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Regular Article |
a Department of Internal Medicine, Section of Andrology, University of L'Aquila, 67100 L'Aquila, Italy
b Centro di Medicina della Riproduzione, 90141 Palermo, Italy
ABSTRACT
There is recent evidence that mouse and human spermatozoa contain constitutive nitric oxide synthase (cNOS) and can synthesize nitric oxide. The aim of this study was to investigate whether the inhibition of human sperm cNOS could affect sperm-oocyte fusion and sperm binding to the zona pellucida (ZP). NG-nitro-L-arginine methyl ester (L-NAME) was used as cNOS inhibitor. Sperm-oocyte fusion was evaluated using the hamster egg penetration test (HEPT). The ZP binding was evaluated using the hemizona assay. L-NAME added from the onset of capacitation strongly inhibited sperm-oocyte fusion. This inhibitory effect was dose dependent, stereospecific, and suppressed by L-arginine in a dose-dependent manner. L-NAME also inhibited sperm-oocyte fusion in the HEPT enhanced with progesterone (P), where P (5 µM) was added for 15 min to capacitated sperm. A lesser but significant inhibition was also observed when sperm suspensions were exposed to L-NAME following capacitation in both versions of HEPT. On the contrary, L-NAME did not affect ZP binding. In conclusion, the present study provides the evidence that cNOS plays a role in the human sperm's capacity to fuse with oocyte but not in the ZP binding.
fertilization, nitric oxide, progesterone, sperm, sperm capacitation/acrosome reaction
INTRODUCTION
The discovery of the key role of nitric oxide (NO) in several biological functions also has recently generated a developing area of research on the reproductive system. Nitric oxide, a labile and diffusible molecule that forms stable metabolites nitrite and nitrate, is produced by the action of NO synthases (NOS), enzymes responsible for the conversion of L-arginine to NO and L-citrulline. To date, three NOS isoforms have been purified, sequenced, and partially characterized: two of them, neuronal NOS (nNOS) and endothelial NOS (eNOS) have been referred to as constitutive (cNOS); they are activated by calcium and calmodulin and are selectively inhibited by NG-nitro-L-arginine methyl ester (L-NAME) and NG-nitro-L-arginine (NO2-Arg). A third, inducible form (iNOS), was first found in macrophages; it is expressed in response to cytokines and lipopolysaccarides, is calcium independent, and is selectively inhibited by aminoguanidine and amidines [1].
Following the recognition of NO as a mediator of penile erection [2], NOS protein and activity have been demonstrated both in male and female reproductive organs [3–7], suggesting an involvement of NO in the physiology of reproduction.
Some observations indicate that NO could modulate sperm functions. Low concentrations of exogenous NO donors have been shown to enhance postthaw human sperm motility and viability [8], human sperm capacitation [9, 10], hamster sperm hyperactivation [11], and human sperm binding to the zona pellucida (ZP) [12]. Conversely, at high concentrations, they decrease human sperm motility and induce sperm toxicity [13, 14]. Therefore, although low amounts of NO, generated under physiological conditions, could be beneficial for sperm functions, excessive generation of NO under pathological conditions, such as infections or endometriosis, could be toxic for sperm. Indeed, higher seminal plasma levels of NO were found in infertile patients, especially those with leukocytospermia, than in fertile controls, and a positive correlation was observed between the concentration of NO and the percentage of immotile sperm [15].
Evidence has been reported that NO can also be generated by spermatozoa. An immunoreactivity for cNOS was observed in mouse [16] and human sperm [16–18]. In mouse sperm, a NOS isoform was revealed at Western blot analysis as a unique band of Mr = 140 kDa, recognized by both eNOS and bNOS antibodies [19] and more recently, in human sperm, as a band of Mr = 145 kDa, recognized by eNOS antibodies [20]. The ability of sperm to synthesize NO during in vitro capacitation was demonstrated in the mouse by measuring L-[14C]citrulline generation [19] and in man, by measuring nitrite accumulation [17] as well as L-[3H]citrulline generation [20]; in the mouse, L-[14C]citrulline generation was inhibited by L-NAME in a dose-dependent manner [19]. Therefore, a role for sperm cNOS in the mammalian sperm acquisition of fertilizing ability might be supposed. Indeed, in mouse sperm, the inhibition of cNOS significantly reduced the in vitro fertilization rate [21], and some data have been reported suggesting an involvement of cNOC in capacitation and acrosome reaction [10, 11, 20, 22].
The aim of this study was to investigate whether the inhibition of cNOS by L-NAME could interfere with human sperm functions involved in fertilization, focusing on the two main steps of the fertilization process, the sperm-oocyte fusion and the zona pellucida (ZP) binding.
MATERIALS AND METHODS
Chemicals
All reagents were from Sigma Chemical Co. (St. Louis, MO). Progesterone was prepared daily as 50 µM stock in dimethyl sulfoxide (DMSO) and diluted in Biggers, Whitten, and Wittingham (BWW) medium to give the final working concentration. L-NAME, D-NAME, and L-arginine were prepared daily as stock solutions in BWW and were diluted in BWW to obtain the working concentration before use.
Spermatozoa
Semen samples were collected according to the World Health Organization (WHO)-recommended procedure [23], by masturbation from healthy normozoospermic donors. All samples were produced into sterile containers and left for at least 30 min to liquefy before processing. Motile sperm suspensions were obtained by swim-up procedure. Briefly, spermatozoa were washed twice (700 x g for 7 min) in BWW medium containing 0.3% BSA. After the second centrifugation, supernatants were removed by aspiration, leaving 0.5 ml on the pellet, and after an incubation time of 30 min, supernatants containing highly concentrated motile sperm were carefully aspirated, and the sperm concentration was adjusted at 1 x 107 per milliliter.
Hamster Egg Penetration Test
Motile sperm suspensions obtained by swim-up procedure were capacitated in BWW with the addition of 0.5% human serum albumin (HSA), fraction V, at 37°C in an atmosphere of 5% CO2/95% air for 5 h.
To evaluate the dose-response effect of L-NAME, motile sperm suspensions were divided into aliquots before capacitation and were exposed to scalar concentrations of L-NAME, as well as to its inactive enantiomer, D-NAME, as control.
To evaluate the effect of L-arginine displacement of L-NAME, motile sperm suspensions were divided into aliquots before capacitation and were exposed to scalar concentrations of L-arginine, added simultaneously with L-NAME (0.6 mM) to the sperm suspensions.
In additional experiments intended to determine whether L-NAME could affect the enhancement of sperm-oocyte fusion induced by progesterone, sperm suspensions were exposed to progesterone (5 µM) or to DMSO (as control) for 15 min at the end of capacitation (5 h). In the same settings, L-NAME or D-NAME (as control) were added to the motile sperm suspensions from the onset of capacitation or to capacitated sperm 30 min before the addition of progesterone (or DMSO). This was done to determine whether sperm NOS was required during the capacitation period.
In all experiments, spermatozoa were always washed and resuspended in fresh BWW medium (1 x 107 per milliliter) before the incubation with oocytes to exclude a possible direct effect of L-NAME on oocytes.
Standard procedures were utilized for the recruitment and processing of hamster oocytes [23]. Twenty zona-free oocytes were added to 1 x 106 sperm in a droplet of 100 µl under paraffin oil. After 3 h of coincubation at 37°C in an atmosphere of 5% CO2, 95% air, oocytes were recovered from the droplets, washed free of loosely adherent spermatozoa, fixed in picric acid and formaldehyde overnight, and stained with Giemsa. Ova were examined at 400x for the evidence of swollen sperm heads. The number of spermatozoa penetrating each egg was assessed and expressed as total number of penetrations per total number of oocytes.
Each experiment was performed three times with different donors in different settings so that about 60 oocytes were used in the assessment of each group of treatment.
Hemizona Assay
Mature human oocytes that showed no evidence of either two pronuclei or cleavage 60 h after in vitro fertilization were placed in 1 M ammonium sulphate solution and stored at 4°C. Prior to each assay, salt-stored oocytes were desalted by washing with BWW containing 0.3% BSA. Oocytes were bisected into equal halves under a stereomicroscope [24].
Motile sperm suspensions obtained by swim-up procedure were divided in two aliquots, exposed to L-NAME (0.6 mM) and to D-NAME (0.6 mM), respectively. One hundred µl (containing 1 x 106 motile sperm per milliliter) were transferred to the center of a Petri dish and covered with warmed (37°C) paraffin oil. One hemizona was added to the drop of L-NAME-exposed sperm and the other hemizona to the drop of D-NAME-exposed sperm (control), followed by incubation at 37°C in an atmosphere of 5% CO2/95% air for 4 h. After incubation, hemizonae were washed in BWW medium, transferred to a 50-µl drop of BWW medium in a Petri dish, and examined under an inverted microscope. The number of sperm bound to each hemizona was determined. Ten pairs of hemizonae were tested with sperm suspensions from three donors.
Sperm Motility Evaluation with Computer-Assisted Semen Analysis
Motility exhibited by sperm suspensions exposed for 5 h to scalar concentrations of L-NAME and to D-NAME, as control, was evaluated with computer-assisted semen analysis (CASA) using ATS20 (JCD, Gauville, France). Determinations were performed on the sperm suspensions used to evaluate the dose-response effect of L-NAME on the hamster egg penetration test (HEPT) in three different settings.
Statistical Analysis
Data were analyzed by the Complete Statistical System for personal computers (CSS/pc), release 2.1, version B640, 1988 (StatSoft Inc., Tulsa, OK ). Differences in the number of penetrations per oocyte between treatment groups were examined with the Mann Whitney U-test. Paired comparisons were conducted using the Wilcoxon matched-pair test. Data were presented as mean values ± SEM.
RESULTS
Dose-Response Effect of L-NAME on Sperm Fusion with Oocytes
The exposure of sperm suspensions to scalar doses of L-NAME from the onset of capacitation produced a significant reduction of penetrations per oocyte, with a clear dose-response effect (Fig. 1). Penetrations per oocyte were 2.5 ± 0.2 in control samples, 1.5 ± 0.1 in samples exposed to L-NAME at 0.1 mM (P = 0.0002), 0.2 ± 0.1 in samples exposed to L-NAME at 0.6 mM (P = 0.0000 vs. L-NAME, 0.1 mM), and no penetration at 1.2 mM. Therefore, L-NAME at 0.6 mM, which produced an inhibition >90% compared with controls, was used in subsequent experiments. This inhibitory effect was not associated with any concomitant change in the percentage of motile spermatozoa, nor in the quality of motility, evaluated with CASA, with respect to controls (Fig. 1) and to basal samples (data not shown). Conventional sperm motility evaluation was always performed in the subsequent experiments, and L-NAME was found to have no effect.
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Effect of L-Arginine Displacement of L-NAME
The inhibition of sperm-oocyte fusion produced by 6 mM L-NAME was reduced by the concomitant addition of 2 mM L-arginine: penetrations per oocyte were 0.3 ± 0.07 vs. 0.9 ± 0.07 (P = 0.00002). The inhibition was completely prevented by the concomitant addition of 6 mM L-arginine, which produced a number of penetrations per oocyte similar to that exhibited by controls exposed to 6 mM D-NAME: 2.0 ± 0.2 vs. 2.0 ± 0.2, but the addition of 6 mM L-arginine to controls treated with 0.6 mM D-NAME had no effect on sperm-oocyte fusion: 1.9 ± 0.2 (Fig. 2). These data indicate that the inhibitory effect of L-NAME was the result of competitive inhibition of cNOS.
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Inhibitory Effect of L-NAME on Progesterone-Enhanced Sperm-Oocyte Fusion
In control samples (exposed to 0.6 mM D-NAME), the exposure to 5 µM progesterone for 15 min at the end of capacitation significantly increased the number of penetrations per oocyte (6.5 ± 0.7 vs. 2.3 ± 0.3; P = 0.0002; Fig. 3), as expected. The exposure to 0.6 mM L-NAME from the onset of capacitation significantly inhibited the oocyte fusion by progesterone-exposed sperm (0.7 ± 0.1; P < 0.00001; Fig. 3). In the same settings, when L-NAME was added to capacitated sperm suspensions 30 min before the addition of progesterone or DMSO, the inhibition was significantly lower than that observed when sperm suspensions were exposed to L-NAME from the onset of capacitation both in progesterone-exposed sperm (2.4 ± 0.3 vs. 0.7 ± 0.1; P < 0.00001) and in DMSO-exposed sperm (1.0 ± 0.2 vs. 0.2 ± 0.1; P = 0.0009; Fig. 3). Nevertheless, it was significant with respect to controls (exposed to D-NAME) both for progesterone (P = 0.0009) and DMSO-exposed sperm (P = 0.0009; Fig. 3).
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Effect of L-NAME on ZP Binding
The exposure to 0.6 mM L-NAME did not inhibit ZP binding. Zona pellucida-bound sperm were 105 ± 16 in samples exposed to L-NAME and were 125 ± 24 in control samples (exposed to D-NAME). The difference was not significant (Fig. 4).
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DISCUSSION
In this study, L-NAME inhibited the capability of human spermatozoa to fuse with oocytes but did not affect the zona pellucida binding. The L-NAME inhibition of sperm-oocyte fusion was dose-dependent and was the result of competitive inhibition of NO-synthase because it was suppressed by L-arginine in a dose-dependent manner. Moreover, in all experiments, L-NAME was matched against its enantiomer D-NAME, used as control, to make sure that the inhibitory effect was stereospecific. A direct effect of L-NAME on the oocytes was also excluded because sperm suspensions were always washed before sperm-oocyte coincubation.
The inhibition of sperm fusion with oocytes was not due to a negative effect of L-NAME on sperm motility because both the percentage of motile sperm and the quality of motility, assessed with CASA, were similar to those of D-NAME-exposed sperm. A reduction of human sperm motility by L-NAME had been reported by Lewis et al. [17], but asthenozoospermic instead of normal sperm samples were tested in their report.
The HEPT monitors the ability of human spermatozoa to exhibit a functional acrosome reaction accompanied by the generation of a fusogenic equatorial segment of the sperm head, which makes spermatozoa able to recognize and fuse with the vitelline membrane of the oocyte [25]. These events are stimulated by micromolar doses of progesterone, which, added to capacitated sperm, enhance sperm-oocyte fusion [26]. Therefore, as expected, a significant increase in the oocyte fusion was exhibited by progesterone-exposed control sperm. A significant inhibition of sperm-oocyte fusion by L-NAME was also observed on the HEPT in this enhanced version. Because the sperm fusion with oocyte as well as its enhancement by progesterone are capacitation dependent [26, 27], the inhibitory effect of L-NAME on sperm-oocyte fusion might be due to an inhibition of capacitation and/or to a direct interference with the dynamics of acrosomal exocytosis and the generation of a fusogenic equatorial segment. Sperm exposure to L-NAME from the onset of capacitation exerted an inhibition significantly higher than the exposure at the end of capacitation in both versions of the HEPT, suggesting an effect of L-NAME on capacitation. Sperm capacitation, whose molecular basis remains largely unknown, is associated with changes of membrane lipid composition, ion channel activation, intracellular second-messenger production, and protein tyrosine phosphorylation [28]. These changes both promote acrosome reactions occurring spontaneously in capacitating media (responsible for the sperm-oocyte fusion in the conventional version of the HEPT) and make the spermatozoa able to undergo the acrosome reaction in response to proper stimuli [29] (responsible for the amplification of the sperm-oocyte fusion in the progesterone-enhanced version of the HEPT). cNOS inhibition of sperm capacitation is supported by previous data. Hyperactivated motility, a capacitation-related phenomenon, was inhibited in hamster sperm [11]. In human sperm, the exposure to cNOS inhibitors throughout capacitation produced a reduction in acrosome reactions induced by follicular fluid and by calcium ionophore A23187 and also decreased the level of tyrosine phosphorylation [10].
However, an adjunctive direct involvement of cNOS in postcapacitation events involved in the fusion with oocytes is also suggested by the present data because a significant reduction in sperm-oocyte fusion was also observed when sperm suspensions were exposed to L-NAME at the end of capacitation. A direct involvement of NOS in the dynamics of progesterone-induced acrosome reactions had been hypothesized by Herrero et al. [22], who reported that in mouse sperm, cNOS inhibition blocked progesterone-induced acrosome reactions, regardless of the time at which mouse sperm suspensions were exposed to NOS inhibitors (from the beginning or at the end of capacitation). Indeed, the evidence has been recently reported, from measuring L-[14]citrulline generation, that progesterone could directly stimulate sperm NO-synthase in capacitated mouse spermatozoa [30]. This stimulation led to an increase in sperm prostaglandin E2 production and in acrosomal exocytosis, whereas cGMP levels were not modified. No other data concerning this issue have been reported until now.
L-NAME did not exert any significant effect on the zona pellucida binding at the dose affecting sperm-oocyte fusion. Although exogenous NO has been shown to enhance human sperm binding to the zona pellucida [12], Herrero et al. [21] reported that in mouse sperm, the inhibition of constitutive NOS significantly reduced the in vitro fertilization rate without impairing sperm-zona pellucida binding, in accordance with the present data. It is not surprising that the L-NAME inhibition of capacitation events required for the sperm-oocyte fusion was not associated with the inhibition of sperm ability to bind to the zona pellucida. L-NAME likely inhibits capacitation events not required for ZP binding.
In conclusion, the present study provides the evidence that in humans, cNOS plays a role in the sperm's ability to fuse with oocyte but does not play a role in the zona pellucida binding. This role is exerted in events that are capacitation dependent and are amplified by progesterone. The capacitation-dependent sperm's ability to undergo functional acrosome exocytosis, accompanied by the generation of a fusogenic equatorial segment, could be the likely target.
FOOTNOTES
First decision: 10 December 1999.
1 This work was supported by MURST-Italy, project "Glycobiology of fertilization" and by CARISPAQ, L'Aquila, Italy. 
2 Correspondence: Felice Francavilla, Dipartimento di Medicina Interna, Università de L'Aquila, Via S. Sisto 22/E, 67100 L'Aquila, Italy. FAX: 39 862 432858; francavi{at}cc.univaq.it
Accepted: March 14, 2000.
Received: November 8, 1999.
REFERENCES
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  J. Lapointe, M. Roy, I. St-Pierre, S. Kimmins, D. Gauvreau, L. A. MacLaren, and J.-F. Bilodeau Hormonal and Spatial Regulation of Nitric Oxide Synthases (NOS) (Neuronal NOS, Inducible NOS, and Endothelial NOS) in the Oviducts Endocrinology, December 1, 2006; 147(12): 5600 - 5610. [Abstract] [Full Text] [PDF]
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 W.C.L. Ford Regulation of sperm function by reactive oxygen species Hum. Reprod. Update, September 1, 2004; 10(5): 387 - 399. [Abstract] [Full Text] [PDF]
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P = 0.0009 vs. control + P; 