to a variability in the intensity of fluorescence emitted by a unit area of sperm surface, as related to the receptor density per unit area [28]. It is therefore possible that some spermatozoa weakly stained with P-BSA-FITC were determined to be positive using flow cytometry but negative using fluorescence microscopy due to difference in sensitivity of the FL-1 detector compared to the naked eye.

C-262- and P-BSA-conjugates have affinity only for the native, not for the denatured progesterone receptor of human and sperm samples. Therefore, we believe that identification of the progesterone receptor is not possible with Western blots of SDS-PAGE-separated sperm protein samples (although similar results were obtained on Western blots of SDS-PAGE human sperm protein samples as described by Sabeur et al. [15]). Another line of evidence for the presence of a progesterone receptor is the fact that for P-BSA-FITC and for C-262/RAM-FITC, 1) fluorescent labeling patterns (Fig. 1) and relative proportions of sperm cell populations with one type of labeling (Fig. 4) were identical and 2) ultrastructural localization was identical (Fig. 3). Furthermore, progesterone competed with P-BSA-FITC for binding to the apical plasma membrane (Fig. 2) and short-time incubations of sperm cells with progesterone or P-BSA-FITC resulted in the induction of the AR (Fig. 4). Therefore, it can be concluded that at least the progesterone receptor of the apical plasma membrane (Figs. 1, A and D, and 3B) is involved in the induction of the AR in stallion sperm (P-BSA-FITC is membrane impermeable). Free progesterone can diffuse over the plasma membrane and thus can bind to the cytosolic progesterone receptors of the postequatorial head region of sperm cells (Figs. 1B and 3B). Most likely the slightly enhanced effect of free progesterone when compared to P-BSA-FITC on the induction of the AR (Fig. 4) is due to a different affinity for the progesterone receptor. However, the alternative explanation, that progesterone binding to the cytosolic receptor has an additive effect on the induction of the AR, cannot be excluded.

Progesterone and zona glycoprotein (ZP3) can induce the AR but do not exert their effects through the same receptor, as far as the receptor-mediated ions fluxes are concerned [17, 35, 36, 39]. Despite the molecular dissimilarity of progesterone and ZP3, each of them can activate at least two different types of receptors responsible for Ca²⁺ channels—one reacting rapidly, and thus probably activated directly by the inducer, and the other responding after some delay and probably regulated by the inducer indirectly via a signaling cascade [34, 36, 40]. The nature of the stallion sperm progesterone receptor as well as the putative Ca²⁺ channel involved in signaling remains to be elucidated. In human spermatozoa, the molecular mass recognized by C-262 was 50–52 kDa protein, which is different from that of the A and B isoforms of human intracellular progesterone receptor, 94 and 120 kDa, respectively [15, 25]. A specific membrane protein with affinity for progesterone has been isolated from rat brain that has an estimated molecular mass of 40–50 kDa [41].

Conclusively, an increasing amount of stallion spermatozoa expose a plasma membrane progesterone receptor during in vitro capacitation treatment. The coupling of progesterone to its sperm receptor is an important step toward the AR induction. Progesterone as an inducer of the AR is an alternative to the zona pellucida [10, 12, 17]. Both inducers are present in the vicinity of the ovulated oocyte. Thus, progesterone might virtually participate in this biologically important acrosomal exocytosis in an additive or synergistic manner, leading to a successful fertilization. The exposure of the progesterone receptor and the subsequent coupling of progesterone are probably involved in sperm binding to the zona pellucida as well as zona-induced AR [12].

	ACKNOWLEDGMENTS

 
We thank Drs. F.M. Flesch, E. Wijnand, Ing. P.J.M. Ursem, A. Marks, A.R. Zandee, and E. Zeinstra for their technical assistance and I. Revah, DVM, PhD, for her critical reading of the manuscript.

	FOOTNOTES

 
¹ Correspondence: Ben Colenbrander, Department of Herd Health & Reproduction, Veterinary Faculty, Utrecht University, Yalelaan 7, 3584 CL Utrecht, The Netherlands. FAX: 31–30 2521887; colenbr{at}bdv.dgk.ruu.nl

Accepted: May 5, 1998.

Received: July 25, 1997.

	REFERENCES

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 

1. Gadella BM, Lopes-Cardozo M, van Golde LMG, Colenbrander B, Gadella Jr TWJ. Glycolipid migration from the apical to the equatorial subdomains of the sperm head plasma membrane precedes the acrosome reaction: evidence for a primary capacitation event in boar spermatozoa. J Cell Sci 1995; 108:935–945.[Abstract]
2. Yanagimachi R. Mammalian fertilization. In: Knobil E, Neill JD (eds.), The Physiology of Reproduction, 2nd ed. New York: Raven Press; 1994: 189–317.
3. Brucker C, Lipford GB. The human sperm acrosome reaction: physiological and regulatory mechanisms, an update. In: Edwards RG (ed.), Human Reproduction Update. Oxford: Academic Press; 1995: 1:51–62.
4. Ellington JE, Ball BA, Yang X. Binding of stallion spermatozoa to the equine zona pellucida after coculture with oviductal epithelial cells. J Reprod Fertil 1993; 98:203–208.[Abstract]
5. Cheng FP, Fazeli AR, Voorhout W, Bevers MM, Colenbrander B. Use of PNA (peanut agglutinin) to assess acrosomal status and the zona pellucida induced acrosome reaction in stallion spermatozoa. J Androl 1996; 17:674–682.[Abstract/Free Full Text]
6. Meyers SA, Liu IKM, Overstreet JW, Vadas S, Drobnis EZ. Zona pellucida binding and zona-induced acrosome reactions in horse spermatozoa: comparisons between fertile and subfertile stallions. Theriogenology 1996; 46:1277–1288.
7. Fehl P, Miska W, Henkel R. Further indications of the multicomponent nature of the acrosome reaction-inducing substance of human follicular fluid. Mol Reprod Dev 1995; 42:80–88.[CrossRef][Medline]
8. Meizel S, Turner OK. Progesterone acts at the plasma membrane of human sperm. Mol Cell Endocrinol 1991; 11:R1-R5.
9. Melendrez CS, Meizel S, Berger T. Comparison of the ability of progesterone and heat solubilized porcine zona pellucida to initiate the porcine sperm acrosome reaction in vitro. Mol Reprod Dev 1994; 39:433–438.[CrossRef][Medline]
10. Roldan ERS, Murase T, Shi QX. Exocytosis in spermatozoa in response to progesterone and zona pellucida. Science 1994; 266:1578–1581.[Abstract/Free Full Text]
11. Meyers SA, Overstreet JW, Liu IKM, Drobnis EZ. Capacitation in vitro of stallion spermatozoa: comparison of progesterone-induced acrosome reactions in fertile and subfertile males. J Androl 1995; 16:47–54.[Abstract/Free Full Text]
12. Cheng FP, Fazeli AR, Voorhout WF, Tremoleda JL, Bevers MM, Colenbrander B. Progesterone in mare follicular fluid induces the acrosome reaction in stallion spermatozoa and enhances in vitro binding to the zona pellucida. Int J Androl 1998; 21:57–66.[CrossRef][Medline]
13. Genuth SM. Female reproduction. In: Berne RM, Levy MN (eds.), Principles of Physiology. London: Wolfe Publishing; 1990: 599–615.
14. Castilla JA, Gil T, Molina J, Hortas ML, Rodriguez F, Torres-Munoz J, Vergara F, Herruzo AJ. Undetectable expression of genomic progesterone receptor in human spermatozoa. Hum Reprod 1995; 7:1757–1760.
15. Sabeur K, Edwards DP, Meizel S. Human sperm plasma membrane progesterone receptor(s) and the acrosome reaction. Biol Reprod 1996; 54:993–1001.[Abstract]
16. Ramirez VD, Zheng J, Siddique KM. Membrane receptors for estrogen, progesterone and testosterone in the rat brain: fantasy or reality. Cell Mol Neurobiol 1996; 16:175–198.[CrossRef][Medline]
17. Meizel S. Amino acid neurotransmitter receptor/chloride channels of mammalian sperm and the acrosome reaction. Biol Reprod 1997; 56:569–574.[Abstract]
18. Wehling M. Specific, nongenomic action of steroid hormones. Ann Rev Physiol 1997; 59:365–393.[CrossRef][Medline]
19. Malmgren L, Op den Kamp B, Wochener A, Boyle M, Colenbrander B. Motility, velocity and acrosome integrity of equine spermatozoa stored under different conditions. Reprod Domest Anim 1994; 29:469–476.
20. Parrish JJ, Susko-Parrish J, Winer MA, First NL. Capacitation of bovine sperm by heparin. Biol Reprod 1988; 38:1171–1180.[Abstract]
21. Borg K, Colenbrander B, Fazeli A, Parlevliet J, Malmgren L. Influence of thawing method on motility, plasma membrane integrity and morphology of frozen-thawed stallion spermatozoa. Theriogenology 1997; 48:531–536.
22. Tao J, Critser ES, Critser JK. Evaluation of mouse sperm acrosomal status and viability by flow cytometry. Mol Reprod Dev 1993; 36:183–194.[CrossRef][Medline]
23. Johnson GD, Gloria M. A simple method of reducing the fading of immunofluorescence during microscopy. J Immunol Methods 1981; 43:349–350.[CrossRef][Medline]
24. Parlevliet JM, Kemp B, Colenbrander B. Reproductive characteristics and semen quality in maiden Dutch Warmblood stallions. J Reprod Fertil 1994; 101:183–187.[Abstract]
25. Weigel NL, Beck CA, Estes PA, Prendergast P, Altmann M, Christensen K, Edwards DP. Ligands induce conformational changes in the carboxyl-terminus of progesterone receptors which are detected by a site-directed antipeptide monoclonal antibody. Mol Endocrinol 1992; 6:1585–1597.[Abstract]
26. Farlin ME, Jasko DJ, Graham JK, Squires EL. Assessment of pisum sativum agglutinin in identifying acrosomal damage in stallion spermatozoa. Mol Reprod Dev 1992; 32:23–27.[CrossRef][Medline]
27. Blackmore PF, Lattanzio FA. Cell surface localization of a novel non-genomic progesterone receptor on the head of human sperm. Biochem Biophys Res Commun 1991; 181:331–336.[CrossRef][Medline]
28. Tesarik J, Mendoza C, Moos J, Carreras A. Selective expression of a progesterone receptor on the human sperm surface. Fertil Steril 1992; 58:784–792.[Medline]
29. Stewart TA, Bellvé AR, Leder P. Transcription and promotor usage of the myc gene in normal somatic and spermatogenic cells. Science 1984; 226:707–710.[Abstract/Free Full Text]
30. Eddy EM, O'Brien DA. The spermatozoon. In: Knobil E, Neill JD (eds.), The Physiology of Reproduction, 2nd edition. New York: Raven Press; 1994: 29–77.
31. Aonuma S, Mayumi T, Suzuki K, Noguchi T, Iwai M, Okabe M. Studies on sperm capacitation. I. The relationship between a given sperm-coating antigen and a sperm capacitation phenomenon. J Reprod Fertil 1973; 35:425–432.[Medline]
32. Oliphant G. Removal of sperm seminal plasma components as a prerequisite to induction of the acrosomal reaction. Fertil Steril 1976; 27:28–38.[Medline]
33. Tesarik J, Mendoza C. Defective function of a nongenomic progesterone receptor as a sole sperm anomaly in infertile patients. Fertil Steril 1992; 58:793–797.[Medline]
34. Tesarik J, Carreras A, Mendoza C. Single cell analysis of tyrosine kinase dependent and independent Ca²⁺ fluxes in progesterone induced acrosome reaction. Hum Reprod 1996; 11:225–232.[Free Full Text]
35. Melendrez CS, Meizel S. Studies of porcine and human sperm suggesting a role for a sperm glycine receptor/Cl^- channel in the zona pellucida-initiated acrosome reaction. Biol Reprod 1995; 53:676–683.[Abstract]
36. Mendoza C, Soler A, Tesarik J. Nongenomic steroid action: independent targeting of a plasma membrane calcium channel and a tyrosine kinase. Biochem Biophys Res Commun 1995; 10:518–523.
37. Oehninger S, Blackmore P, Morshedi M, Sueldo C, Acosta AA, Alexander NJ. Defective calcium influx and acrosome reaction (spontaneous and progesterone-induced) in spermatozoa of infertile men with severe tetratozoospermia. Fertil Steril 1994; 61:349–354.[Medline]
38. Keij JF, Herweijer H. Flow cytometric analysis and sorting of viable cells. In: Mason WT (ed.), Fluorescent and Luminescent Probes for Biological Activity. Cambridge: Academic Press; 1993: 310–319.
39. Wistrom C, Meizel S. Evidence suggesting involvement of an unique human sperm steroid receptor/Cl^- channel complex in the progesterone-initiated acrosome reaction. Dev Biol 1993; 159:679–690.[CrossRef][Medline]
40. Florman HM. Sequential focal and global elevations of sperm intracellular Ca²⁺ are initiated by the zona pellucida during acrosomal exocytosis. Dev Biol 1994; 165:152–164.[CrossRef][Medline]
41. Tischkau SA, Ramirez VD. A specific membrane binding protein for progesterone in rat brain: sex differences and induction by estrogen. Proc Natl Acad Sci USA 1993; 90:1285–1289.[Abstract/Free Full Text]