Modulation of human sperm function by peritoneal fluid

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1 FERTILITY AND STERILITY VOL. 80, NO. 4, OCTOBER 2003 Copyright 2003 American Society for Reproductive Medicine Published by Elsevier Inc. Printed on acid-free paper in U.S.A. Modulation of human sperm function by peritoneal fluid María José Munuce, Ph.D., a,b Clara I. Marín-Briggiler, Ph.D., c Adriana M. Caille, M.Sc., a César L. Berta, M.D., b Patricia S. Cuasnicú, Ph.D., c and Lida Morisoli, Ph.D. a Laboratorio de Estudios Reproductivos, Servicio de Reproducción Humana y Planificación Familiar, Universidad Nacional de Rosario, Rosario, and Instituto de Biología y Medicina Experimental, Buenos Aires, Argentina Received September 13, 2002; revised and accepted March 7, Supported by a Re-Entry Grant from the UNDP/ UNFPA/WHO/World Bank Special Programme of Research, Development and Research Training in Human Reproduction, World Health Organization (no ). Presented at the Seventh World Congress of Gynecological Endocrinology, Buenos Aires, Argentina, April 25 28, Reprint requests: María José Munuce, Ph.D., Laboratorio de Estudios Reproductivos, Cátedra de Bioquímica Clínica, Facultad de Ciencias Bioquímicas y Farmacéuticas, Universidad Nacional de Rosario, Suipacha 531, (2000) Rosario, Argentina (FAX: ; reprolab@citynet.net.ar). a Laboratorio de Estudios Reproductivos, Cátedra de Bioquímica Clínica, Facultad de Ciencias Bioquímicas y Farmacéuticas, Universidad Nacional de Rosario. b Servicio de Reproducción Humana y Planificación Familiar, Cátedra de Ginecología, Escuela de Medicina, Universidad Nacional de Rosario. c Instituto de Biología y Medicina Experimental, CONICET-UBA, Buenos Aires, Argentina /03/$30.00 doi: /s (03) Objective: To examine the effect of peritoneal fluid on various parameters of sperm function in vitro. Design: Prospective study. Setting: Basic research laboratory. Patient(s): Semen samples were obtained from normozoospermic volunteers (n 43). Peritoneal fluids were aspirated laparoscopically from women with unexplained infertility (n 14). Follicular fluid and oocytes were collected from patients undergoing IVF-ET. Intervention(s): Sperm incubated under capacitating conditions were exposed to peritoneal fluid, and functional variables were evaluated in vitro. Main Outcome Measure(s): Sperm viability and motility, follicular fluid and calcium ionophore-induced acrosome reactions, protein tyrosine phosphorylation, expression of D-mannose binding sites, and ability of sperm to interact with zona pellucida. Result(s): Exposure of sperm to peritoneal fluid for up to 6 hours did not affect sperm viability or motility. Unlike follicular fluid, peritoneal fluid did not induce the acrosome reaction. Moreover, incubation of sperm with 20% v/v peritoneal fluid for 1 hour prevented the follicular fluid and the ionophore-induced acrosome reaction. Although treatment with peritoneal fluid allowed protein tyrosine phosphorylation during capacitation, it resulted in a significant decrease in the expression of D-mannose binding sites and sperm zona pellucida binding. Conclusion(s): Peritoneal fluid maintains sperm survival and decreases sperm ability to respond to inducers of the acrosome reaction and bind to the zona pellucida in vitro, indicating that this fluid might modulate sperm function in vivo. (Fertil Steril 2003;80: by American Society for Reproductive Medicine.) Key Words: Peritoneal fluid, capacitation, acrosome reaction, sperm, fertilization Mammalian sperm cannot fertilize the oocyte immediately after ejaculation. This ability is acquired after a series of physiologic and functional changes, collectively known as capacitation, which occurs as sperm pass through the female genital tract (1). During their ascent towards the oocyte, sperm interact with female genital tract fluids of multiple origin and complex composition; these fluids modulate spermfertilizing ability by currently unknown mechanisms (2). The oviduct provides the microenvironment for gamete storage and fertilization (3). The lack of a physical barrier separating the infundibular portions of the fallopian tubes and the peritoneal cavity allows peritoneal fluid to enter into the oviduct. At ovulation, peritoneal fluid might also be introduced by the waving action of the fimbriae that helps to capture the released ovum from the peritoneal cavity (4). Therefore, the microenvironment in which gamete interactions occur in vivo is composed of not only oviductal secretions and the follicular fluid embedded in the cumulus oocyte complex but also peritoneal fluid. Moreover, sperm that reach the ampulla before ovulation may interact with this fluid for hours before being exposed to follicular fluid, particularly in humans, among whom intercourse may occur at any time in the reproductive cycle. Whereas numerous reports have demonstrated that peritoneal fluid from infertile pa- 939

2 tients with endometriosis adversely affects several sperm variables (5 7), much less is known about the effect of peritoneal fluid on sperm function in the absence of endometriosis (8 10). We sought to examine the effect of peritoneal fluid from women with unexplained infertility on variables known to reflect the functional status of human sperm. MATERIALS AND METHODS The study was approved by the Institutional Review Board of the School of Medicine of Rosario, Argentina. All human samples were obtained after the donors gave written consent. Recovery and Preparation of Peritoneal and Follicular Fluids Peritoneal fluid was collected from the pouch of Douglas of patients scheduled for laparoscopy as part of the investigation of their cause of infertility. Samples were collected during the periovulatory phase ( days), as calculated from menstrual data. Only patients with unexplained infertility were included ( years). Peritoneal fluids were aspirated into sterile plastic tubes, and after microscopic assessment, blood-free samples were immediately transported to the laboratory (n 14). Follicular fluid was obtained during egg retrieval from six women participating in an IVF program. Superovulation was induced by daily IM injection of 150 IU of hmg (Serono Laboratories, Buenos Aires, Argentina) starting from day 3 of the cycle. Follicular growth was monitored by daily transvaginal ultrasonography and serum E 2 measurement. On the day that the leading follicles reached 18 mm, 10,000 IU of hcg (Serono Laboratories) was administered IM. Fluid was collected only from follicles with a diameter of 18 to 19 mm that had no blood contamination and contained an oocyte. Peritoneal and follicular fluids were centrifuged for 10 minutes at 600 g to remove cellular debris. Samples were filtered through a m membrane (Millipore Corp., Bedford, MA) and stored at 20 C until use. Fluids were tested for antisperm antibodies by using the indirect Mar test (Mar- Screen; Fertility Technologies, Inc., Natick, MA), and only antibody-free fluids were included. Follicular fluids were pooled for testing, whereas peritoneal fluid samples were tested individually. Semen Samples and Sperm Processing Semen samples were obtained from 43 normozoospermic donors ( years). The samples were collected by masturbation after 3 to 5 days of sexual abstinence. After complete liquefaction, semen analysis was performed according to World Health Organization guidelines (11) and morphology strict criteria (12). Semen samples were tested for antisperm antibodies by using the direct Mar test, and only antibody-free semen samples were included. Because each semen sample was used only once, the total number of samples used is the same as the number of experiments performed in each study. Seminal plasma was removed by layering 1 ml of semen on top of a discontinuous 90%-70%-50% Percoll gradient (Sigma Chemical Co., St. Louis, MO). Each tube was centrifuged for 20 minutes at 275 g. The bottom gradient, containing the motile sperm fraction, was then washed for 10 minutes with 2 ml of human tubal fluid (HTF) medium (GIBCO BRL, Life Technologies, Inc., Grand Island, NY). Sperm concentration was adjusted to 2 to /ml, and cells were incubated for capacitation in HTF supplemented with bovine serum albumin, 35 mg/ml (bovine serum albumin fraction V; Sigma Chemical Co.) for several time periods (see below), at 37 C and 5% CO 2 in air. For the D-mannose binding assay (D-mannose binding assay; Fertility Technologies, Inc.), sperm were incubated according to the manufacturer s instructions in HTF medium supplemented with HEPES buffer (GIBCO BRL, Life Technologies, Inc.) containing human serum albumin, 5 mg/ml (Sigma Chemical Co.). Postincubation sperm viability was assessed by mixing one drop of sperm suspension with one drop of eosin Y solution (0.5% in PBS) on a slide and examining 100 sperm at 400 magnification, as described elsewhere (11). Sperm Motion Analysis Sperm motility was evaluated by using a fully automated system (MEII; RoyCo S.A., Madrid, Spain). In brief, a 5- L drop of sperm suspension was placed in a Makler chamber (Sefi-Medical Instruments, Haifa, Israel), and sperm movement was captured by a video camera using a phase-contrast microscope (Olympus Optical Co., Ltd., Tokyo, Japan) with 200 magnification. Curvilinear velocity, progressive velocity, percentage of linearity (curvilinear velocity/progressive velocity 100), and lateral head displacement were measured in at least 100 sperm cells. Acrosome Reaction Induction To determine the ability of peritoneal fluid to induce the acrosome reaction, sperm were incubated for 22 hours under capacitating conditions and divided into several aliquots that were exposed for 1 hour to 20% or 50% v/v peritoneal fluid, 20% v/v follicular fluid, or medium alone, at 37 C and 5% CO 2 in air. In another set of experiments, after 22 hours of incubation, cells were exposed for 1 hour to peritoneal fluid (final concentrations of 0, 1%, 5%, 20%, and 50% v/v) and incubated with 20% v/v follicular fluid for an additional 1 hour. To evaluate the effect of peritoneal fluid on the calcium ionophore-induced acrosome reaction, sperm incubated in capacitating medium for 3 hours were exposed to 50% v/v peritoneal fluid or HTF for 1 hour at 37 C and 5% CO 2 in air. Each sample was then divided into two aliquots and 940 Munuce et al. Peritoneal fluid and human sperm function Vol. 80, No. 4, October 2003

3 exposed for 30 minutes to 10 M of calcium ionophore A23187 or medium (Sigma Chemical Co.), according to WHO guidelines (11). After the acrosome reaction induction procedure, sperm were washed twice with 1 ml of phosphate buffer (PBS; Sigma Chemical Co.) by centrifugation at 3000 g for 10 minutes. Cells were placed on a microscope slide until dry and were then fixed in absolute cold methanol for 30 seconds. The permeabilized cells were stained for 30 minutes with the fluorescent probe fluorescein isothiocyanate labeled Pisum sativum lectin (50 g/ml; Sigma Chemical Co.) at room temperature. Smears were examined at 1000 magnification by using an epifluorescence microscope (Olympus Optical Co., Ltd.). Two hundred sperm were counted for each individual experiment, and the percentage of acrosome reacted cells (with staining restricted to the equatorial segment) was determined. The acrosome reaction after ionophore challenge (ARIC) score was calculated as the percentage of acrosome reaction in the presence of ionophore minus the percentage of acrosome reaction in the absence of this compound (11). Electrophoresis and Western Blot Assay Proteins from noncapacitated cells and from sperm incubated for 3 hours under capacitating conditions and then exposed for 1 hour to 50% v/v peritoneal fluid or HTF were analyzed by SDS-PAGE and Western blot assay. Sperm were washed twice with PBS and resuspended in Laemmli sample buffer (13) containing deoxyribonuclease (DNAse), 5 mg/ml. After 15 minutes of incubation at room temperature, samples were centrifuged for 5 minutes at 6000 g, and the resulting supernatants were removed and heated for 5 minutes at 100 C in the presence of 70 mm of 2- mercaptoethanol. After centrifugation, the supernatants were stored at 20 C until use. Solubilized proteins (corresponding to sperm per lane) were separated on 7% SDS-polyacrylamide gels (13). Prestained molecular weight standards (GIBCO BRL, Life Technologies, Inc.) were run in parallel. Electrophoretic transfer of proteins to nitrocellulose (Sigma Chemical Co.) was performed according to the method of Towbin et al. (14) at 35 V and 4 C for 18 hours. The membranes were first blocked with 2% dry skim milk in PBS plus 0.1% Tween 20 (blocking solution) and were incubated for 1 hour with a monoclonal antiphosphotyrosine antibody (catalog number ; Upstate Biotechnology Inc., New York, NY) diluted 1:10,000 in blocking solution. Following 4 washings with PBS - 0.1% Tween 20, antimouse peroxidase conjugated IgG (catalog number ; Jackson ImmunoResearch Laboratories Inc., West Grove, PA) 1:5000 in blocking solution was added. After 1 hour of incubation, the membranes were extensively washed, and reactive bands were detected by enhanced chemioluminiscence using the ECL kit (Amersham Life Science, Inc., Oakville, Ontario, Canada) according to the manufacturer s instructions. All incubations were performed at room temperature. In some experiments, the antiphosphotyrosine antibody was blocked with 40 mm of O-phosphotyrosine (Sigma Chemical Co.) for 1 hour with constant rotation before use in immunoblotting. D-Mannose Binding Assay The D-mannose binding assay was performed on noncapacitated sperm or cells incubated for 3 hours under capacitating conditions, followed by 1 hour of exposure to 50% v/v peritoneal fluid or medium. Sperm were then incubated for 90 minutes with an -D-mannosylated biotin albumin solution (1 mg/ml in HTF plus HEPES buffer; Sigma Chemical Co.) at 37 C, and washed twice with PBS by centrifugation at 250 g for 5 minutes. A 10- L drop of the sample was placed on a slide and air-dried. A solution of avidin peroxidase (0.6 mg/ml in PBS; Sigma Chemical Co.) was added and incubated for 30 minutes at 37 C in a moist humidity chamber, followed by incubation with a drop of an urea diaminobenzidine solution (Sigma Chemical Co.). After 3 minutes, excess reagent was removed, a coverslip was placed on the slide, and at least 200 sperm were evaluated under 1000 magnification. Sperm showing a brown precipitate on all or part of the head were considered to have tested positive (i.e., they expressed the D-mannose receptor). Hemizona Binding Assay Human unfertilized oocytes donated by women undergoing an assisted reproduction program were stored in a salt solution containing 1.5 M of MgCl 2, 0.1% polyvinylpyrrolidone (molecular weight 36000), and 40 mm of HEPES in PBS (ph, 7.2). On the day of the experiment, the oocytes were washed for 15 minutes in HTF medium at room temperature, immobilized with a holding pipette, and bisected into equal halves by using a microscalpel attached to a micromanipulator and an inverted microscope. Each hemizona was placed in a 100- L oil overlay droplet containing 50% v/v peritoneal fluid or HTF (supplemented with 35 mg/ml bovine serum albumin; control hemizona), and inseminated with progressively motile sperm/ml. After 4 hours of incubation in 5% CO 2 in air, the hemizona were washed by repeated vigorous pipetting and the number of tightly bound sperm was counted under 400 magnification by using an inverted microscope. Results were also expressed as the hemizona index [(number of sperm bound in peritoneal fluid-treated hemizona)/(number of sperm bound in control hemizona) 100]. Statistical Analysis Statistical analysis was performed by using the GraphPad InStat program (GraphPad Software, San Diego, CA). Differences between treatments were determined by one-way analysis of variance and the Tukey Kramer multiple comparison post-test. The ARIC scores were analyzed by using FERTILITY & STERILITY 941

4 TABLE 1 Effect of peritoneal fluid on sperm viability and motility. Variable 0 hour 2 hours 6 hours Viable sperm (%) Control PF Progressively motile sperm (%) Control PF Curvilinear velocity ( m/s) Control a PF a Progressive velocity ( m/s) Control a PF a Linearity (%) Control PF Lateral head displacement ( m) Control PF Note: Results are means ( SE) (n 3); PF peritoneal fluid. a P.05 vs. 0 hour, by one-way analysis of variance and Tukey Kramer multiple comparison post-test. FIGURE 1 Effect of peritoneal fluid (PF) on the acrosome reaction. Overnight-capacitated sperm were exposed for 1 hour to human tubal fluid medium (control reaction [C]), 20% or 50% v/v peritoneal fluid, or 20% v/v follicular fluid (FF). The percentage of acrosome-reacted sperm was determined by using Pisum sativum agglutinin staining. Results are means ( SE) (n 5). a P.01 vs. other conditions. the Student t-test. The Wilcoxon signed-rank test was used to compare the number of sperm bound to each hemizona. Data are expressed as means ( SE). P.05 is considered significant. RESULTS Effect of Peritoneal Fluid on Sperm Viability and Motility To exclude a toxic effect of peritoneal fluid on sperm function, motility-enriched sperm samples were incubated under capacitating conditions in the presence or in the absence (control) of 50% v/v peritoneal fluid. Sperm viability and motility variables were evaluated at 0, 2, and 6 hours. Exposure of sperm to peritoneal fluid did not affect the percentage of viable sperm, progressive motility, or motility parameters (curvilinear velocity, progressive velocity, percentage of linearity, and lateral head displacement) at any time point studied (Table 1). After 2 hours of incubation, curvilinear velocity and progressive velocity increased significantly (P.05) in control sperm experiments (n 3) and in peritoneal fluid-treated sperm. Effect of Peritoneal Fluid on Induced Acrosome Reaction To investigate whether peritoneal fluid can induce the acrosome reaction, sperm incubated overnight in capacitating conditions were exposed to peritoneal fluid or follicular fluid as a positive control. Whereas 20% v/v follicular fluid significantly induced the acrosome reaction in these cells (n 5; P.01), peritoneal fluid did not increase the rate of acrosome reaction at either 20% or 50% v/v (Fig. 1). Because in vivo, sperm might be first exposed to peritoneal fluid and then to the follicular fluid in the vicinity of the oocyte, we performed experiments in which capacitated cells were sequentially incubated with peritoneal fluid followed by follicular fluid. Sperm that were treated with peritoneal fluid at concentrations 5% v/v and then exposed to follicular fluid had a significantly increased percentage of acrosome-reacted cells (n 5; P.001) (Fig. 2). Preincubation with peritoneal fluid at concentrations 20% v/v for 1 hour, however, prevented a follicular fluid-induced acrosome reaction (Fig. 2). To further investigate the effect of peritoneal fluid on acrosome reaction induction, sperm were incubated with peritoneal fluid before calcium ionophore challenge. Preincubation with peritoneal fluid significantly inhibited the ionophore-induced acrosome reaction observed under control conditions (Fig. 3). When the percentages of acrosome reaction were used to calculate the ARIC scores for each sample, all samples incubated in medium alone had values higher than 15% (the cut-off level according to the World Health Organization), but only 23% of samples had scores greater than 15% after exposure to peritoneal fluid (n 13) 942 Munuce et al. Peritoneal fluid and human sperm function Vol. 80, No. 4, October 2003

5 FIGURE 2 Effect of pretreatment with peritoneal fluid on follicular fluid (FF)-induced acrosome reaction. Overnight-capacitated sperm were exposed for 1 hour to peritoneal fluid (PF) at concentrations of 0, 1%, 5%, 20%, and 50% v/v and then to follicular fluid 20% v/v for 1 additional hour. An aliquot incubated for the whole period in human tubal fluid medium served as control (C). The percentage of acrosome-reacted sperm was determined by using Pisum sativum agglutinin staining. Results are means ( SE) (n 5). a P.001 vs. control reaction, PF 20%,and PF 50%. FIGURE 3 Effect of pretreatment with peritoneal fluid on the calcium ionophore-induced acrosome reaction. Capacitated sperm were exposed 1 hour to medium or 50% v/v peritoneal fluid, then divided into two aliquots and exposed for 30 minutes to medium or 10 M of calcium ionophore. The four aliquots obtained were called: C, C I, PF, and PF I, respectively. The percentage of acrosome-reacted sperm was determined by Pisum sativum agglutinin staining. Results are means ( SE) (n 13). a P.001 vs. other conditions. (mean values, 38.0% 4.8% vs. 13.0% 4.3%, respectively; P.001). Effect of Peritoneal Fluid on Sperm Protein Tyrosine Phosphorylation To investigate whether the inhibition of acrosome reaction induction by peritoneal fluid was due to an effect of this fluid on capacitation, sperm incubated for 3 hours under capacitating conditions were exposed to 50% v/v peritoneal fluid for an additional 1 hour and the pattern of protein phosphorylation on tyrosine residues was analyzed by Western blot assay. Incubation under capacitating conditions enhanced sperm protein tyrosine phosphorylation compared with that in fresh, noncapacitated cells (Fig. 4, lane b vs. a). When sperm incubated for 3 hours were exposed for 1 hour to 50% v/v peritoneal fluid, the phosphotyrosine signal was similar to that obtained in the absence of the fluid (Fig. 4, lane c vs. b). Phosphorylation was specific for tyrosine residues, as indicated by the fact that immunoreactivity was eliminated when the antibody was previously blocked with O-phosphotyrosine (data not shown). Effect of Peritoneal Fluid on Expression of Sperm D-Mannose Binding Sites Expression of sperm D-mannose binding sites on the sperm surface has been related to capacitation (15). To examine whether peritoneal fluid affects the appearance of these molecules on sperm, cells incubated under capacitating conditions were exposed to peritoneal fluid (50% v/v) and binding of D-mannose was evaluated by using the D-mannose binding assay. Figure 5 shows that whereas capacitated sperm had a significant increase in expression of D-mannose receptors compared with noncapacitated cells, the percentage of sperm exposed to peritoneal fluid that could bind to D-mannose residues was significantly decreased (n 8; P.05). Effect of Peritoneal Fluid on Sperm Zona Pellucida Binding To examine whether the reduction in the level of D- mannose receptors produced by peritoneal fluid was also reflected in a lower ability of sperm to recognize and bind to the zona pellucida, sperm were incubated for 4 hours with human hemizona in capacitating medium alone or medium containing 50% v/v peritoneal fluid. Peritoneal fluid resulted in a significant reduction in the number of sperm per hemizona that were tightly bound compared with the control reaction (n 6; P.01) (Fig. 6). Under these conditions, the mean hemizona index was 54.5% 5.5%. DISCUSSION During their transit through the female tract, sperm interact with different reproductive fluids. The peritoneal fluid FERTILITY & STERILITY 943

6 FIGURE 4 Protein tyrosine phosphorylation patterns in noncapacitated sperm (lane a) and cells incubated for 3 hours under capacitating conditions followed by 1 hour incubation in either medium alone (lane b) or 50% v/v peritoneal fluid (lane c). Sperm protein patterns were analyzed by Western blot assay using a monoclonal antiphosphotyrosine antibody. Molecular weight standards (Mr 10 3 ) are shown on the left side of the panel. Results of a typical experiment are shown. Each experiment was performed three times with different peritoneal fluid; similar results were obtained each time. FIGURE 5 Effect of peritoneal fluid on D-mannose binding assay. Noncapacitated sperm or 3 hour capacitated cells exposed for 1 hour to peritoneal fluid 50% v/v or control medium (C) were treated with an -D-mannosylated biotin albumin solution, and the presence of binding sites was recorded microscopically. Results are means ( SE) (n 8). a P.05 vs. other conditions. bathes the fimbrial end of the fallopian tubes and mixes with the follicular and oviductal fluids, contributing to the milieu in which fertilization takes place. Peritoneal fluid or some of its constituents may therefore participate in events that directly or indirectly influence sperm function (2). We examined the effect of this fluid on different variables that reflect the functional status of human spermatozoa. Although the effects of peritoneal fluid on sperm motility have been previously investigated, the results are contradictory. Although motile sperm have been laparoscopically recovered from the peritoneal cavity of infertile patients (16), several in vitro studies showed that peritoneal fluid from both fertile and infertile women can inhibit sperm motility (8, 17, 18). In view of this, we considered it important to first examine any possible toxic effect of our peritoneal fluid samples on sperm viability and motility. Sperm incubated for up to 6 hours in a capacitating medium containing peritoneal fluid at a high concentration (50% v/v) retained their viability, and their motility variables did not differ from those of control sperm. FIGURE 6 Effect of peritoneal fluid on sperm zona pellucida interaction. The hemizona binding assay was performed in the presence of peritoneal fluid, 50% v/v, or control medium (C), and the number of sperm tightly bound to the outer face of each hemizona was counted. The horizontal lines indicate mean values (n 6). a P.01 vs. control reaction. 944 Munuce et al. Peritoneal fluid and human sperm function Vol. 80, No. 4, October 2003

7 Although some investigators have found that fluids aspirated from hormonally stimulated patients maintain sperm motility (9), our observations indicate that even in the absence of stimulation, peritoneal fluid would be a favorable environment for sperm survival. This conclusion is in agreement with the success of pregnancies resulting from direct intraperitoneal sperm insemination (19, 20). The inhibitory effect described in other studies could be due to differences in the experimental protocols used to prepare the samples; early centrifugation of peritoneal fluid is required for removal of macrophages and lymphocytes, which may secrete toxic or immobilizing substances (21). Because the acrosome reaction is necessary for fertilization, we examined the effect of peritoneal fluid on this exocytotic event. Unlike follicular fluid, which is a physiologic acrosome reaction inducer (22, 23), peritoneal fluid did not induce the acrosome reaction, even at a concentration higher (50% v/v) than that assayed for follicular fluid (20% v/v). Using peritoneal fluid from hormonally stimulated patients, Revelli et al. (10, 24) reported similar results. However, in these cases, the lack of effect of peritoneal fluid on the acrosome reaction might be attributed to use of both a capacitating period and a sperm concentration not appropriate for examining acrosome reaction induction (23). Our experiments, which were performed under the optimal conditions for such observation, confirm that peritoneal fluid is not an acrosome reaction inducer. Given that P has been shown to induce the acrosome reaction (25), the inability of peritoneal fluid to produce this response might be attributed to its low P content (100-fold lower than that of follicular fluid) (26; unpublished data). Since human sperm can be exposed to peritoneal fluid during the entire menstrual cycle before reaching the follicular fluid containing cumulus, we investigated the effect of previous incubation with peritoneal fluid on the follicular fluid-induced acrosome reaction. Results of sequential exposure of sperm to peritoneal and follicular fluid, which was intended to mimic in vivo conditions, differed from those of previous studies in which sperm were directly incubated for several hours in the presence of both fluids simultaneously (24). We found that concentrations of peritoneal fluid of 5% v/v or less had no effect, whereas incubation of capacitated sperm with 20% v/v or more of peritoneal fluid for only 1 hour significantly reduced the incidence of follicular fluidinduced acrosome reaction. We also found that exposure of sperm to peritoneal fluid significantly inhibited the ionophore-induced acrosome reaction and decreased ARIC scores, variables that may be used to assess the ability of capacitated sperm to undergo the acrosome reaction and to evaluate the fertilizing ability of sperm (27, 28). These findings indicate that peritoneal fluid not only lacks the ability to induce the acrosome reaction but could also prevent its induction, similar to findings that have been reported for human oviductal fluid (29) or tubal cell secretions (30). Except for the few sperm that undergo the acrosome reaction in the oviduct, cells keep their acrosomes intact until they come into contact with the oocyte (1). It is possible that peritoneal fluid, as part of the oviductal milieu, regulates the number of sperm that undergo the acrosome reaction by stabilizing the acrosome and preventing a premature acrosomal loss. Considering that only fully capacitated sperm can undergo the acrosome reaction (1), it can be hypothesized that peritoneal fluid inhibits induction of the acrosome reaction by affecting sperm capacitation. Since capacitation involves an increase in protein tyrosine phosphorylation (31), we examined the possible effect of peritoneal fluid on this specific event. Our findings showed that incubation with peritoneal fluid did not affect the pattern of tyrosine phosphorylated proteins. Thus, the decreased ability of sperm to respond to acrosome reaction inducers after exposure to peritoneal fluid would not be due to an effect of the fluid on the signal transduction pathways leading to protein tyrosine phosphorylation. The possibility that peritoneal fluid components affect subsequent capacitation events, however, cannot be excluded. In this regard, it has been reported that D-mannose binding sites are expressed during capacitation and that their appearance correlates with the success of IVF (15, 32). When we examined the effect of peritoneal fluid on this functional event, results indicated that peritoneal fluid significantly reduced expression of D-mannose binding sites on the sperm surface. Considering that D-mannose residues participate in sperm zona pellucida binding (33), these results suggested that peritoneal fluid-incubated sperm might also be less able to recognize and bind to the zona pellucida. This possibility was supported by subsequent experiments indicating that peritoneal fluid significantly reduced the number of sperm bound to the human hemizonae compared with control reactions. At the end of the assay, motility levels were the same as in controls (Table 1), indicating that this inhibition is not due to an effect of the fluid on the sperm capacity to reach the zona pellucida. To our knowledge, these are the first studies indicating that peritoneal fluid from patients with unexplained infertility affects sperm zona pellucida binding capacity, an important variable in sperm function. The inhibition of the ability of sperm to respond to acrosome reaction inducers, together with the lower expression of D-mannose binding sites and the reduced sperm zona pellucida binding ability, strongly suggests that peritoneal fluid can modulate sperm function. Because sperm fertilizing capacity wanes relatively soon after the acrosome reaction, peritoneal fluid, as part of the oviductal environment, may retain the fertilization potential of sperm by maintaining motility and stabilizing the acrosome. The ability of peritoneal fluid to allow sperm survival might be important in FERTILITY & STERILITY 945

8 cases in which sperm must stay in the ampulla for hours before meeting the oocyte; this particularly pertains to humans, because intercourse may occur at any time during the menstrual cycle. After ovulation, follicular fluid mixes with the oviductal and peritoneal fluids already present in the oviduct. Thus, the ultimate effect of peritoneal fluid on sperm function in vivo depends on the relative contribution of this fluid to the tubal environment, which changes during the cycle (26). The mechanisms by which the peritoneal fluid exerts these effects are unknown. Peritoneal fluid may contain some components that interfere with the expression of sperm zona pellucida receptors during capacitation or that directly block or mask these receptors, leading to a decreased sperm ability to bind to zona pellucida and to respond to acrosome reaction inducers. The presence of such factors has been reported for other fluids in the oviduct that also inhibit the zona pellucida binding capacity of human sperm (34). Further studies are required to elucidate the mechanisms by which peritoneal fluid modulates sperm function during fertilization. Acknowledgment: The authors thank Fabiana Pauluzzi for technical assistance. References 1. Yanagimachi R. Mammalian fertilization. In: Knobil E, Neill JD, eds. The physiology of reproduction. New York: Raven Press, 1988: Barratt CL, Cooke ID. Sperm transport in the human female reproductive tract a dynamic interaction. Int J Androl 1991;14: Suarez SS. Sperm transport and motility in the mouse oviduct: observations in situ. Biol Reprod 1987;36: Leese HJ. The formation and function of oviduct fluid. J Reprod Fertil 1988;82: Syrop CH, Halme J. Peritoneal fluid environment and infertility. Fertil Steril 1987;48: Coddington C, Oehninger S, Cunningham DS, Hansen K, Sueldo C, Hodgen GD. 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