Instituto de Histología y Embriología (IHEM), Centro Universitario, Facultad de Ciencias Médicas, Universidad Nacional de Cuyo—CONICET, Parque Gral, San Martín, Casilla de Correo 56, Mendoza 5500, Argentina (e-mail: mfornes@ fmed2.uncu.edu.ar).
ABSTRACT: The goals of the present study were to determine the availability of progesterone (P4) receptor (P4r) in mouse sperm during maturation and capacitation and to make the first steps toward a characterization of P4r. It has been proposed that P4 is able to induce an acrosomal reaction (AR) by using a membrane P4r. This induction was verified in sperm isolated from the cauda epididymis (fully mature) when incubated in specific conditions that capacitate sperm. First, we set up the conditions in our laboratory to induce an AR in mature and capacitated sperm triggered by P4 that was detected by a chlortetracycline (CTC) assay. Then, we examined sperm isolated from the caput epididymis (immature) incubated under conditions that support cauda sperm capacitation and found that the AR could not be detected. Moreover, P4 was unable to induce the AR when it was applied to sperm isolated from either region and incubated under conditions that did not support capacitation. These results can be explained by changes in P4r availability. A suitable marker for P4r is the gold (Au)-P4 complex. This marker shows a binding capacity that can be visualized directly by electron microscopy (EM) and indirectly by silver-enhanced methods with light microscopy. The Au-P4 complex was localized in capacitated cauda sperm at the dorsal edge of the head. Using these techniques, we observed a significant decrease in both noncapacitated cauda sperm and caput sperm (whether incubated in capacitating media or not). Genomic P4r could be responsible for the signal detected, but antibodies against the P4 nuclear receptor did not recognize any sites in the sperm by immunostaining methodology. Instead, a 44-kd protein band was detected in the sperm by a ligand blot assay. In conclusion, P4 promotes the AR in capacitated cauda sperm but is unable to do so in noncapacitated or immature sperm because the availability of P4r increases during epididymal transit and after capacitation. The P4r responsible for this behavior is different from a classical nuclear receptor—on the basis of the immunostaining results—and is probably a protein close to 44 kd—on the basis of the ligand assay results.
Progesterone (P4) has been proposed as a trigger for the acrosomal reaction (AR) directly or in association with the zona pellucida protein (ZP3)-priming mechanism (Roldan et al, 1994). Mouse sperm react in the presence of isolated ZP3 or P4 (Roldan et al, 1994) as do human (Sueldo et al, 1993; Meizel, 1997), hamster (Llanos and Anabalon, 1996), pig (Melendrez et al, unpublished data), and horse (Meyers et al, unpublished data; Cheng et al, 1998a,b) spermatozoa. In human and stallion sperm, there is evidence for a P4 receptor (P4r) at the sperm surface (Blackmore et al, 1991; Cheng et al, 1998b). In human sperm, P4 induces a rapid influx of calcium, calcium-dependent phosphoinositide hydrolysis, an increase in tyrosine phosphorylation of sperm proteins, and chloride efflux, which ultimately leads to the stimulation of an AR (Baldi et al, 1998). In mouse sperm, an increase in intracellular calcium was also observed after the addition of P4 to the culture medium preceding the acrosomal exocytosis (Kobori et al, 2000).
Some controversy exists about the specific but nongenomic actions of steroid hormones, as documented in a recent literature review (Wehling, 1997; Revelli et al, 1998; Maller, 2001). There is well-characterized nongenomic P4 action in amphibian oocytes. During the resumption of meiosis, P4 activates a signal transduction pathway that is related to germinal vesicle breakdown (Palmer and Nebreda, 2000; Maller, 2001). Regardless of the signal transduction pathway activated by P4—whether it is a nuclear receptor or a distinct receptor located on the cell membrane—there is evidence that P4 induces an AR in sperm that lack the nuclear receptors acquired by a nongenomic pathway (Meyers et al, unpublished data; Meizel and Turner, 1991; Sabeur and Meizel, 1996).
Finally, using a ligand blot analysis, P4-binding proteins have been reported in capacitated human spermatozoa. It has also been reported that the membrane fraction analysis shows the existence of P4-binding proteins (Luconi et al, 1998) and that sperm lack genomic P4rs (Castilla et al, 1995).
In this study, we describe a modification in the P4 membrane receptor of mouse spermatozoa that takes place during the epididymal transit and the capacitation process. Through an immunostaining method, we also present evidence for the absence of a P4 nuclear receptor, and we take the first steps toward a characterization of the P4-binding protein by ligand blot analysis.
Materials and Methods
Reagents were obtained from Sigma Chemical Company (St Louis, Mo), except for the electron microscopy (EM) reagents that were obtained from Pelco (Redding, Calif) and the antibodies that were obtained from Dako Corporation (Carpinteria, Calif).
Collection of Gametes and Capacitation
Mouse sperm were obtained by cutting the caput or cauda epididymis from mature BALB/c outbred mice in several places and isolating them under HM medium for 15 minutes. HM was made by thawing 5 mL of 3× HM stock and adding the following reagents: sodium pyruvate (1.65 mg), 17 mM of CaCl2 (1.5 mL), and 8.5 mL of double-distilled water. Finally, the pH was adjusted to 7.3 with 10 M of NaOH (HM 1× consists of 25 mM of HE-PES, 109 mM of NaCl, 4.77 mM of KCl, 1.19 mM of MgSO4 0.7 H2O, 1 mg/mL of glucose, 3.7 μL of Na-lactate (60% syrup), and 1.19 mM of KH2PO4) (Visconti et al, 1995). After 15 minutes, the epididymides were removed, and the remaining “caput” or “cauda” spermatozoa were washed in HM by a centrifugation (600 × g for 10 minutes)-resuspension protocol. Final pellets were resuspended and adjusted to 1 × 106 sperm/mL in HMB medium and incubated for 90 minutes in an incubator (NAPCO, Winchester, Va) at 37°C and 5% CO2 in air. HMB was made from 15 mL of HM media along with 25 mM of NaHCO3 and 3 mg/mL of BSA (pH 7.3) (Visconti et al, 1995). The resultant sperm were considered capacitated (C) only in the case of cauda sperm or just incubated (I) in the case of the caput epididymis because caput sperm cannot be capacitated. Some pellets from caput and cauda sperm were suspended in HM— which does not support capacitation—and immediately processed to avoid any incubation time. These sperm are called noncapacitated (non-C) for cauda sperm or nonincubated (non-I) for caput sperm. These 2 experimental conditions—1) non-C = HM and nontime, and 2) C = HMB and time—produced 4 types of sperm: cauda capacitated (C), caput incubated (I), cauda noncapacitated (non-C), and caput nonincubated (non-I).
Several times during each experiment, the motility of the sperm was checked using 40 μL of sperm suspension viewed at 400× with a light microscope. This permitted a quick check of the viability of the sperm cells. At the end of incubation period, 20 μL of sperm suspension was mixed with 20 μL of 1% eosin Y in phosphate-buffered saline (PBS: 20 mM of phosphate buffer and 150 mM of NaCl, pH 7.4; Sigma tablets), and vitality was checked again.
Induction of the AR by P4
Capacitated sperm were induced to react by adding the following reagents to the sperm suspension for an additional 15 minutes: 10 μM of P4 or 10 μM of calcium ionophore A23187 (A23187) (final concentrations). P4 was solubilized in dimethylsulfoxide (DMSO; 50 mM as stock solution), aliquoted, and stored at −18°C. Ionophore was solubilized in DMSO (100 mM as stock solution), aliquoted, and also stored at −18°C. Controls were performed by using no induction reagents. The doses used in this study were selected on the basis of information gathered from reviewed literature or from preliminary experiments. The acrosomal status was evaluated by the chlortetracycline (CTC) method (see below). Non-C sperm were exposed to the same reagents and conditions.
It has been postulated that caput sperm are not capable of undergoing capacitation and for this reason do not undergo an AR (Visconti et al, 1995; Kopf et al, 1998). Because of this, P4 was added to the I or non-I sperm suspensions, and the AR was scored. As a control, A23187 was also added to parallel tubes.
The CTC assay was performed following the technique of Kholkute et al (1995). Briefly, the CTC solution was made by dissolving CTC-HCl at a concentration of 500 μM in a buffer containing 20 mM of Tris HCl, 130 mM of NaCl, and 5 mM of cysteine for a final pH of 7.8. Fresh CTC solution was made before each assay. Fifty microliters of sperm suspension from the 4 types of sperm was mixed with 50 μL of CTC solution. After a few seconds, the sperm were fixed by the addition of 8 μL of 12.5% glutaraldehyde in PBS. The sperm suspension was washed with PBS by centrifugation at 120 × g for 10 minutes. The pellets were resuspended in 100 μL of PBS, and 7 μL of this suspension was placed on a clear slide with a drop of 10 mg/mL of n-propylgallate and 50% glycerol (all in PBS). Spermatozoa were examined for CTC fluorescence at a magnification of 600× on a microscope (Optiphot-2, excitation at 400–440 nm and emission at 455 nm; Nikon Instruments Inc, Melville, NY). A total of 100 spermatozoa were scored on each slide. As reported in a previous study (Pietrobon et al, 2001), 2 CTC-staining patterns were observed in C sperm: 1) head fluorescence with a fluorescence-free band in the postacrosomal region, which is characteristic of capacitated and nonreacted sperm; and 2) a variation in head fluorescence between a weakly homogeneous fluorescence to an absence of fluorescence, which is characteristic of acrosome-reacted cells (data not shown). Non-C and consequently nonreacted sperm showed an intensely homogeneous head fluorescence, without differences between acrosomal and postacrosomal regions. In contrast, the whole head of caput sperm was moderately stained with CTC under all conditions, without differences between acrosomal and postacrosomal regions except in a few sperm cells that had presumably undergone an AR and showed an absence of fluorescence (data not shown).
Detection of Surface P4r
P4-Gold (Au) Complex
P4-binding sites were detected by a conjugate formed with Au-P4. This complex was obtained by the method described by Bendayan (1989). Briefly, 1, 1.5, 2, 2.5, 3, 3.5, and 4 μL of 30 μM P4 in HM were added to several tubes containing a suspension of 100 μL of Au obtained from a stock solution (see below). After 30 minutes, 100 μL of 10% NaCl was added, and the tubes that acquired a wine-red color were kept, and the ones that appeared as a dark violet/blue color were discarded, because the Au complex was unstable. To recover the complex, the wine- red—colored tubes were ultracentrifuged (30 minutes at 45 000 × g using a Ti50 rotor in a Beckman ultracentrifuge at 4°C). The dark red sediment corresponding to the P4-Au complex was kept at 4°C. The dark sediment corresponding to metallic Au and the supernatant containing the unbounded P4 were carefully harvested and discarded.
The Au stock solution was made with 25 μL of 20% AuCl3 in 50 mL of double-distilled water stabilized with 1.6 mL of 1% sodium citrate. Au obtained by this method produces Au particles with a diameter of 10 to 20 ηm (Handley, 1989).
P4-Au Complex as a Marker
The P4-Au complex was used to detect binding sites on the 4 types of sperm surfaces with the following protocols.
Sperm cells obtained from all conditions were coincubated in the presence of P4-Au (1 × 105 to 106 cells/mL in a 1:1 ratio of P4-Au) for 60 minutes at room temperature. Then, they were washed twice with PBS by centrifugation (750 × g for 15 minutes) to eliminate the unbound P4-Au. Sperm pellets were resuspended in 2% glutaraldehyde in PBS. All sperm samples were split, and one was processed for transmission electron microscopy, and the other was dried onto a slide for light microscopy.
First, the 4 types of sperm derived from the 2 experimental conditions were initially incubated with increasing concentrations of P4 (10, 30, 100, and 200 μM in HM) and then exposed to the P4-Au complex (competition assays). They were processed under the same conditions for both light microscopy and transmission electron microscopy. Statistical analysis was performed only on the light microscopy results. 2) The 4 types of sperm cells were also incubated with colloidal Au only to control for any nonspecific absorption by sperm cells and were then processed as described above. 3) Finally, the 4 types of sperm were exposed only to the reagent from the silver (Ag)-enhancement kit (see below) also to control for the presence of unspecified random material that stained with Ag.
Slides with sperm samples processed as described above were washed and rehydrated by immersion in PBS, and then drops of an Ag enhancement kit (Kit SE-100, Sigma) were applied following the manufacturer's instructions. After 8 minutes, slides were washed with double-distilled water and immersed in a thiosulfate solution (2.5% [wt/vol] in double-distilled water) for 2 minutes. Finally, they were washed twice in double-distilled water, dehydrated, and mounted. Slides were observed and photographed with a Nikon Optiphot-2 microscope. One extra control was performed only for light microscopy. Slides with sperm from all of the experimental protocols were fixed and dried without incubation with P4-Au or Au and developed in the same way (Hacker, 1989).
Transmission Electron Microscopy
The fixed sperm suspensions were washed twice by centrifugation (750 × g for 10 minutes) in PBS, and the final pellets were suspended in 200 μL of PBS and an equal volume of 2% OsO4 overnight at 4°C. The cells were then centrifuged (750 × g for 10 minutes), and the pellets were dehydrated in ethanol-acetone and finally embedded in Epon 812 (Pelco). Thin sections were obtained with an Ultracut ultramicrotome (Leica, Bannockburn, Ill), stained by routine uranyl-Pb techniques, and observed with an Elmiskop I transmission electron microscope (Siemens, Munich, Germany).
Detection of Nuclear P4r
The immunocytochemical procedure was performed using a highly sensitive method (Universal Dako Corporation EnVision System using peroxidase-labeled secondary antibodies) following the protocol suggested by the manufacturer. The first antibody used was a monoclonal antibody (clone 1A6) against the P4 nuclear receptor (dilution, 1:1000; Dako).
An antigen-retrieval protocol with citrate buffer (pH 6) was used to unmask the antigen (Shi et al, 1995; Vargas et al, 1999). Specimens were incubated with the primary antibody (1:100) overnight at 4°C in a humid chamber. Diaminobenzidine (0.5 mg/mL) and hydrogen peroxide (0.01%) were used as the chromogen substrate. Slides were counterstained with 0.5% methyl green to show nuclei. Biopsies of human breast tumor and mouse ovary were used as a positive control for the P4r.
Detection of P4r Proteins by Ligand Blot Analysis
Total proteins were obtained from the 4 types of sperm described above. Briefly, sperm cells—after the incubation/capacitation period—were washed with HM by centrifugation (700 × g for 10 minutes). The pellets were resuspended at a concentration of 20–30 × 106 sperm/mL in cold lysis buffer (20 mM of Tris HCl buffer, 150 mM of NaCl, 0.25% Tween 20, 1 mM of Na3VO4, and 1 mM of phenyl methyl sulfonyl fluoride, pH 7.4) for 1 hour at 4°C. Sperm cells were disrupted with a probe-type ultrasonicator (Bransonic, Branbury, Conn) with 8 bursts for 10 minutes in 3 cycles. The resultant samples were centrifuged (10 000 × g for 15 minutes at 4°C). The supernatants were retained and mixed with a protease inhibitor cocktail (P8340, Sigma, for use with mammalian cell and tissue extracts), boiled with an equal volume of 2× Laemmli sample buffer without mercaptoethanol for 10 minutes, and stored at −20°C. Proteins were analyzed with 7%–10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), blotted onto a nitrocellulose membrane, and developed with the peroxidase-P4 complex (Sigma) following the protocol of Luconi et al (1998). Controls were performed with an excess of free P4 until application of the peroxidase-P4 conjugate.
All statistical analyses and graphics were performed with SigmaPlot software (Version 1.02a, Jandel Scientific Corporation, San Rafael, Calif). Differences were considered statistically significant when P was less than .05. The values reported in the text and figures are means plus or minus standard deviations (range). Comparisons between any 2 of the conditions shown in “Results” were made by the paired Student's t test.
Experiments in which the fractions of motile cells or living cells—eosin tested—were less than 70% were not considered.
Induction of the AR by P4
P4 as well as A23187 promoted an increase in the percentage of the AR in C sperm compared with control and non-C sperm (Figure 1A). Non-C sperm did not react in the presence of P4; however, a small, statistically insignificant increase was detected with A23187 (Figure 1A). A significant increase can also be noted when compared to both controls (C and non-C). The percentage of the AR in caput sperm was always very low for any experimental condition (Figure 1B). A small increase in the AR was observed under incubated conditions with stimulation by A23187, although the percentage of the AR was low (Figure 1B). These results indicate that mouse sperm cells must undergo a maturation and capacitation process before P4 can stimulate the mechanisms leading to an AR, such as binding to P4r.
Detection of Surface P4r
Studies by a number of different laboratories have attempted to localize the P4-binding zone in different species. A study to locate the P4r/binding zone in stallion spermatozoa was carried out by indirect immunofluorescence using a P4-BSA-fluorescein isothiocyanate conjugate, which is a large nonhydrophilic complex (Cheng et al, 1998b). In this study, another conjugate with similar characteristics, P4-Au, was used. Au is a classical ultrastructural marker for use with EM with the added advantage that it can also be used to make observations with light microscopy (for counting large numbers of sperm) if the image is enhanced by development with an Ag method. In order to analyze the effect of maturation and the capacitation process, sperm collected from the caput and cauda epididymides—capacitated/incubated or not— were exposed to P4-Au.
It is well established that Ag-enhanced colloidal Au has been used as a marker in light microscopy to aid in image interpretation (Hacker, 1989). In this case, an intense dark deposition over the acrosomal region of the C sperm head was detected that corresponded to the P4-binding zone. But the number of stained sperm varied from none in non-I sperm isolated from the caput epididymis to a high percentage of stained sperm in capacitated cauda sperm (Figure 2, cf A through D; results are summarized in Figure 3A). However, a small number of incubated caput sperm and non-C cauda sperm showed a small area with very low Ag deposition (Figure 2, Panels B and C, arrow; results are also summarized in Figure 3A). Increasing the amount of P4 during a preincubation step resulted in a decrease in Ag staining (Figure 3B). Au alone or a treatment using Ag without preincubation with P4-Au resulted in no staining (Figure 3B). Together, these experiments demonstrate that the P4-binding zone is located over the dorsal edge of the sperm head and that binding site activity depends on the maturity and capacitation status of the mouse spermatozoa.
Transmission Electron Microscopy
P4-Au conjugates were located at the dorsal edge of the capacitated mouse sperm head (Figure 4). The amount of conjugate bonded to noncapacitated sperm or caput sperm— with or without incubation—is very low (Figure 4A) when compared to C sperm (Figure 4B). A substantial reduction in the number of Au particles in the sperm preincubated with P4 alone was detected (cf Figure 4C with 4B, preincubation with P4 = 30 μM; also see Figure 3B). P4-Au conjugates located over a swollen vesiculated acrosome were also observed in capacitated sperm (Figure 4D). Incubation with colloidal Au alone (control 2) showed a very low nonspecific absorption (data not shown). These data suggest that the P4-binding zone is recognized specifically by the P4-Au conjugate and with high affinity in capacitated cauda sperm, as proposed above.
Detection of Nuclear P4r
Nuclei of cells from human mammary glands or the mouse ovary showed an intense brown nuclear staining consistent with a nuclear location for the P4 nuclear receptors (Figure 5A). In contrast, capacitated sperm cells did not show any brown staining in the nuclear zone or over the plasma membrane (Figure 5B).
Detection of P4r Proteins by Ligand Blot Analysis
Luconi et al (1998) identified P-binding proteins using ligand blot analysis in human spermatozoa. They found a 54- and 57-kd protein band recognized by a P4-peroxidase conjugate in a total lysate of capacitated human sperm that showed a molecular weight similar to that of proteins recognized in an immunoblot analysis using the monoclonal antibody c262 (Luconi et al, 1998). In this study, a total lysate of caput (with or without incubation) and cauda (capacitated or not) spermatozoa was analyzed using a similar ligand blot analysis that showed a protein band of 44 kd in cauda sperm (capacitated or not; Figure 6) that was absent in the control blot ligand assay (data not shown). These results show that the 44-kd protein is specific for P4 in capacitated (or not) cauda sperm.
It has been demonstrated that P4 promotes the AR in mouse cauda epididymal spermatozoa (Roldan et al, 1994; Cheng et al, 1998b). The present study confirms that P4 induces the AR in cauda epididymal mouse sperm—incubated under capacitating conditions—as well as in ionophore A23187. Conversely, cauda epididymal sperm under noncapacitating conditions exposed to P4 did not undergo the AR, even in the presence of A23187 alone. These results suggest that the capacitation period promotes sperm mechanisms involved in the AR, causing them to become more reactive to P4 than noncapacitated sperm. For instance, it has been demonstrated in stallion sperm that membrane P4rs increase during the capacitation period (Cheng et al, 1998a,b). In this study, we have demonstrated that the number of bound P4-Au conjugates—or the intensity of Ag staining—increases after the capacitation period, which indicates that this could be directly related to the unmasking of the receptor/binding zone. However, we cannot discard alternative mechanisms such as changes in signal transduction or membrane modifications. During the capacitation period, cholesterol is released from the plasma membrane (Visconti et al, 1999). Preincubation in a medium containing cholesterol inhibits the AR induced by P4 (Cross, 1996). A decrease in cholesterol causes an increase in the fluidity of the membranes, which might be involved in increasing receptor/binding zone availability. More effort must be made to determine the mechanisms involved.
Another result presented is that caput sperm did not react in the presence of P4, regardless of whether they were subjected to capacitating conditions or not. This could indicate that immature sperm are not able to undergo the AR because the P4r/binding zone is not available. In this study, we demonstrated that the surface P4r/binding zone is less available in caput sperm than in cauda sperm. Some Ag staining was observed after an incubation period, possibly due to the unmasking of receptors or changes in the membrane composition. Regardless, the intensity in Ag staining or the amount of bonded Au conjugate did not equal that of the capacitated cauda sperm. The unmasking of surface molecules by proteolysis has been proposed to explain the changes observed in this type of cell in which no protein synthesis could be verified (Phelps et al, 1990; Rochwerger and Cuasnicu, 1992; Kholkute et al, 1995). The ability to obtain a low but significant AR with incubated sperm in the presence of A23187 could indicate that the mechanisms necessary for an AR are present but are in some way inhibited.
Some physiological interpretation can be made from the fact that an interaction with the P4-Au conjugate was frequently observed over a swollen vesiculated acrosome in capacitated sperm. If the binding of P4-Au with P4r was not altered by colloidal Au, then the AR was consequently promoted. This idea was corroborated by the micrographs obtained by EM.
Another aspect is the distribution of the P4r. It was localized at the surface of the sperm head, specifically at the plasma membrane over the acrosome, but not in other membrane domains such as the postacrosomal region or tail.
Despite our results or those of previous studies, the nature of the existence of the P4 membrane/surface receptor continues to be debated. For many years, it has been thought that the P4r should be classed as a nuclear receptor, but there have been suggestions that some of its actions are mediated at the plasma membrane (Wehling, 1997; Revelli et al, 1998; Maller, 2001). In cells other than sperm, better evidence for a surface receptor has been reported, such as in the amphibian oocyte (Palmer and Nebreda, 2000) or for the neurons of the central nervous system (Burt and Kamatchi, 1991). In this study, we have presented evidence that the P4r/binding zone is probably located at the cell surface in the mouse sperm because the conjugate cannot cross the plasma membrane to react with a cytoplasmic or nuclear receptor. Additional support for a nonnuclear P4r was found in the speed of the AR, which was verified within minutes and is consistent with the actions of a surface receptor rather than with slower nuclear functions. To support this, a nuclear receptor was not detected in our immunohistological experiment. Furthermore, in the blotting experiments, a protein of 44 kd was found to interact with P4. We also present results from specific competition assays showing that the Au conjugates are binding to the P4rs rather than to a nonspecific absorption of Au conjugates. Despite this evidence, more effort must be made to conclusively demonstrate the existence of a surface receptor in mouse sperm.
In conclusion, P4 promotes the AR in mouse spermatozoa. In all likelihood, mouse spermatozoa possess a P4 membrane receptor/binding zone that increases in reactivity during epididymal transit and the capacitation process.
It is distinct from a nuclear receptor and possesses a molecular weight close to 44 kd.
The authors thank to Drs L. Vargas and D. Ciocca (IMBECU, CRICYT, Mendoza) for helping with the detection of the nuclear P4r and Dr Sean Patterson for English editing. Special thanks to A. Vincenti for her technical assistance and to R. Yunes and A. Penissi for their critical reading.
This research was supported by SECyT-UNC (Science Secretary of National University of Cuyo) and CONICET (National Research Council of Argentina).