Biol Reprod Keystone Symposia Conference on Frontiers in Reproductive Biology & Regulation of Fertility.
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Biology of Reproduction 67, 1360-1365 (2002)
© 2002 Society for the Study of Reproduction, Inc.

Inhibition of In Vivo and In Vitro Fertilization in Rodents by Gonadotropin-Releasing Hormone Antagonists1

Patricio Morales2,a, Consuelo Pastena, and Eduardo Pizarroa

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


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
We have examined the effect of two GnRH antagonists, Ac-D-Nal1-Cl-D-Phe2-3-Pyr-D-Ala3-Arg5-D-Glu(AA)6-GnRH (Nal-Glu) and Ac3,4-dehydro-Pro1,-p-fluoro-D-Phe2,D-Trp3,6-GnRH (4pF), on in vivo and in vitro fertilization in rodents. Female rats were treated in the afternoon of proestrus with 2 µl of Nal-Glu or 4pF (0.5 and 5 mM) injected directly into one oviductal horn (experimental); saline was injected into the contralateral horn (control). Females were then mated and the oviducts were perfused for egg and sperm recovery. The results indicate that both antagonists inhibited in vivo fertilization. Thus, the percentage of fertilized eggs in control oviducts ranged from 92% ± 5% to 100% ± 0%, whereas in treated oviducts, fertilization ranged from 25% ± 6% to 73% ± 5%. GnRH antagonists did not interfere with the process of ovulation, sperm migration to the site of fertilization, or early embryo development. In additional experiments with mice, GnRH antagonists inhibited in vitro fertilization. One fertilization event that was specifically inhibited by GnRH antagonists was the process of sperm binding to the zona pellucida. This step was precisely monitored using the hemizona assay. GnRH antagonists did not affect sperm movement or acrosomal status. These observations indicated that local treatment with GnRH antagonists inhibit in vivo fertilization and give additional support to the idea that endogenous GnRH may play an important role during fertilization by increasing the efficiency of sperm-zona binding.

female reproductive tract, fertilization, gamete biology, gonadotropin-releasing hormone, sperm motility and transport


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
All mammalian eggs are surrounded by the zona pellucida (ZP), an extracellular coat that is synthesized by the oocyte [1]. The ZP is the site at which the initial interaction between the spermatozoa and the egg takes place. This interaction includes species-specific sperm-ZP binding and induction of the acrosome reaction, events that are essential for fertilization success [2]. Thus, the search for molecules with the capacity to modify gamete interaction is of physiological relevance because these molecules could alter the probability of conception. In the last few years, several molecules with the capacity to modify sperm-ZP binding have been studied (see the review in [3]). GnRH is one of them [4].

GnRH is a hypothalamic decapeptide, the function of which is to produce the pulsatile release of FSH and LH from pituitary gonadotrophs [5]. The presence of GnRH, or one or more GnRH-like peptides, has been described in many extrahypothalamic places, including the reproductive tract [5]. Thus, reports exist that describe the presence of GnRH in follicular fluid [6], seminal plasma [7, 8], uterus, and fallopian tubes [9, 10]. In addition, receptors for GnRH (GnRH-R) have been described in the reproductive tract, including testes and ovaries [11, 12]. Recently, it was reported that human sperm possess GnRH-R in their plasma membrane [13] and the mRNA for the GnRH-R has been found in the testicular germ cells of rats and mice [14].

It has been reported that GnRH treatment increases sperm-ZP binding in humans [4] and in vitro fertilization (IVF) in bovines [15], and treatment of spermatozoa with GnRH has resulted in a several-fold increase in the number of zona-bound sperm. This effect was specific to spermatozoa because treatment of the ZP did not modify sperm binding, and it was not related to changes in the pattern of sperm movement or acrosomal status [4, 16]. Moreover, the effect of GnRH was totally blocked by GnRH antagonists. In a separate study, we found that GnRH antagonists decreased the ability of the sperm to initiate binding to the human ZP in a dose-dependent manner without affecting the pattern of sperm movement, frequency of sperm-zona collisions, or acrosomal status [16].

These observations have led us to hypothesize that spermatozoa might interact with GnRH (or a GnRH-like peptide) during their journey through the male and female genital tracts. Interaction with GnRH might confer greater zona-binding capabilities on spermatozoa. Conversely, prevention of this interaction might render spermatozoa unable to initiate binding to the ZP and decrease or inhibit the possibility of conception. In this work we studied the effect of GnRH antagonists on in vivo and in vitro fertilization in rodents. We evaluated several aspects of the fertilization process in an attempt to unravel the specific step during fertilization at which GnRH antagonists act.


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

All reagents were obtained from Sigma Chemical Company (St. Louis, MO) unless otherwise indicated. Two GnRH antagonists were used in this study, Ac-D-Nal1-Cl-D-Phe2-3-Pyr-D-Ala3-Arg5-D-Glu(AA)6-GnRH (Nal-Glu) and Ac-3,4-dehydro-Pro1,-p-fluoro-D-Phe2,D-Trp3,6-GnRH (4pF). GnRH antagonist 4pF was obtained from Bachem Bioscience Inc. (King of Prussia, PA).

In Vivo Experiments

Animals We used Sprague-Dawley rats that had been bred in-house. The animals were kept under controlled temperature (22–24°C), and lights were on from 0700 to 1900 h. Water and pelleted food were supplied ad libitum. Males were 4–5 mo old, weighed 400–450 g, and were of proven fertility. Females weighing 200–250 g were selected from among those that had had at least two regular cycles of 4 days. The regularity of these cycles was verified by daily vaginal smears.

In vivo fertilization Cycling females in the afternoon of proestrus were used in these experiments. To test the effect of GnRH antagonists on IVF, females were treated by delivering 2 µl of a solution containing the antagonist directly into the lumen of one oviduct; the contralateral oviduct received 2 µl of the solvent of the GnRH antagonist (saline). In some animals, GnRH antagonists were injected intrabursa; saline was injected into the contralateral bursa. In additional control animals, one oviduct received 2 µl of saline and the contralateral oviduct was sham-injected.

Intraoviductal injection technique To perform these procedures, animals were anesthetized with ether at 1600–1700 h on the day of proestrus. Two small incisions were made directly above the fat pad of the left and right ovaries. Each oviduct and ovary were externalized through the respective incision, and the intraoviductal injection was performed under microscopic magnification by inserting a 30-gauge needle fitted to a 5-µl Hamilton syringe (Hamilton Company, Reno, NV) through the ovarian bursa into the infundibulum of the oviduct. Intrabursa injection was performed by inserting the 30-gauge needle into the ovarian bursa. The organs were then returned to the peritoneal cavity, and muscles and skin were sutured. Animals were initially placed in separate cages until normal behavioral activity was resumed, and were then caged with males of proven fertility for 12–14 h. The next morning, mating was verified by observation of spermatozoa in the vaginal smears. This day was considered Day 1 of pregnancy.

Oocyte/embryo recovery During the morning on Day 2 of pregnancy, females were killed with an overdose of ether, and the oviducts were removed and freed of fat tissue. Each oviduct was flushed separately with saline. The flushings were examined under low-power magnification and the number of two-cell embryos was scored [17]. The eggs that were not divided by Day 2 of pregnancy were fixed in 1:3 acetic acid:ethanol, stained with 1% orcein, and observed under 100x phase contrast microscopy to establish the presence of pronulei [18].

Sperm migration In additional experiments we evaluated the effect of GnRH antagonists on sperm migration to the site of fertilization. During the afternoon of proestrus, females were treated by injecting 2 µl of a solution containing the antagonist directly into the lumen of one oviduct; the contralateral oviduct received 2 µl of the solvent of the GnRH antagonist (saline). After the animals recovered from surgery, they were caged with males of proven fertility. The next morning, approximately 6–8 h after coitus, females were killed with an overdose of ether and the oviducts were removed and freed of fat tissue. The number of sperm recovered from each oviduct was evaluated essentially as described by Smith and Yanagimachi [19] and modified by Orihuela et al. [20]. In brief, after extracting the oviducts, they were divided into ampulla and isthmus (containing the intramural segment), and each segment was flushed twice, the first time with 50 µl of saline and the second time with 50 µl of saline + 0.5% Triton X-100. This was done to remove the spermatozoa that were free in the lumen and those that were adhering to the oviductal epithelium, respectively. The sperm in each flushing were counted using brightfield microscopy at a magnification of 20x.

In Vitro Experiments

Animals We used CF1 mice that had been bred in-house. The animals were maintained at 22–24°C on a 12L:12D cycle. Water and pelleted food were supplied ad libitum. Males were 10–12 wk of age and of proven fertility. Females were 4–6 wk of age.

Oocyte collection Females were superovulated by an i.p. injection of 5 IU of eCG (Folligon; Intervet, Boxmeer, Holland) followed by an i.p. injection of 5 IU hCG (Chorulon; Intervet) given 48 h later. Animals were killed by ether overdose 14 h after hCG injection, and cumulus-enclosed oocytes were collected from the oviducts. The entire oviduct was dissected into M2 medium [21, 22] maintained at 37°C. The ampullae were opened by tearing them with a 25-gauge needle, and cumulus-oocyte masses were released into M2 medium at 37°C and 5% CO2 in air. Ten minutes before use, the oocytes enclosed in cumulus masses were transferred to T6 medium [21, 22] at 37°C and 5% CO2 in air. The T6 medium consisted of 99.4 mM NaCl, 1.42 mM KCl, 0.47 mM MgCl2, 0.36 mM Na2HPO4, 1.78 mM CaCl2, 25 mM NaHCO3, 24.9 mM sodium lactate, 0.47 mM sodium pyruvate, 5.56 mM glucose, 100 U/ml penicillin, 50 µg/ml streptomycin sulfate, 0.001% (w/v) phenol red, and 20 mg/ml BSA (A4378; Sigma). The M2 medium consisted of 94.7 mM NaCl, 4.78 mM KCl, 1.19 mM MgSO4, 1.19 mM KH2PO4, 1.71 mM CaCl2, 4 mM NaHCO3, 21 mM Hepes, 23.3 mM sodium lactate, 0.33 mM sodium pyruvate, 5.56 mM glucose, 100 U/ml penicillin, 50 µg/ml streptomycin sulfate, 0.001% (w/v) phenol red, and 4 mg/ml BSA (A4378; Sigma).

Sperm preparation Two males were used for each experiment. The caudae epididymides were removed from each male, minced, placed into 0.5-ml drops of T6 medium under oil in sperm dispersion dishes (Falcon 1008; Fisher Scientific, Pittsburgh, PA), and the residual tissue was discarded. The dispersion dishes were placed in 5% CO2 and air at 37°C for 30 min to allow the sperm to disperse. Then, the sperm suspension drops were twice diluted 3:2 with T6 medium and placed into 0.5-ml drops in fertilization dishes (3801; Falcon) under oil. The final sperm concentration was adjusted to 4 x 106 cells/ml and the suspension was incubated for an extra 60 min to allow capacitation. Fifteen minutes before the end of capacitation, different concentrations of GnRH antagonists were added to the sperm drops. Control drops received the solvent of the antagonists (saline). During this period, sperm aliquots were taken to evaluate sperm motility.

In vitro fertilization In vitro fertilization was carried out in 0.5-ml drops of T6 medium under mineral oil as described [21, 22]. After capacitation, oocyte-cumulus complexes were transferred to the 0.5-ml sperm suspension drops in the fertilization dishes. Incubation was allowed to proceed for 4.5 h at 37°C in a 5% CO2/air incubator. At the end of this period, the inseminated ova were fixed in 1:3 acetic acid:ethanol, stained with 1% orcein, and observed under 100x phase contrast microscopy [18]. The presence of two pronuclei with a visible second polar body was evidence of fertilization. In each experiment, 30–50 oocytes per experimental condition were used, and each experiment was replicated five times.

Acrosome reactions The percentage of acrosome reactions after GnRH antagonist treatment was measured using Coomassie blue G-250 as described by Larson and Miller [23]. Briefly, mouse sperm were capacitated as described above, and GnRH antagonists were added for 15 min. Some sperm aliquots were treated with ionomycin (5 µM final concentration). Then, the cells were fixed with 7.5% formaldehyde in PBS for 10 min, and the sperm were centrifuged and washed twice in 0.1 M ammonium acetate pH 9. Fifty microliters of the sperm suspension was spread on to poly-L-lysine-coated slides, and air-dried. The slides were then stained with freshly made Coomassie stain (0.22% Coomassie blue G-250, 50% methanol, 10% glacial acetic acid, and 40% water) for 2 min, gently rinsed with deionized water, air dried, and then mounted with Permount. Stained sperm were examined under brightfield microscopy at 400x.

Sperm-zona binding We previously showed that GnRH antagonists block sperm-zona binding in humans [16]. To determine the effect of GnRH antagonists on sperm-zona binding in the mouse, the hemizona assay was carried out, essentially as it was described for humans [4, 24]. Briefly, cumulus-enclosed mouse oocytes, obtained as described above, were deposited in Dulbecco PBS containing 300 IU of hyaluronidase for 30–40 sec to remove cumulus cells. The oocytes were washed five times in fresh M2 medium and then cut to obtain two equal halves or hemizonae. Sperm were capacitated for 1 h at 4 x 106 cells/ml and 5% CO2 in air at 37°C under mineral oil (see above). Fifty-microliter sperm drops were incubated for 15 min with different concentrations of GnRH antagonists (test) or with the solvent of the antagonist (saline, control). One hemizona was then added to the control sperm drop and the matching hemizona was added to the test sperm drop. Control and test sperm drops 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 bound to the outer surface of each hemizona were counted under a phase-contrast microscope. In additional experiments, the ability of GnRH to overcome the inhibitory effect of the GnRH antagonist on sperm-zona binding was assessed. Sperm aliquots were treated for 15 min with 100 µM 4pF alone or followed by 1 µM, 100 µM, or 500 µM GnRH for an extra 10 min. The hemizona assay was used to compare the number of zona-bound sperm in the different groups.

Statistics

A one-way analysis of variance with the Tukey-Kramer multiple comparisons test was used to compare the number of fertilized eggs between treatments. The nonparametrics Kruskal-Wallis test, followed by the Mann-Whitney U-test, was used to analyze sperm numbers in the oviducts, because these data exhibited nonnormal distribution. To compare the number of zona-bound spermatozoa in the control and treated groups, the paired t-test, the Dunnett multiple comparison test, or both were used. Differences were considered significant at the 0.05 level of confidence. All results are expressed as means ± SEM.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
In Vivo Experiments

Intraoviductal administration of GnRH antagonists to female rats during the afternoon of proestrus significantly decreased the percentage of eggs that were fertilized after mating (Table 1). Both GnRH antagonists were equally effective at inhibiting fertilization, and inhibition was higher with the higher dose of antagonist used. The intrabursal injection of GnRH antagonists also inhibited fertilization in relation to injection of saline. However, the inhibition was less effective than when the antagonists were delivered intraoviductally (Table 1). Thus, when 0.5 mM Nal-Glu was injected intraoviductally, the percentage of fertilized eggs was 43% ± 1%, and when the same dose was injected intrabursa, the percentage of fertilized eggs was 73% ± 0.4% (Table 1). The same was true when 0.5 mM 4pF was administered intraoviductally and intrabursally. Intraoviductal administration of saline did not have any effect on the percentage of fertilized eggs in comparison with sham-injected oviducts (Table 1).


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  		TABLE 1. Effect of GnRH antagonists on in vivo fertilization in the rat

The inhibitory effect of GnRH antagonists was not due to an impairment of ovulation. In effect, the total number of recovered eggs did not differ between control and treated oviducts (Table 1). The total number of eggs recovered from control oviducts ranged from 6.0 ± 0.3 to 6.9 ± 0.5, whereas in oviducts treated with GnRH antagonists they ranged from 5.9 ± 0.5 to 7.0 ± 0.4 (Table 1). In addition, there was no difference in the total mean number of eggs recovered per rat in each experimental group, which ranged from 12 ± 0.4 to 14 ± 1.

In a different set of animals, sperm migration to the site of fertilization after treatment with GnRH antagonists was evaluated. Intraoviductal injection of 5 mM Nal-Glu or 5 mM 4pF did not alter the number of sperm that reached the oviduct compared with saline treatment (Fig. 1). This was true whether the number of sperm flushed with saline or with saline + Triton X-100 was counted (data not shown).



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  		FIG. 1. Effect of GnRH antagonists on sperm migration into the rat oviduct. Female rats were treated by delivering 5 mM Nal-Glu (A) or 5 mM 4pF (B) into one oviduct (experimental) and saline in the contralateral (control) oviduct. Females were then mated, and the next morning the oviducts were flushed and all sperm were recovered from the isthmic and ampullary portion and counted. Data are the mean (±SEM) number of sperm recovered from each oviductal segment

In Vitro Experiments

In an effort to elucidate what specific step of the fertilization process GnRH antagonists affected, we performed IVF in mice. Treatment with both GnRH antagonists significantly inhibited IVF and this effect was dependent on the dose of antagonists used (Fig. 2). For Nal-Glu, the percentage of IVF inhibition was 0.1 µM = 29% ± 13% (P < 0.05), 1 µM = 37% ± 4% (P < 0.05), 100 µM = 61% ± 9% (P < 0.005), and 500 µM = 70% ± 11% (P < 0.005). For 4pF, the percentage of IVF inhibition was 0.1 µM = 16% ± 7% (not significant), 1 µM = 45% ± 6% (P < 0.05), 100 µM = 68% ± 10% (P < 0.005), and 500 µM = 90% ± 5% (P < 0.001).



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  		FIG. 2. Effect of GnRH antagonists on in vitro fertilization in the mouse. Sperm suspensions were treated with different concentrations of the GnRH antagonists Nal-Glu or 4pF. Then, 30 to 50 cumulus-enclosed oocytes were added to each sperm suspension. After 4.5 h of incubation, eggs were evaluated to establish fertilization. Data are the mean (±SEM) number of eggs fertilized in each treatment. *Significantly different from its respective control (P < 0.01)

We also tested the effect of GnRH antagonists on the ability of mouse sperm to bind to the mouse zona pellucida using the hemizona assay (Fig. 3). All concentrations of antagonists produced a significant decrease in the number of zona-bound sperm in relation to control sperm suspensions. This inhibition was dependent on the dose of antagonist used (Fig. 3) and the two compounds showed a similar magnitude. For Nal-Glu experiments the average number (±SEM) of zona-bound sperm in the control was 49 ± 7, and for 4pF it was 68 ± 8. The percentage of sperm-zona binding inhibition reached a maximum with 100 µM Nal-Glu (77% ± 7%) and with 100 µM 4pF it was 76% ± 9%.



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  		FIG. 3. Effect of GnRH antagonists on the number of sperm bound to hemizona pellucidae in the mouse. Data are expressed as a percentage (mean ± SEM) of the control hemizona. Sperm suspensions were incubated for 15 min with different concentrations of the GnRH antagonists Nal-Glu or 4pF prior to the hemizona assay. *Significantly different from its respective control (P < 0.01)

In addition, when sperm previously exposed to 100 µM 4pF were treated with increasing concentrations of GnRH, the inhibitory effect of the GnRH antagonist was completely reversed (Fig. 4). The ability of GnRH to reverse the inhibitory effect of 100 µM 4pF was related to the dose of GnRH used.



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  		FIG. 4. Effect of GnRH on the inhibitory action of the GnRH antagonist 4pF. Data are expressed as a percentage (mean ± SEM) of the control hemizona. Sperm suspensions were treated with 100 µM of the GnRH antagonist 4pF for 15 min. Then, a different GnRH concentration was added to each sperm suspension before the hemizona assay. *Significantly different from its respective control (P < 0.01)

Finally, incubation of spermatozoa with GnRH antagonists during the last 15 min of capacitation did not have any effect on the percentage of motile spermatozoa (data not shown) or on the percentage of acrosome reactions. The percentage of acrosome reactions was 29% ± 3% in the control group and 31% ± 2% and 28% ± 4% in the groups treated with Nal-Glu and 4pF (500 µM), respectively. Sperm treated with 5 µM ionomycin exhibited 66% ± 2% acrosome reactions.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Fertilization in mammals involves several levels of interaction between the sperm and the egg, during their travel through the female genital tract. Leaving aside the interactions that may take place between the gametes and the genital tract, once spermatozoa are inside the female genital tract, the fertilizing spermatozoon must first reach the ampulla of the oviduct where fertilization might take place if an oocyte is present [2]. The fertilizing spermatozoon must first traverse the cumulus oophorus and then bind to the ZP. Once it has bound to the zona, the fertilizing spermatozoon will enter the acrosome reaction and begin to penetrate the zona thickness. Finally, it will bind and fuse with the oocyte plasma membrane. Several molecules present in the female genital tract have been described as having an effect on this process (see review in [3]). Because GnRH is present in seminal plasma, uterus, oviduct, and follicular fluid [6–10], its participation in the fertilization process has been investigated [4, 15, 16]. The concentration of GnRH in each segment of the reproductive tract, however, is unknown.

In this study we present evidence that GnRH antagonists exert an inhibitory effect on fertilization, both in vivo and in vitro. For in vivo experiments, treatment with GnRH antagonists reduced the number of fertilized eggs in the rat oviduct in a dose-dependent manner. The evidence suggests that this effect was specific to fertilization and was not related to sperm migration to the site of fertilization, impairment of ovulation, or delayed embryo development. Fertilization, however, is a complex process that operationally ends when the sperm head is visible within the egg cytoplasm, and when sequential interactions take place between the sperm and the cumulus oophorus, ZP, egg plasma membrane, and egg cytoplasm. To address the issue of what specific interaction was affected by GnRH antagonists, we carried out in vitro assays.

The present results allow us to exclude an effect on sperm migration to the site of fertilization by treatment with GnRH antagonists. In effect, the number of sperm recovered from control and treated oviducts was not significantly different. This was true for both GnRH antagonists tested and for both regions of the oviduct (i.e., isthmus and ampulla). The numbers of sperm that we found in the oviduct and the presence of significantly more sperm in the isthmus than in the ampulla are in good agreement with what other investigators have found [20, 25, 26]. The latter observation is consistent with the idea that the isthmus of the oviduct may act as a sperm reservoir [20, 25, 26]. Although there were fewer sperm in the ampulla than in the isthmus, they were adequate in number to have accomplished fertilization [25]. Therefore, these results and the direct observation of mouse sperm movement after GnRH antagonist treatment allow us to suggest that GnRH antagonists did not have an effect on sperm movement parameters. The same observation was reported for human sperm [4].

The present results also allow us to suggest that ovulation was not affected by GnRH antagonist treatment. It has been well documented that ovulation is suppressed and therefore contraception is achieved by chronic and systemic administration of GnRH antagonists ([27] and references therein). However, intraoviductal injection of GnRH antagonists in the afternoon of proestrus did not interfere with the process of ovulation, as was evident when the number of eggs recovered from control and treated oviducts was analyzed. The lack of an effect of GnRH antagonists on the process of ovulation support the idea that their action involved a local effect, manifested only on the side of the genital tract in which they were delivered.

Finally, the present results also suggest that fertilization and embryo development was not delayed by treatment with GnRH antagonists. In fact, when the eggs were evaluated at Day 2 of pregnancy, either they were at the two-cell stage or they were not fertilized. No evidence of fertilization was apparent in the egg that did not have two blastomeres. Therefore, after GnRH antagonist treatment, either the eggs were fertilized at the normal time or they were not fertilized at all.

Therefore, the question of what specific step of the fertilization process was affected by GnRH antagonists is still open, although it has been narrowed down to, at a minimum, sperm-cumulus oophorus interaction, sperm-zona binding and penetration, and sperm-egg fusion. To shed light into this query, we carried out in vitro experiments using the mouse as the animal model. GnRH antagonists inhibited in vitro fertilization and the specific step that was affected was the process of sperm binding to the ZP. This process was directly monitored using the hemizona assay. Moreover, treating the sperm with increasing concentrations of GnRH reversed the inhibitory effect of GnRH antagonists. GnRH antagonists did not affect mouse sperm acrosome reactions, as was evident in humans [16]. In effect, it was previously reported with human sperm that GnRH and GnRH antagonists did not induce the acrosome reaction [4] and that GnRH antagonists did not prevent follicular fluid-induced acrosome reactions [16].

We still do not know how GnRH antagonists inhibit sperm-zona binding in these rodent species. However, we also showed that GnRH antagonists inhibited the ability of human sperm to initiate binding to the ZP. In humans, treatment with GnRH antagonists drastically reduced the efficiency of the sperm-ZP binding process [4, 16]. In the in vitro conditions that were used for human experiments, the efficiency of sperm-zona binding was low. In control conditions, the proportion of sperm that remained bound to the ZP in regard to those that collided with the ZP was 1 in 30 (3.3%) [4]. Treatment with GnRH antagonists reduced this ratio even further. Only 1 of 78 (1.3%) sperm that collided with the ZP remained bound to it [16].

It has been demonstrated that GnRH is present at the site and time of fertilization [9, 10] and that sperm possess GnRH-R on their plasma membrane [13]. Therefore, it is possible that the fertilizing spermatozoon may interact with GnRH during its transit to the site of fertilization. This interaction may confer on the sperm greater capacity to initiate binding to the ZP. Binding of GnRH to its sperm receptor could increase the affinity of zona ligands on the spermatozoa or it could expose zona ligands that had been previously masked. These changes on zona ligands could be due to direct configuration changes in the ligand itself or to the production of second messengers in the sperm cells. A previous report indicated that GnRH increases the intracellular Ca2+ concentration through Ca2+ influx [28] and provided additional support to this suggestion. The mechanism of action of GnRH antagonists on pituitary gonadotrophs is based on their capacity to bind to GnRH receptors on these cells and directly compete with endogenous GnRH. GnRH antagonists exert a rapid (within minutes) inhibitory effect of the secretion of LH and FSH, and they remain bound to the membrane of the gonadotrophs for a long time [29–32]. Thus, binding of GnRH antagonists to the sperm GnRH-R would prevent their interaction with endogenous GnRH and might render the spermatozoa less capable of initiating binding to the ZP and therefore inhibit the possibility of conception.

This explanation, however, is not entirely satisfactory for in vitro experiments. The main criticism of these experiments is related to the uncertainty of the presence of a GnRH-like peptide in the fertilization dish. One could hypothesize that the inhibitory effect of GnRH antagonists on IVF is due to a block of the stimulatory effect that native GnRH has on fertilization. Genital tract GnRH (or a GnRH-like peptide) would still have to be bound to the sperm GnRH-R even after the sperm have been removed from the genital tract in order for this explanation to be correct. There is no evidence that this may be the case. Notwithstanding, the presence of GnRH on the oocyte-cumulus complex should be considered [6, 32]. Alternatively, or in addition, GnRH antagonists may have a direct inhibitory effect on sperm-zona binding that is not related to a competition with endogenous GnRH. Such direct effects of GnRH antagonists have been reported regarding inhibition of proliferation of normal granulosa cells through apoptosis [33], and inhibition of proliferation of some cancer cell lines [34–38] through effects on a different set of intracellular signals as those used by GnRH in the pituitary [39, 40]. These are all cells that express the normal pituitary GnRH-R and the action of the GnRH antagonists is mediated by binding to these receptors [33, 35, 39]. The latter explanation is to be taken with caution and by no means as a suggestion that sperm may behave like cancer cells, but that GnRH antagonists may have more actions than the sole occupancy of GnRH receptors, by competing with endogenous GnRH. In conclusion, GnRH antagonists inhibit sperm-zona binding and therefore fertilization, by a still unknown mechanism.


   FOOTNOTES
 
1 This work was financed by Fondecyt 197 1243. The investigation received financial support through project A05139 from the Special Programme of Research, Development, and Research Training in Human Reproduction sponsored by the United Nations Development Programme, United Nations Population Fund, World Health Organization, and the World Bank. C.P. and E.P. are recipients of the PROGRESAR fellowship. Back

2 Correspondence: Patricio Morales, Unit of Reproductive Biology, Faculty of Health Sciences, University of Antofagasta, P.O. Box 170, Antofagasta, Chile. FAX: 5655 637 802; pmorales{at}uantof.cl Back

Received: 11 December 2001.

First decision: 8 January 2002.

Accepted: 29 May 2002.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

  1. Wassarman PM. Zona pellucida glycoproteins. Annu Rev Biochem 1988 57:414-442
  2. Yanagimachi R. Mammalian fertilization. In: Knobil E, Neill JD (eds.), The Physiology of Reproduction. New York: Raven Press; 1994: 189–317
  3. Morales P, Llanos MN. Human sperm interaction with the zona pellucida and the mechanism of the acrosome reaction. Front Biosci 1996 1:146-160
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