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Biology of Reproduction 67, 1327-1336 (2002)
© 2002 Society for the Study of Reproduction, Inc.

Progesterone Receptor as an Indicator of Sperm Function

Sushama Gadkara, C.A. Shaha, Geetanjali Sachdevaa, Urmila Samantb, and C.P. Puri1,a

a Institute for Research in Reproduction, Indian Council of Medical Research, Parel, Mumbai 400 012, India b Tata Memorial Hospital, Parel, Mumbai 400 012, India


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Expression of progesterone receptor (PR) localization on spermatozoa was determined in men with normal and abnormal spermiograms. Studies were also carried out to evaluate the potential of PR as a marker of sperm function. Progesterone receptor expression on spermatozoa from men with normozoospermia (n = 8), oligozoospermia (n = 7), asthenozoospermia (n = 8), oligoasthenozoospermia (n = 7), and teratozoospermia (n = 11) was analyzed using an immunocytochemical method with monoclonal antibodies against PR, and flow cytometry using a cell-impermeable fluorescein-tagged progesterone coupled to BSA complex (P-FITC-BSA). Both methods revealed significantly fewer (P < 0.05) PR-positive spermatozoa in men with oligozoospermia, asthenozoospermia, oligoasthenozoospermia, and teratozoospermia compared with men with normozoospermia, thereby suggesting that down-regulation of PR expression in spermatozoa may be one of the causes of male infertility. Spermatozoa from men with normozoospermia (n = 12), oligozoospermia (n = 12), asthenozoospermia (n = 12), oligoasthenozoospermia (n = 9), and teratozoospermia (n = 10) were exposed to low osmotic conditions in the hypoosmotic swelling (HOS) test and then analyzed for PR expression using P-FITC-BSA complex. A significantly higher percentage (P < 0.05) of spermatozoa with physiologically active plasma membrane (HOS+) lacked PR expression (HOS+PR-) in all categories of men with infertility, thereby suggesting that compared to the HOS test, PR expression is a better indicator of sperm function. Furthermore, PR expression in spermatozoa showed a strong (P < 0.05) positive correlation with their ability to undergo an in vitro acrosome reaction. This was observed in all study groups (i.e., normozoospermia, r = 0.8545; oligozoospermia, r = 0.8711; asthenozoospermia, r = 0.7645; oligoasthenozoospermia, r = 0.9003; and teratozoospermia, r = 0.8676). This suggests a potential role for PR in the events leading to the acrosome reaction in sperm.

acrosome reaction, progesterone receptor


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
A reliable tool to assess the fertilizing potential of human spermatozoa is not yet available. Therefore, it is not surprising that the causes of infertility remain undetectable in a large percentage of men. Routine semen analyses that include estimation of sperm concentration, motility, and morphology have limitations as fertility indicators [1–3]. These analyses lack the potential to ascertain the functional capacity of spermatozoa, and they cannot predict the possible occurrence of both in vivo and in vitro conception [4–6]. In the last few years, efforts have been directed toward the development of various functional tests such as the zona-free hamster oocyte sperm penetration test [7, 8], the hypoosmotic swelling (HOS) test [9], and the acrosome reaction (AR) [10] to assess the sperm's functional capacity.

The zona-free hamster oocyte sperm penetration test [11–13] has been developed as an indicator of the ability of human spermatozoa to capacitate, acrosome react, and fuse with the vitelline membrane of the oocyte [14, 15]. However, some reports have suggested that assessment of sperm fertilizing capacity using this test does not have significant clinical benefit in diagnosing infertility [7, 16]. Dead or immotile spermatozoa also penetrate zona-free hamster eggs under certain conditions [15, 17]. Furthermore, this assay is time-consuming, complex, and subject to many variables such as capacitation time [18], sperm concentration and incubation conditions [19], medium composition [15], and the age of the ovum after ovulation [20].

The HOS test can predict the fertilizing capacity of human spermatozoa [9, 21, 22]. A positive HOS test is an indicator of the functional integrity of the sperm membrane as judged by a swollen head and curled tail of spermatozoa when exposed to hypo-osmotic conditions [9]. A decrease in the number of HOS-positive forms in the ejaculates of men with oligozoospermia, asthenozoospermia, and oligoasthenozoospermia compared with men with normozoospermia has been reported [23]. Positive correlation was also observed between HOS and the fertilizing ability of sperm [21, 22, 24–26], however, others have not found a significant correlation between HOS and in vitro fertilization (IVF) outcome [27–30]. Thus, the clinical utility of the HOS test in assessment of sperm function is still debatable.

The AR, which is one of the prerequisites for successful fertilization, seems to be a more relevant parameter for assessing sperm function [2, 7, 31–33]. AR is an irreversible, exocytotic, postcapacitational event that involves fusion and fenestration of the outer acrosomal membrane with the plasma membrane [10]. It has been demonstrated that progesterone facilitates the AR [34–37]. Studies have also been carried out to determine whether an assessment of a sperm's responsiveness to progesterone may predict its fertilizing ability in vitro [38–44]. A significant correlation has been found between the outcome of IVF and progesterone-stimulated Ca2+ influx [45, 46]. A physiological role for progesterone in AR has also been suggested by other reports demonstrating a relationship between the ability of sperm to respond in vitro to progesterone and male infertility [47–49].

The mechanism by which progesterone elicits an AR and subsequent events probably involves its interaction with a cell surface receptor on spermatozoa [50–52]. The presence of membrane receptors that specifically bind to progesterone has been demonstrated on human spermatozoa [51, 53, 54], and there is evidence to suggest that blocking these surface receptors inhibits progesterone-induced AR [55, 56]. This prompted us to investigate the possibility of an aberration in the expression of progesterone receptor (PR) on spermatozoa in men with an abnormal spermiogram and whether it has any effect on sperm functions (i.e., the AR). Studies were also conducted to compare the predictive value of PR with that of other tests such as HOS for sperm function.

This is the first report to assess the characteristics of HOS and positive PR in the same sperm samples. To our knowledge, no such attempt has been made until now to analyze the expression of a surface protein of potential functional relevance on spermatozoa, which is categorized on the basis of membrane activity. This is also the first study in which PR expression in spermatozoa from fertile and infertile groups of men was evaluated by both immunocytochemistry and flow cytometric analysis.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Samples

Semen samples were collected by masturbation after 36–48 h of sexual abstinence from healthy and proven fertile (n = 20) volunteers. These fertile men had fathered a child within the last 12 mo. Semen samples were collected from infertile (n = 76) men attending the infertility clinic at Wadia Maternity Hospital, Mumbai. The wives of the volunteers had normal hormonal levels, menstrual cycle length, ovulation, and endometrial function. Informed consent was obtained from each subject before samples were collected.

Samples were classified in accordance with World Health Organization guidelines [6] by a trained andrologist. They were categorized either as normozoospermic (n = 20; count >=20 million/ml, progressive motility >=50%, morphology >=30% normal forms), oligozoospermic (n = 19; count <20 million/ml, progressive motility >=50%, morphology >=30% normal forms), asthenozoospermic (n = 20; count >=20 million/ml, progressive motility <50%, morphology >=30%), oligoasthenozoospermic (n = 16; count <20 million/ml, progressive motility <50%, morphology >=30% normal forms), or teratozoospermic (n = 21; count >=20 million/ml, progressive motility >=50%, morphology <30% normal forms).

Spermatozoa from men with normozoospermia (n = 8), oligozoospermia (n = 7), asthenozoospermia (n = 8), oligoasthenozoospermia (n = 7), and teratozoospermia (n = 11) were analyzed for PR expression by flow cytometric and immunocytochemical methods. Spermatozoa from men with normozoospermia (n = 12), oligozoospermia (n = 12), asthenozoospermia (n = 12), oligoasthenozoospermia (n = 9), and teratozoospermia (n = 10) were used in the HOS test followed by PR localization and AR analysis.

Each ejaculate was allowed to liquefy at room temperature for 30 min, washed twice with 1x PBS pH 7.4, centrifuged at 980 x g for 10 min, and assayed by all three techniques.

Collection of Follicular Fluid

Follicular fluid was obtained by aspiration of antral follicles from women undergoing laparoscopy for IVF and embryo transfer. Follicular fluids were pooled and centrifuged at 2800 x g at 4°C to remove cellular debris, and progesterone content was measured by radioimmunoassay [57]. Follicular fluid was then aliquoted and stored at -20°C until used.

Preparation of Stock Solutions

Progesterone 3-(O carboxymethyl) oxime:BSA conjugate (5–10 mol of progesterone per mole of BSA) labeled with fluorescein isothiocyanate (FITC; P-FITC-BSA; Sigma, St. Louis, MO) was stripped of free progesterone using dextran-coated charcoal [58]. FITC was conjugated with BSA according to the method described by Goding [59]. The conjugate solution was lyophilized and reconstituted in PBS to obtain a solution of 1 mg/ml. FITC-conjugated Pisum sativum lectin (Sigma) was dissolved in PBS at 1 mg/ml and stored frozen (-20°C) until used.

Immunocytochemistry

Approximately 1 x 106 spermatozoa in 10 µl of PBS were smeared on clean, grease-free slides, air-dried, and fixed in chilled methanol for 30 min. The slides were washed twice with 1x PBS and once with PBS-T (0.5% Tween 20 in 1x PBS) for 10 min at room temperature. After washing, the sperm membrane was permeabilized with 0.1% sodium deoxycholate in 1x PBS at 4°C for 30 min. Smears were washed with PBS-T for 5 min, and blocked in 1% BSA in 1x PBS at room temperature for 20–25 min. Slides were incubated with mouse antisera (Affinity Bioreagents, Golden, CO) against the carboxyl terminus of PR (clone PR-AT4.14) diluted 1:100 in 1x PBS at 4°C for 18–20 h. The slides were washed with PBS-T for 5 min and then incubated with biotin-conjugated goat anti-mouse secondary antibody diluted 1:500 in 1x PBS for 1 h at room temperature. The slides were washed twice with 1x PBS and incubated with avidin-biotin complex (Vectastain kit, Vector Laboratories, Burlingame, CA) for 30 min at room temperature. The slides were then washed twice with 1x PBS at room temperature for 30 min each, followed by treatment with 0.05% diaminobenzidine in 1x PBS with 0.06% H2O2 for 10 min. The slides were counterstained with 1% hematoxylin for 2 min, dehydrated in ascending grades of alcohol for 10 min each, and kept in xylene for 2 h followed by mounting in DPX. Specificity of the staining was evaluated by running a negative control (1% BSA without antibody).

The slides were examined using the 100x objective of a brightfield microscope (Olympus, Tokyo, Japan) with a double-blind technique. At least 200 spermatozoa were counted in 20 microscopic fields selected at random. Counterstained spermatozoa showing a brown color at the head region were counted as PR-positive. Spermatozoa that did not react with the antibodies against PR had no brown precipitates and appeared blue throughout due to counterstaining and were counted as PR-negative.

Direct Fluorescence

Four million sperm were incubated with 50 µl of 0.1% digitonin in PBS at 4°C for 30 min. These samples were washed twice with 1x PBS and centrifuged at 1000 x g for 10 min. Washed pellets were incubated with either 0.1 µM P-FITC-BSA or FITC-BSA in 50 µl of PBS at 4°C for 16–18 h. Unbound fluorescein-labeled compounds were removed by washing with 1x PBS and centrifuged at 1000 x g. The pellets were suspended in 1 ml of 1x PBS. Ten microliters of each cell suspension was processed for direct fluorescence analysis and the remaining for flow cytometry.

Ten microliters of cell suspension was smeared on a clean, grease-free slide, mounted, and visualized with a 100x objective of the fluorescence microscope (Olympus) using a triple-band filter (Cube U-MNIBA, DM-505, BP-470-550, and BA-515-550). Positive staining following incubation with P-FITC-BSA resulted in a green color at the acrosomal region, whereas negative staining as well as incubation of spermatozoa with FITC-BSA resulted in a red color.

Flow Cytometric Analysis

Approximately 4 million plain or digitonin-treated spermatozoa were incubated with P-FITC-BSA or FITC-BSA as mentioned in the previous section. The samples were washed and suspended in 1 ml of PBS. These samples were analyzed using a FACScan cytometer (Becton Dickinson, San Jose, CA) equipped with a 15 mW air-cooled 488 nm argon-ion laser. FL1 (FITC) signals were detected through a 530/30 nm bandpass filter. To distinguish spermatozoa from debris, a dot plot distribution of spermatozoa according to a forward angle light scatter (correlating with cell size) and right angle light scatter (correlating with cell density) were used to determine a "sperm window" as described by Haas and Cunningham [60]. The fluorescence intensity of 10 000 spermatozoa within the sperm window was computed twice in list mode and analyzed using Lysis II software (Becton Dickinson).

Intensity of the fluorescence of stained cells was presented in log scale on the X-axis and number of cells showing fluorescence on the Y-axis in flow cytograms. Channels M1, M2, and M3 represented low, moderate, and high intensity staining, respectively. Flow cytometry data for PR positivity were compared with the data for PR positivity and evaluated with the immunocytochemical method.

Localization of PR on Sperm Subjected to the HOS Test

The HOS test was performed on 0.1 ml of the original ejaculate mixed with 1 ml of hypo-osmotic solution of sodium citrate and fructose (150 mOsm/L) [9]. After incubation for 40 min at 37°C, 200 spermatozoa were observed at a magnification of 400x with a phase contrast microscope. The number of spermatozoa with head and tail changes typical of swelling (positive for hypoosmotic swelling) was counted [9]. Spermatozoa subjected to HOS were pelleted by centrifugation at 1000 x g for 5 min and smeared on clean, dry, grease-free slides. Smears were fixed in methanol for 10 min at 4°C. Smears were treated with 0.1% digitonin at 4°C for 30 min and washed three times with 1x PBS. The spermatozoa were incubated with 0.1µM P-FITC-BSA or FITC-BSA at 4°C for 16 h and washed three times with 1x PBS to remove unbound stain. The smears were then mounted with glycerol in PBS (1:9). Stained slides were observed using a 100x objective of a fluorescence microscope (Olympus). Spermatozoa subjected to HOS showing green color at the acrosomal or equatorial region were counted as PR-positive spermatozoa. PR staining was assessed in both HOS-positive and HOS-negative spermatozoa from fertile and infertile men, respectively. This evaluation of PR positivity was made by two different observers in a blinded manner. For quantitative assessment of PR at least 200 spermatozoa were examined in randomly selected 20 fields.

Acrosome Reaction

Semen samples were suspended in Biggers Whitten Whittingham (BWW) medium [61] and separated from seminal plasma by centrifugation at 1000 x g at room temperature. Washed sperm (1–2 x 106) were overlaid with 1 ml of BWW medium supplemented with 3.5% human serum albumin (HSA-BWW) and incubated at 37°C in 5% CO2 and 95% air for 6 h to allow capacitation [62]. Capacitated spermatozoa were divided into two equal aliquots and centrifuged at 1000 x g. The acrosome reaction was induced by incubating the capacitated spermatozoa in 20 µl of 20% follicular fluid in HSA-BWW for 20 min at 37°C in 5% CO2 and 95% air. In another aliquot, 20 µl of HSA-BWW instead of follicular fluid was used to take into account the number of spermatozoa undergoing a spontaneous AR or an unstimulated AR. The sperm cells were then separated from the medium by mild centrifugation at 1000 x g to visualize the AR.

Visualization of Acrosomal Status

Separated sperm cells were diluted (approximately 1 x 106/ml) in BWW medium and smeared on clean, dry, grease-free slides. The smears were fixed with methanol for 10 min, washed with PBS, and allowed to react with FITC-labeled Pisum sativum lectin (1 µg/ml) for 16–18 h at 4°C. Excess stain was removed by washing with PBS. The smears were then mounted with glycerol in PBS (1:9) and examined under a fluorescence microscope (Olympus) using an oil immersion objective.

Two hundred spermatozoa per sample were counted to determine whether they were intact (stained acrosomal cap) or acrosome reacted (patchy and equatorially stained and acrosome lost). The percentage of AR was expressed as the difference between stimulated and unstimulated (spontaneous) acrosome reacted sperm population.

Statistical Analysis

All statistical analyses were performed using the Statistical Program for the Social Sciences (SPSS; Chicago, IL), version 9. Percentages of PR-positive sperm evaluated by immunocytochemistry and flow cytometry were compared with one-way ANOVA.

Percentages of HOS+PR+, HOS+PR-, HOS-PR- and acrosome-reacted sperm populations in different study groups were also compared via one-way ANOVA. The coefficients of correlation between %HOS, %HOS+PR+, %HOS+PR-, and %AR were determined with SPSS version 9.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
PR Expression on Spermatozoa

Immunocytochemical localization using antibodies against the conventional PR demonstrated localization of PR, as evidenced by brown staining at the acrosomal region (Fig. 1). Aliquots of the samples processed for flow cytometric analysis were also subjected to direct fluorescence using fluorescein-tagged ligand (P-FITC-BSA). This also demonstrated a similar pattern of PR localization (i.e., at the acrosomal region) in spermatozoa from both fertile and infertile groups (Fig. 2).



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  		FIG. 1. Detection of PR using antibodies against conventional PR in spermatozoa from men with normozoospermia (b), oligozoospermia (c), asthenozoospermia (d), oligoasthenozoospermia (e), and teratozoospermia (f). Negative control (no antibody) is shown in a. Brown color indicates positive staining for PR. Blue color appears because of counterstaining with hematoxylin. Spermatozoa were visualized with an x100 oil immersion objective



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  		FIG. 2. Localization of PR using P-FITC-BSA on spermatozoa from men with normozoospermia (b), oligozoospermia (c), asthenozoospermia (d), oligoasthenozoospermia (e), and teratozoospermia (f). Negative control (FITC-BSA) is shown in a. Green color indicates positive PR and red color indicates negative staining. Spermatozoa were visualized with an x100 oil immersion objective

Immunocytochemical and flow cytometric analysis revealed a significant decrease (P < 0.05) (Table 1) in the number of PR-positive spermatozoa in infertile groups compared with those that were fertile (Figs. 1 and 3).


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  		TABLE 1. Percentage of PR-positive spermatozoa in fertile and infertile groups assessed by immunocytochemical localization and flow cytometry.a



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  		FIG. 3. Flow cytometric analysis to quantitate the number of PR-positive spermatozoa in samples from men with normozoospermia (a), oligozoospermia (b), asthenozoospermia (c), oligoasthenozoospermia (d), and teratozoospermia (e). Respective controls (samples stained with FITC-BSA) are shown in the left panel. Comparison of PR positivity for each category was done by comparing the fluorescence in M2 channel

PR was also localized to spermatozoa with abnormal morphology (Fig. 4). It was interesting that the number of PR-positive spermatozoa was higher in men with teratozoospermia than it was in men with other types of infertility. Very weak positive correlation was observed between PR expression and the traditional semen parameters such as motility and morphology (data not shown).



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  		FIG. 4. Immunolocalization of PR on human spermatozoa with abnormal morphology. Spermatozoa with head defects (c–f), midpiece defects (g and h), and tail defects (i and j) were stained with antibodies against the conventional PR. Brown staining indicates PR positivity and blue color appears due to counterstaining with hematoxylin. Spermatozoa with normal morphology are shown in b. Negative control (without antibody) is shown in a. Sperm were visualized with an x100 oil immersion objective

HOS Positivity in Spermatozoa

A significant decrease in the percentage of HOS-positive spermatozoa (P < 0.05) was observed in all infertile groups, except in the teratozoospermic group, compared with those that were fertile (Table 2).


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  		TABLE 2. Spermatozoa showing positivity for hypoosmotic swelling test and acrosome reaction in fertile and infertile men.a

PR Expression and HOS Positivity in Spermatozoa

Spermatozoa subjected to hypoosmotic conditions revealed three patterns of PR expression (Fig. 5): 1) HOS+PR+: HOS positive spermatozoa with a functionally active plasma membrane demonstrating PR expression at the equatorial or acrosomal region. 2) HOS+PR-: HOS-positive spermatozoa that lacked PR expression. 3) HOS-PR-: Spermatozoa that lacked a functionally active membrane as well as PR expression.



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  		FIG. 5. PR localization on spermatozoa exposed to hypoosmotic conditions. a) Double positive, HOS+PR+, coiled tail (indicative of HOS positivity), and green color (indicative of PR positivity); b) Single positive, HOS+PR-, coiled tail, and red color (PR negativity); c) double negative, HOS-PR-, uncoiled tail, and red color. Sperm were visualized with an x100 oil immersion objective

A significant decrease (P < 0.05) in HOS+PR+ and a significant increase (P < 0.05) in HOS+PR- spermatozoa was observed in all infertile groups compared with normozoospermic men (Table 3; Fig. 6). However, no significant change was observed in the number of HOS-PR- spermatozoa in infertile men except in those with oligoasthenozoospermia compared with fertile men.


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  		TABLE 3. Percentage of spermatozoa with different patterns of PR expression detected following HOST.a



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  		FIG. 6. Detection of PR on spermatozoa previously exposed to hypoosmotic conditions in men with normozoospermia (b), oligozoospermia (c), asthenozoospermia (d), oligoasthenozoospermia (e), and teratozoospermia (f). Negative control (FITC-BSA) is shown in a. Green-yellow color indicates PR positivity and red indicates negative staining

PR Expression and AR in Spermatozoa

Significantly fewer (P < 0.05) spermatozoa underwent an in vitro AR in all infertile groups compared with those in the normozoospermia group (Table 2).

A weak positive correlation was found between the percentage of HOS-positive forms and the percentage of AR spermatozoa in all groups (normozoospermia, r = 0.4982; oligozoospermia, r = 0.4958; asthenozoospermia, r = 0.4680; oligoasthenozoospermia, r = 0.5800), whereas a negative correlation (r = -0.2204) was observed in men with teratozoospermia (Table 4). In contrast, a very strong positive correlation was observed between the percentage of HOS+PR+ and the percentage of AR spermatozoa in men with normozoospermia (r = 0.8545), oligozoospermia (r = 0.8711), asthenozoospermia (r = 0.7645), oligoasthenozoospermia (r = 0.9003), and teratozoospermia (r = 0.8676) (Table 4). No strong positive correlation was found between the percentage of HOS+PR- and the percentage of AR spermatozoa in any group (Table 4).


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  		TABLE 4. Coefficient of correlation between % HOS+/% HOS+PR+/% HOS+PR- and % AR spermatozoa in fertile and infertile men


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Culmination of the sperm-egg interaction into an event of successful fertilization requires sperm to undergo the AR, which is an irreversible, exocytotic, postcapacitational event. This consists of fusion and fenestration of the outer acrosomal membrane with the plasma membrane. AR dysfunctions have been proposed to be one of the causes of reduced fertility in men. The assessment of AR in vitro is thus expected to yield additional information on the fertilization potential of spermatozoa, which has not been derived from conventional semen analysis [63].

Progesterone, which is secreted by cumulus cells, has been indicated as a physiological stimulus or costimulus for initiating the AR in spermatozoa. It is speculated that progesterone-induced effects (i.e., an increase in calcium influx, hyperactive motility, zona pellucida binding, and zona-free oocyte penetration) are mediated via distinct, nongenomic PR [55, 56]. It is also speculated that these sperm functions probably involve three different types of receptors—a plasma membrane Ca2+ ion channel, a GABAA receptor, and a membrane-associated protein tyrosine kinase [64]. Our previous studies on the biochemical and molecular characterization of these membrane-bound progesterone binding sites on human spermatozoa have revealed similarities as well as dissimilarities of these receptors with the conventional intracellular PR [54, 65]. These studies demonstrated that these progesterone binding sites on spermatozoa are membrane-bound, heat-labile, and localized at the acrosomal region [54]. These sites were found to be masked in spermatozoa of ejaculates from normal men. It has been speculated that the reshuffling of membrane components during in vivo capacitation, the AR, or both facilitates unmasking of these progesterone binding sites. The present study was undertaken to investigate whether PR expression on spermatozoa has any significance for evaluating infertility in men.

In contrast to other reports demonstrating that only 11% of sperm in ejaculates from fertile men bear PR [47, 66], its expression was observed in 70%–80% of spermatozoa from fertile men in the present study. Our results are in agreement with a report demonstrating significant responsiveness to progesterone in 93% of spermatozoa from fertile donors [67]. It can be hypothesized that either the coating of the sperm PR with proteins of testicular or epididymal origin or masking of these sites may stearically hinder the binding of progesterone to its receptor. It is likely that treatment of spermatozoa with mild detergents (i.e., digitonin) as carried out in this study unmasks these sites and thereby allows detection of a higher percentage of PR-positive cells. In the present study, the functional assessments (acrosome reaction) of live spermatozoa were conducted in the absence of detergent because these events led to reshuffling of membrane components in spermatozoa and probably subsequent unmasking of PR. However, for localization of PR, fixed sperm smears were treated with mild detergent (either 0.1% digitonin or 0.01% sodium deoxycholate).

Evidence supporting a physiological role for progesterone in the AR comes from reports suggesting a relationship between male infertility and the inability of spermatozoa to respond to progesterone in vitro [47–49]. However, these studies were conducted on spermatozoa from selected groups (either patients with unexplained infertility or oligozoospermia or teratozoospermia). The present study was undertaken to analyze PR expression in spermatozoa from men with various seminal abnormalities.

A significant decrease in the number of PR-positive spermatozoa was detected in infertile men in the present study. To our knowledge, this is the first study in which two methods (flow cytometry and immunocytochemistry) were used to evaluate PR expression in spermatozoa from fertile and infertile men. Percentages of PR-positive spermatozoa using both immunocytochemical and flow cytometric methods were found to be similar. These two techniques were used to rule out the possibilities of measuring postacrosomal fluorescence (due to diffusion of P-FITC-BSA into dead cells) and of manual bias in counting the positive cells, respectively. A slightly higher percentage of PR-positive spermatozoa was recorded by flow cytometry in some infertile groups compared with that assessed by immunocytochemistry. This may arise because of the cumulative fluorescence (postacrosomal, acrosomal, and equatorial) measured by the flow cytometer, whereas the data collected using immunocytochemistry took into account only those cells that showed PR localization at the acrosomal region. The failure to express PR by spermatozoa from infertile men may reflect an underlying pathological mechanism.

It was intriguing to find that only 24% of spermatozoa from normal men undergo inducible AR. This may be attributed to the possibility of incomplete unmasking of PR on human spermatozoa or the requirement of additional inducers for AR. The other plausible reason for the observed difference in the numbers of PR-positive spermatozoa and in vitro acrosome-reacted spermatozoa is that plain washed (not digitonin-treated) spermatozoa were subjected to the AR. It is also likely that the concentration of AR stimulator (follicular fluid in the present study) may not precisely mimic the effects of physiological inducers. In the present study, the use of follicular fluid was preferred over chemical inducers (i.e., calcium ionophore), because these chemical inducers are known to be cytotoxic. Although follicular fluid has other AR-inducing components in addition to progesterone, and it would have been more appropriate to evaluate only progesterone-induced AR and rule out the contribution of other follicular fluid components to AR, follicular fluid is an AR inducer that closely mimics physiological conditions. Further, the same batch of follicular fluid was used to stimulate the AR in spermatozoa from both fertile and infertile groups.

In the present study, a significant decrease in the percentage of acrosome-reacted spermatozoa was observed in men with oligozoospermia, asthenozoospermia, oligoasthenozoospermia, and teratozoospermia compared with those with normozoospermia. These results are in agreement with the findings of Fuse et al. [40]. A decrease in the percentage of spermatozoa with the potential to undergo the AR in infertile groups is suggestive of aberrations in the cascade of events leading to the AR. This may arise because of the lower PR expression on spermatozoa in infertile men observed in this study.

The potential role of PR in the AR is also indirectly evident by the observation of a higher percentage of both acrosome-reacted as well as PR-positive spermatozoa in men with teratozoospermia compared with those with oligozoospermia, asthenozoospermia, and oligoasthenozoospermia. It is interesting that no strong correlation was found between PR expression and other seminal parameters such as motility and morphology (data not shown). In light of these observations, it is not surprising that spermatozoa with abnormal morphology retain their fertilization potential as evident by in vitro fertilization studies [49, 68], and abnormal morphology is not necessarily accompanied by a failed AR [63].

Attempts have been made in the past to use the functional activity of the plasma membrane (i.e., the HOS test) as an indicator of sperm function [21, 22, 25, 26, 69]. However, its diagnostic usefulness is still debatable [27–30]. The ability of the sperm tail to swell in the presence of a hypoosmotic solution is an indicator of the membrane activity. In the present study, significantly fewer HOS+ spermatozoa were found in men with oligozoospermia, asthenozoospermia, and oligoasthenozoospermia than in men with normozoospermia, thereby indicating an impaired membrane integrity of spermatozoa in men with infertility. However, HOS positivity alone failed to serve as an indicator of sperm function in men with teratozoospermia. Further, when an attempt was made to find a correlation between HOS positivity and AR in spermatozoa, only a weak correlation was found. Although the HOS test and the AR require a functionally active sperm membrane, these seem to be independent events. In contrast, a strong positive correlation was found between the percentage of HOS+PR+ and the percentage of AR sperm cells. Significantly fewer HOS positive spermatozoa from men with normozoospermia lacked PR expression compared with those in men with abnormal spermiograms. This probably suggests that not only the integrity of the plasma membrane, but that PR expression, are both required for the AR. This is the first study in which the expression of a surface antigen or membrane-bound molecule was analyzed in spermatozoa exposed to hypoosmotic conditions.

Evaluation of PR expression on spermatozoa may give insight into the functional competence of sperm because the PR expression on sperm bears a strong correlation with its ability to undergo in vitro AR. Further studies need to be undertaken to understand the mechanism of progesterone-induced AR through PRs. Nevertheless, the present study demonstrates that compared to HOS, PR expression seems to be a better predictor of sperm function. In men with an abnormal spermiogram, a higher percentage of HOS-positive spermatozoa lacked PR expression. Further, the detection of PR on human spermatozoa is fast, easy to perform, and does not require expensive equipment. It is possible to analyze the slides, and they can be permanently preserved and reassessed. Hence, analysis of PR expression on spermatozoa is a useful method for evaluating sperm function.


   ACKNOWLEDGMENTS
 
The authors acknowledge Dr. Vandana Walvekar, the dean of Wadia Maternity Hospital, Mumbai, for providing the semen samples. We appreciate the suggestions by Dr. Geeta Vanage and Dr. Usha Natraj (assistant directors, Institute for Research in Reproduction, Parel, Mumbai) for immunocytochemical studies. The authors also thank Dr. M. Hansotia of the Fertility Clinic in Mumbai for providing human follicular fluid. The authors also thank Mr. D. Balaiah (assistant director, Institute for Research in Reproduction, Parel, Mumbai) for assisting in statistical analysis.


   FOOTNOTES
 
1 Correspondence. FAX: 91 22 4139412, 4964853; vichin{at}vsnl.com and dirirr{at}vsnl.com Back

Received: 29 January 2002.

First decision: 15 February 2002.

Accepted: 28 May 2002.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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