Gonadotropin-Releasing Hormone Increases Ability of the Spermatozoa to Bind to the Human Zona Pellucida¹

^a Unit of Reproductive Biology, Faculty of Health Sciences, University of Antofagasta, Antofagasta, Chile

	ABSTRACT

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Sperm-zona pellucida binding, a crucial step in the process of fertilization, takes place in vivo in the upper portion of the fallopian tube. The presence of GnRH-like peptides in the female and the male genital tract has been described. In this work, the effect of GnRH and related peptides upon sperm-zona pellucida binding ability was studied. Sperm aliquots, capacitated for 4.5 h, were incubated for 5 min with saline (control) or 20 nM of GnRH, C-terminal (1-5) or N-terminal (5-10) fragments of GnRH, Substance P, dynorphin, bombesin, or mixed GnRH (a synthetic peptide with the same amino acids as GnRH but in different order). Sperm were also incubated with the GnRH antagonist Ac-3,4-dehydro-Pro¹, -p-fluoro-D-Phe², D-Trp^3,6-LHRH, alone or before adding GnRH. The sperm were then tested using the hemizona assay. After 10 min, the number of zona-bound sperm was determined. In addition, the effect of GnRH upon the acrosome reactions, sperm movement characteristics, and sperm-zona collisions was evaluated. Sperm treated with GnRH bound in higher numbers to the zona than did control sperm (p < 0.005). The GnRH fragments, the GnRH antagonist, and related peptides did not have any effect on sperm-zona interaction; however, the GnRH antagonist totally blocked the stimulatory effect of GnRH. GnRH did not affect the percentage of acrosome-reacted sperm, pattern of sperm movement, or frequency of sperm-zona collisions. I suggest that the increased ability of the sperm to bind to the zona may be due to exposure and/or change of affinity of zona receptors on the sperm plasma membrane.

	INTRODUCTION

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Sperm-zona pellucida interaction represents a highly specific, effective cellular recognition system in nature and has been the subject of intense research. Significant progress has been made in the study of the mammalian zona pellucida, particularly concerning its macromolecular composition, structure, biosynthesis, and function [1]. In contrast, our knowledge about sperm plasma membrane components is still sparse [2].

It is generally accepted that the acrosome reaction, a crucial event for the success of fertilization, occurs when the fertilizing spermatozoon reaches the surface of the zona pellucida. Moreover, it has been reported that the acrosome reaction can be induced by some component(s) of the zona pellucida [1]. Thus, factors that enhance or inhibit sperm-zona pellucida binding acquire special relevance since they could alter the probability of conception. These factors may be present in the fluids that bathe the gametes before their encounter and/or in the microenvironment in which gamete interaction occurs in vivo.

GnRH-like material has been detected in follicular fluid [3] and human seminal plasma [4, 5]. This material is probably synthesized in the gonads [6-9]. Recently, GnRH and GnRH agonists have been shown to enhance bovine in vitro fertilization through an effect on cumulus-oocyte complexes [10]. In addition, a tripeptide of prostatic origin that is structurally related to thyrotrophin-releasing hormone, pyroglutamylglutamylproline-amide, has been implicated in the process of mouse sperm capacitation [11, 12].

The present work was undertaken to study the effect of GnRH upon the ability of human sperm to bind to the human zona pellucida. Additionally, the effect of GnRH upon the human sperm acrosome reaction, pattern of sperm movement, and frequency of sperm-zona pellucida collisions was evaluated.

	MATERIALS AND METHODS

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Reagents

GnRH, Ac-3,4-dehydro-Pro¹, -p-fluoro-D-Phe², D-Trp^3,6-LHRH (pF-antagonist), and the C-terminal fragment (1-5) of GnRH (pGlu-His-Trp-Ser-Tyr-NHNH₂) were obtained from Bachem Bioscience Inc. The N-terminal fragment (5-10) of GnRH (Tyr-Gly-Leu-Arg-Pro-Gly-NH₂) was a gift from Dr. Jean Rivier, Salk Institute, La Jolla, California. The synthetic peptide pGlu¹-Pro²-Arg³-Gly⁴-Tyr⁵-Leu⁶-Gly⁷-Trp⁸-His⁹-Ser¹⁰-NH₂, which possesses the same amino acids as GnRH but in mixed order (mixed GnRH), was obtained from Bios Chile (Santiago, Chile). All other reagents were obtained from Sigma Chemical Co. (St. Louis, MO).

Source and Preparation of Biological Material

Human zonae pellucidae. The zonae pellucidae used in this study were recovered from human cadaveric ovarian tissue as described previously [13, 14]. Briefly, ovarian tissue was placed on ice and dissected immediately according to the protocol of Overstreet et al. [15]. Zona-intact, immature oocytes were denuded of granulosa cells, placed in capillary tubes containing 2 M dimethylsulfoxide in PBS, and stored at -80°C. Before use, the oocytes were cut to obtain two equal halves or hemizonae (see below).

Human spermatozoa. Samples were obtained from six different men with normal semen according to WHO guidelines [16]. Motile spermatozoa were obtained using a two-step Percoll gradient as previously described [14, 17]. The sperm were then resuspended at 10⁷ cells/ml and incubated in modified Tyrode's medium [14] supplemented with 2.6% BSA, at 5% CO₂ in air.

Sperm-Zona Pellucida Binding Assay

The effect of GnRH and related peptides upon sperm-zona binding ability was tested with the hemizona assay (HZA) [18]. In the HZA, 100-µl droplets of control and test sperm suspensions were held under oil in a plastic Petri dish. One hemizona was added to the control sperm droplet, and the matching hemizona was added to the test sperm droplet. Control and test sperm droplets containing hemizonae were incubated for 10 min at 37°C in 5% CO₂ in air. After incubation, each hemizona was removed and gently washed with a wide-bore pipette. The sperm that were tightly bound to the outer surface of each hemizona were counted under a phase-contrast microscope.

Effect of GnRH and Related Peptides upon the Gametes

General procedures. Droplets (95-µl) of sperm suspended in modified Tyrode's medium containing 2.6% BSA were treated by adding 5 µl of GnRH, GnRH-related peptides, or saline.

Sperm-zona pellucida binding. Sperm aliquots capacitated for 4.5 h were treated with 20 nM GnRH or its vehicle (saline) for 5 min and then tested in the HZA. In addition, the effect of C-terminal and N-terminal fragments of GnRH and of the pF-antagonist were tested. For the antagonist, the sperm were treated with either 1) 20 nM pF-antagonist for 5 min or 2) 20 nM pF-antagonist for 5 min followed by 5 min with 20 nM GnRH. Then the sperm were tested in the HZA. In control experiments, sperm were treated with 20 nM of Substance P, dynorphin, bombesin, or mixed GnRH for 5 min and then tested in the HZA. To verify whether GnRH has an effect on the zona pellucida, the test hemizonae were incubated with 20 nM GnRH, and the control hemizonae with saline for 15 min. Then they were washed in cultured medium and tested in the HZA using untreated sperm. To verify whether GnRH had to be present during gamete interaction, some sperm suspensions were treated with 20 nM GnRH for 5 min. Then the sperm were washed with fresh medium before the HZA.

Acrosome reactions. Sperm aliquots capacitated for 4.5 h or 20 h were treated with 20 nM GnRH or saline for 5 min. As a positive control, sperm aliquots were incubated with 20% follicular fluid, as previously described [19]. After 10 min, sperm viability and acrosomal status were evaluated using the supravital dye Hoechst 33258 and the fluorescein isothiocyanate (FITC)-labeled Pisum sativum agglutinin, respectively [19, 20].

Pattern of sperm movement. Motile sperm suspensions, obtained as described above, were treated with 20 nM GnRH or saline for 10 min. Then the sperm movement was recorded using positive phase contrast optics at 37°C. Videotape recording was carried out as described previously [21]. The videotapes were analyzed manually frame by frame. Twenty-five sperm were analyzed for each condition. Each spermatozoon was analyzed for straight-line velocity, linearity of the trajectory, rolling frequency of the sperm head, flagellar beat frequency and amplitude, and flagellar curvature ratio. A detailed description of this procedure has been published previously [21, 22].

Sperm-zona collision versus binding. To determine whether the effect of GnRH involved an increase in the number of sperm-zona collisions or in the effectiveness of sperm-zona collisions (defined as number of zona-bound sperm divided by number of sperm colliding with the zona x 100), the following experiments were designed. Aliquots of GnRH-treated and control sperm suspensions, obtained as described before, were added to a hemizona in a plastic Petri dish and incubated for 10 min at 37°C in 5% CO₂ in air, under oil. The sperm-hemizona interaction was recorded at a rate of 60 fields/sec by a high-speed video camera (Tritronics, Burbank, CA). A record of elapsed time in 0.01-sec intervals, generated by a video time generator (For A, Los Angeles, CA), was simultaneously recorded on a video cassette recorder (Victor Co., Tokyo, Japan). Details of these procedures have been published previously [21, 22]. At the end of the 10-min recording, the videotapes were analyzed manually frame by frame to determine 1) the number of sperm-zona collisions and 2) the number of zona-bound sperm.

Statistics. Bartlett's test for homogeneity, followed by the F test and then the paired t-test and/or Dunnett's multiple comparison tests were used to compare the number of bound spermatozoa in the control and treated groups. Differences were considered significant at the 0.05 level of confidence.

	RESULTS

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GnRH treatment increased the number of sperm bound to the zona pellucida (61.5 ± 9 control, 221 ± 20 GnRH, mean ± SEM). This increase represents an average of 365 ± 15% more sperm bound to the zona in the GnRH group than in the control group (p < 0.005) (Fig. 1). The stimulatory effect of GnRH was still evident after the sperm were washed and resuspended in fresh Tyrode's medium before the HZA (41.3 ± 7 and 124.3 ± 18 zona-bound sperm in the control and GnRH-treated groups, respectively; Fig. 1). Treatment with C-terminal or N-terminal fragments of GnRH had no effect on the number of sperm bound to the zona pellucida (Fig. 1). Nor did treatment of the hemizonae with GnRH increase the number of sperm bound to them (Fig. 1).

View larger version (16K): [in this window] [in a new window]   FIG. 1. Effect of GnRH on the number of spermatozoa bound to the human hemizona pellucida. Data are expressed as a percentage (mean ± sem) of the control hemizonae. Sperm suspensions were incubated for 5 min with saline (control), 20 nM GnRH, 20 nM GnRH and then washed and resuspended in fresh medium (Washed GnRH), 20 nM GnRH fragment 1 to 5 (F1-5), or 20 nM GnRH fragment 5 to 10 (F 5-10) before the hemizona assay. In some experiments, the hemizonae were treated with 20 nM GnRH and then tested in the hemizona assay using untreated sperm (Zona). The number within the bars indicates the number of experiments in each group. *Significantly different from groups not treated with GnRH (p = 0.0015).

Other peptides of size and/or structure similar to that of GnRH did not have any significant effect on the sperm-zona binding ability (Fig. 2). This was the case even when the sperm were incubated with the "mixed GnRH" peptide, which possesses the same amino acids as GnRH but in a different order (Fig. 2).

View larger version (14K): [in this window] [in a new window]   FIG. 2. Effect of various peptides on the number of spermatozoa bound to the human hemizona pellucida. Data are expressed as a percentage (mean ± sem) of the control hemizonae. Sperm suspensions were incubated for 5 min with saline (control) or with 20 nM GnRH, mixed GnRH, Substance P, dynorphin, or bombesin before the hemizona assay. The number within the bars indicates the number of experiments in each group. *Significantly different from all other groups (p = 0.001).

The stimulatory effect of GnRH on sperm-zona pellucida binding depended upon the concentration of the decapeptide used (Fig. 3). The curve of dose-response of GnRH has an inverted "U" shape, with maximum activity between 10 nM and 75 nM. Lower and higher concentrations of GnRH did not have any significant effect on the number of zona-bound sperm (Fig. 3).

View larger version (14K): [in this window] [in a new window]   FIG. 3. Effect of different doses of GnRH on sperm binding to the human zona pellucida. Data are expressed as a percentage (mean ± sem) of the control hemizonae at a given GnRH concentration. Sperm suspensions were incubated for 5 min with saline (open circle) or with various concentrations of GnRH (closed circles) before the hemizona assay.

The next experiments were conducted to test the effect of a GnRH antagonist upon the stimulatory effect of GnRH. The pF-antagonist by itself had no effect on the number of sperm bound to the zona (Fig. 4). However, sperm treated with the pF-antagonist before the addition of GnRH exhibited a zona-binding ability not different from that of the control and significantly lower than that of sperm treated with GnRH alone (Fig. 4).

View larger version (16K): [in this window] [in a new window]   FIG. 4. Effect of a GnRH antagonist on the number of spermatozoa bound to the human hemizona pellucida. Sperm suspensions were incubated for 5 min with saline (Control), 20 nM GnRH, 20 nM GnRH antagonist (pF), or 20 nM of the antagonist followed by 20 nM GnRH (pf + GnRH) before the hemizona assay. The number within the bars indicates the number of experiments in each group. *Significantly different from respective control (p = 0.014).

There are at least two possible means by which GnRH could stimulate sperm-zona binding: 1) by increasing the frequency of sperm-zona collisions, perhaps through a change in the pattern of sperm movement, or 2) by increasing the effectiveness of sperm-zona collisions. These possibilities were explored in the following experiments.

The analysis of video tapes revealed that treatment with GnRH did not modify the pattern of sperm movement. None of the sperm movement parameters measured differed for control and GnRH-treated sperm suspensions (data not shown). In addition, significant differences were not detected between the two groups in the number of sperm colliding with the zona pellucida (Fig. 5A). This was true whether the numbers of sperm-zona collisions per minute (data not shown) or the cumulative frequencies of collisions were compared (Fig. 5A). At the end of the 10-min recording period, the mean (± SEM) number of cumulative sperm-zona collisions was 261 ± 37 for the control and 287 ± 41 for the GnRH-treated group. At the same time, however, the mean number of zona-bound sperm was 6.8 ± 1 for the control and 26.4 ± 3 for the GnRH group (p < 0.001; Fig. 5B). Thus, in the control group, 38 sperm had to collide with the zona in order to obtain 1 zona-bound sperm. In contrast, in the GnRH-treated group only 11 sperm had to collide with the zona to obtain 1 sperm bound to it. Therefore, the effectiveness of sperm-zona collision was increased from 2.4% in the control to 9.5% in the GnRH-treated group.

View larger version (14K): [in this window] [in a new window]   FIG. 5. Frequency of sperm-zona pellucida collisions versus number of zona-bound sperm. A) Mean number of cumulative sperm-zona collisions per minute. Sperm suspensions were incubated for 5 min with saline (open circles) or 20 nM GnRH (closed circles) before sperm-zona interaction was recorded. Each symbol represents the mean ± sem of 5 experiments. B) Number of zona-bound sperm (mean ± sem). Data are expressed as a percentage (mean ± SEM) of the control hemizonae. Sperm suspensions were incubated for 5 min with saline (control) or 20 nM GnRH before the hemizona assay. The number within the bars indicates the number of experiments in each group. *Significantly different from respective control (p < 0.01).

GnRH did not have any significant effect on the percentage of acrosome reactions or on the viability of the human spermatozoa. This was the case whether the sperm were capacitated for 4.5 or 20 h (Table 1). In contrast, follicular fluid treatment induced a significantly higher percentage of acrosome reactions without a change in sperm viability (Table 1).

	DISCUSSION

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In this report, evidence has been presented that GnRH has a direct stimulatory effect upon human sperm. Treatment with GnRH increased almost 4-fold the number of sperm bound to the human zona pellucida, in comparison to the control. This effect was direct and specific upon the sperm cells and was not mediated by an increase in the acrosome reaction or by a change in the pattern of sperm movement. This is the first time that a direct effect of GnRH upon the sperm cells has been described.

The experimental evidence that supports the assumption that the effect of GnRH was specific upon sperm is as follows: 1) GnRH increased zona-binding ability only when the sperm were incubated with the peptide. Incubation of the zona pellucida with GnRH did not stimulate sperm binding. 2) The effect required the intactness of the GnRH molecule; GnRH fragments were unable to mimic the stimulatory effect of the decapeptide. Moreover, a synthetic peptide, constructed using the same 10 amino acids of native GnRH but in mixed order, was unable to mimic the effect of GnRH on the sperm. 3) Other natural peptides, of molecular structure similar to that of GnRH, did not have any significant effect on the sperm. 4) The stimulatory effect of GnRH was inhibited by incubating the sperm with a GnRH antagonist. The pF-antagonist, used at the same concentration of GnRH, had no effect by itself, but it effectively blocked the stimulatory action of GnRH. The development of powerful GnRH antagonists has provided an important tool to prevent the action of GnRH. The antagonists occupy the GnRH receptors without inducing conformational changes [23]; therefore, the cellular response is blocked. Moreover, they have greater affinity for the receptor than does the native molecule.

Whether there are GnRH receptors on the sperm cells is still an open question, although the present results strongly suggest this possibility. GnRH receptors have been demonstrated in the testis [24, 25] and in the ovary [24, 26]. In the testis, the receptors seem to be located in the Leydig cells [27–31]. In the ovary, they are located in the granulosa and theca cells [27, 32]. In addition, local production of a GnRH-like material has been demonstrated in the testis and in the ovary, in which Sertoli and granulosa cells, respectively, may secrete it [33, 34]. The GnRH-like material in the gonads is immunologically different from hypothalamic GnRH; it is sensitive to elevated temperatures that do not affect GnRH, and its chromatographic behavior differs from that of GnRH [6, 35, 36]. Finally, GnRH-like material has been detected in human seminal plasma [4, 5]. Accordingly, there are several possible places along the male and female genital tract where sperm might interact with GnRH (or GnRH-like material). For instance, GnRH produced by Sertoli cells may influence sperm during spermatogenesis. During ejaculation, the sperm cells may interact with GnRH present in seminal plasma. Finally, during their transit throughout the oviduct, the sperm cells may interact with GnRH transferred by the products of ovulation, i.e., follicular fluid and granulosa cells. The presence of GnRH-like material in the oviductal fluid, however, has not been investigated.

The observations reported here also provide important basic information about the process of sperm-zona pellucida interaction. Without exogenous GnRH, sperm did bind to the zona pellucida, although with remarkably low efficiency. Only about 2% of the sperm that collided with the zona pellucida were able to bind to it. GnRH increased the efficiency of this process to about 10%. In other words, treatment with GnRH increased the proportion of sperm capable of binding to the zona pellucida. Although the exact mechanism by which GnRH accomplished this is unknown, a change in the pattern of sperm movement or in the percentage of acrosome-reacted sperm were not involved. Therefore, I suggest that the effect of GnRH may be due to exposure and/or change of affinity of receptors for the zona pellucida on the sperm plasma membrane. These possibilities are now under investigation in our laboratory.

In summary, this study has demonstrated that GnRH increases sperm binding to the zona pellucida in vitro, that this effect depends upon the integrity of the peptide structure, and that a GnRH antagonist inhibits this effect. It has also demonstrated that the effect of GnRH was not mediated by a change in the percentage of acrosome-reacted sperm or by a change in the pattern of sperm movement. Additional work is needed before it can be concluded whether GnRH is acting in vivo and whether there are GnRH receptors in the sperm plasma membrane.

	FOOTNOTES

 
¹ This work was financed by Fondecyt 197 1243.

² Correspondence: Patricio Morales, Unit of Reproductive Biology, Faculty of Health Sciences, University of Antofagasta, P.O. Box 170, Antofagasta, Chile. FAX: (5655) 24 74 83; pmoral;cactcreuna.cl

Accepted: April 3, 1998.

Received: December 29, 1997.

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