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Journal of Clinical Endocrinology & Metabolism , doi:10.1210/jc.2006-0599
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The Journal of Clinical Endocrinology & Metabolism Vol. 91, No. 12 4969-4975
Copyright © 2006 by The Endocrine Society

Expression and Localization of {delta}-, {kappa}-, and µ-Opioid Receptors in Human Spermatozoa and Implications for Sperm Motility

Ekaitz Agirregoitia, Asier Valdivia, Arkaitz Carracedo, Luis Casis, Javier Gil, Nerea Subiran, Carmen Ochoa and Jon Irazusta

Department of Physiology (E.A., L.C., J.G., N.S., J.I.), Faculty of Medicine and Dentistry, University of the Basque Country, Leioa, 48940 Bizkaia, Spain; Department of Nursing II (A.V.). School of Nursing, University of the Basque Country, Donostia, 20014 Gipuzkoa, Spain; Department of Biochemistry and Molecular Biology I (A.C.), School of Biology, Complutense University, Madrid, 28040 Spain; and Laboratory of Seminology and Clinical Embryology (C.O.), Euskalduna Clinic, Bilbao, 48080 Bizkaia, Spain

Address all correspondence and requests for reprints to: Ekaitz Agirregoitia, Department of Physiology, Faculty of Medicine and Dentistry, University of the Basque Country, PO Box 699, Bilbao, 48080 Bizkaia, Spain. E-mail: e.agirregoitia{at}ehu.es.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Context: Endogenous opioid peptides signal through {delta}-, {kappa}-, and µ-opioid receptors. Some of these peptides such as endorphins and enkephalins are present in the male reproductive tract, but the presence of the corresponding receptors in human sperm cells has not yet been reported.

Objective: Our objective was to study the expression and localization of {delta}-, {kappa}-, and µ-opioid receptors on human spermatozoa and the implication in sperm motility.

Methods: The expression of receptors was studied by RT-PCR, Western blot, and immunofluorescence techniques. We evaluated the effects of activation of each opioid receptor by specific agonist and antagonist.

Results: Human spermatozoa express {delta}-, {kappa}-, and µ-opioid receptors. These receptors were located in different parts of the head, in the middle region, and in the tail of the sperm. Progressive motility of spermatozoa, an important parameter to evaluate male fertility, was found to be significantly reduced after incubation with the µ-receptor agonist morphine, whereas this effect was antagonized in the presence of the corresponding antagonist naloxone. The {delta}-receptor antagonist naltrindole significantly reduced progressive motility immediately after its addition. However, the {delta}-receptor agonist DPDPE had no significant effect. Finally, neither the {kappa}-receptor agonist U50488 nor its antagonist nor-binaltorphimine significantly affected the progressive motility of human spermatozoa.

Conclusion: We report for first time the presence of functional {delta}-, {kappa}-, and µ-opioid receptors in human sperm membranes. These findings are indicative of a role for the opioid system in the regulation of sperm physiology.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
ENDOGENOUS OPIOID PEPTIDES are known to participate in the regulation of reproductive physiology at multiple sites, operating as a multimessenger system (1). Opioids exert their effects by binding to membrane receptors. Nowadays, three main types of opioid receptors have been identified. These are all seven transmembrane, G protein-coupled receptors that have been termed {delta}-, {kappa}-, and µ-receptors (DOR, KOR, and MOR, respectively). Each of them seems to be involved in the regulation of different aspects of reproduction (2, 3).

One of the best known effects of opioid peptides on the reproductive system is their inhibitory role on GnRH secretion in the central nervous system (CNS). There is also evidence of opiate action in the pituitary gland (4) and testis (5). However, these peptides may also have important direct effects on sperm cells because large amounts of opioid peptides (endorphins and enkephalins) have been found in seminal fluid or in sperm cells (6, 7). In addition, the activity of the enkephalin-metabolizing enzyme has been reported to be higher in semen than in other tissues (8).

The effect of opiates on spermatozoa is incompletely understood and in some aspects is still controversial. Thus, contradictory effects of opioids on sperm motility have been reported (9, 10, 11). Nevertheless, it is quite probable that the opioid system participates in the control of the sperm movement because a reduced motility (asthenozoospermia) is a common abnormality found in opiate drug addicts (12). In addition, the activity of the enkephalin-metabolizing enzyme aminopeptidase N was reported to be lower in males with asthenozoospermia (13). Enkephalins are also expressed in male germ cells, and a possible role of these pentapeptides in spermatogenesis has been suggested (7, 14).

In 2004, the localization of {delta}-, {kappa}-, and µ-opioid receptors was reported on sperm cells of two fishes (15), and during the preparation of this manuscript, the expression of the µ-opioid receptor has been reported in equine spermatozoa (11). However, the presence of opioid receptors on human sperm cells is currently unknown. Therefore, the aim of this study was to characterize the expression and distribution of the three types of opioid receptor in human spermatozoa. In addition, because opioids and opiates seem to play a role in the motility of sperm cells, we also evaluated the effect of different opioid agonists and antagonists on sperm motility.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Reagents

Morphine hydrochloride was from Alcaliber S.A. (Madrid, Spain). All other reagents were purchased from Sigma-Aldrich (St. Louis, MO).

Sperm preparation

Human semen was obtained by masturbation after 2–3 d of abstinence. All the donors (aged 25–40 yr) were healthy and normozoospermic according to World Health Organization standards (16) and had no previous history of opiate drug consumption. Ethical approval was obtained from the Ethics Committee of the University of the Basque Country and from the Cruces Hospital Ethics Committee. Informed consent was obtained from all donors. Samples were ejaculated into sterile containers and allowed to liquefy at 37 C for 30 min before processing. Semen volume, as well as sperm concentration and motility, were measured for each sample.

A swim-up technique was applied to semen samples to remove all nonmotile cells, such as round cells. We used multiple tubes with small volumes of fresh semen (250 µl) and 500–600 µl tyrode-modified noncapacitating medium (TNC), described by Flesch and Gadella (17), per tube. This modified medium, containing 7 mg/ml BSA, was made by omitting bicarbonate. After 60 min incubation at 37 C, most of the upper TNC layer was removed from each tube. The resulting sample was placed in a 15-ml centrifuge tube and centrifuged at 2400 rpm for 10 min. The pellet was resuspended in TNC containing 0.5% polyvinyl alcohol for the estimation of sperm concentration and motility. Only sperm cells collected by means of this swim-up technique were used in subsequent procedures. All samples were checked visually under the microscope to verify the absence of round cells.

Preparation of sperm membranes

Sperm membranes were prepared as previously described (18), modifying the lysis buffer [PBS and 1% (vol/vol) Triton X-100, containing protease inhibitor cocktail].

SDS-PAGE and immunoblotting

Membrane pellets were suspended in lysis buffer, and then protein extracts were diluted in Laemmli sample buffer (19) containing ß-mercaptoethanol (5% vol/vol) and boiled for 5 min. Proteins (sperm, ~500 x 106 cells/ml; cerebral cortex, 30–50 µg) were loaded onto 12% resolving gels and separated by one-dimensional SDS-PAGE. Proteins were then transferred to polyvinylidene fluoride membranes using the Mini Trans-Blot electrophoretic transfer system (Bio-Rad Laboratories, Hercules, CA). Blotted membranes were treated and revealed as previously reported (20). Dilutions of polyclonal rabbit anti-MOR antiserum (1:2500; Chemicon International, Temecula, CA), anti-DOR antiserum (1:2500; Chemicon), or anti-KOR antiserum (1:300; Santa Cruz Biotechnology, Inc., Santa Cruz, CA) were used.

RT-PCR analysis

The RNA of swim-up spermatozoa was isolated with the RNeasy Protect kit (QIAGEN, Valencia, CA), including a DNase digestion step using an RNase-free DNase kit (QIAGEN) to exclude possible contamination by genomic DNA. cDNA was obtained with Transcriptor reverse transcriptase (Roche, Indianapolis, IN). Primers used for PCR were as follows: human {delta}-opioid receptor, 5'-ACGTGCTTGTCATGTTCGGCATCGT-3' and 5'-ATGGTGAGCGTGAAGATGCTGGTGA-3' (located on different exons to avoid amplification of genomic DNA; 222-bp product); human {kappa}-opioid receptor, 5'-AGATACACAAAGATGAAGACAGCAACCAAC-3' and 5'-TCCCTGACTTTGGTGCCTCCAAGGACTATT-3' (352-bp product); human µ-opioid receptor, 5'-GCAGATGCCTTAGCCACCAGTA-3' and 5'-GAGGCGCAAGATCATCAGTCCATA-3' (located on different exons to avoid amplification of genomic DNA; 440-bp product); and human/mouse GAPDH, 5'-GGGAAGCTCACTGGCATGGCCTTCC-3' and 5'-CATGTGGGCCATGAGGTCCACCAC-3' (322-bp product) used as endogenous control.

PCRs were performed using the following parameters: 95 C for 5 min and 40 cycles at 95 C for 30 sec, 58 C for 30 sec, and 72 C for 1 min, followed by a final extension step at 72 C for 5 min. The mixture was electrophoretically separated on a 2% agarose gel.

Real-time quantitative PCR

The TaqMan real-time PCR system is based on a three-primer system. Two of the primers are designed following the classical PCR concept (upper and lower primers). The third primer has a fluorescent group that, when incorporated into the amplification, increases the fluorescence emission. This system enhances the amplification specificity (three primers are used) and avoids nonspecific double-stranded DNA detection because the fluorescence is bound to one of the primers. cDNA was obtained using a Transcriptor kit (Roche). Intron spanning TaqMan probes were designed following the Roche Universal Probe Library method (www.roche-applied-science.com). Amplifications were run in a 7900 Real-Time PCR System (Applied Biosystems, Foster City, CA). The following primer pairs were used: human {delta}-opioid receptor, upper primer, 5'-TTGTCATGTTCGGCATCG-3', and lower primer, 5'-AAGGCCAGGTTGAAGATGTAGA-3' (probe: human no. 49); human {kappa}-opioid receptor, upper primer, 5'-CCTTGAAGGCAAAGATCATCA-3', and lower primer, 5'-TGCAAGGAGCACTCAATGAC-3' (probe: human no. 71); and human µ-opioid receptor, upper primer, 5'-CGGCCAATACAGTGGATAGAA-3', and lower primer, 5'-GTTAGGGCAACGGAGCAGT-3' (probe: human no. 38).

Immunofluorescence

To localize the three receptors immunocytochemically, sperm were isolated using swim-up as described above, suspended in PBS, and smeared onto a slide coated with poly-L-lysine. Duplicate slides were prepared for each sample, which was fixed with 3% paraformaldehyde for 10 min. Opioid receptors were immunocytochemically detected in cells that had been treated in the presence or absence of 0.5% Triton X-100 (10 min) to detect the presence of intracellular receptors and surface expressed receptors, respectively.

Slides were then washed three times in PBS and incubated for 20 min in PBS/10% (vol/vol) bovine fetal serum. For indirect immunofluorescence staining, slides were incubated with anti-DOR, anti-KOR, and anti-MOR antisera at a dilution of 1:800 ({delta} and µ) and 1:50 ({kappa}) overnight at 4 C. Slides were then washed in PBS three times, incubated with Alexa Fluor 488 goat antirabbit IgG secondary antibody (Molecular Probes, Eugene, OR) for 2 h at 37 C in the dark, washed in PBS three times (in some cases, we stained the nuclei with Hoechst 33342 during the second wash), assembled with Fluoromount G (EMS, Hatfield, UK), and finally examined by confocal microscopy. Negative controls were performed in the same way, except for omission of the primary antibody before secondary antibody addition.

Incubation medium

Spermatozoa were separated by swim-up, washed, and resuspended to approximately 30 x 106 cells/ml. They were then incubated (3.5 h at 37 C) with different agonists and antagonists (duplicate incubations for each condition). The basal solution and vehicle control in all cases was TNC containing 0.5% polyvinyl alcohol. The following drugs were assayed: the µ-agonist morphine and antagonist naloxone, the {delta}-agonist DPDPE and antagonist naltrindole, and the {kappa}-agonist U-50488 and antagonist nor-binaltorphimine. All media were made on the day of use and maintained at an osmolarity of 300 mOsm/kg at pH 7.4 and 37 C.

We evaluated the effects of activation of each opioid receptor by specific agonist at concentrations of 10–5, 10–6, and 10–7 M. Some experiments were preceded by sperm preincubation with the specific receptor antagonist for 30 min before agonist addition. We assayed antagonism with antagonist doses of 10–4, 10–5, and 10–6 M plus agonist doses of 10–5, 10–6, and 10–7 M, respectively. Finally, parallel control experiments were performed to evaluate whether the highest dose (10–4 M) of specific antagonist, alone, induces some effect on sperm motility.

Motility analysis

Motility analysis was conducted by computer-assisted sperm analysis (Sperm Class Analyzer; Microptic, Barcelona, Spain) at time 0 and 0.5, 1, and 3.5 h after drug addition to the medium. Setting parameters and the definition of measured sperm motion parameters for computer-assisted sperm analysis were established by the manufacturer: number of frames to analyze, 25; number of frames/sec, 25; straightness threshold, 80%; cell size range (low), 2; cell size range (high), 60; volume, at least 3.0 ml; sperm concentration/ml, at least 20 x 106; forward motility, at least 60%. To measure both sperm concentration and motility, aliquots of semen samples (7.5 µl) were placed into a prewarmed (37 C) Makler counting chamber (Sefi Medical Instruments, Haifa, Israel). A minimum of 100 sperm from at least two different drops of each sample was analyzed from each specimen. Percent motile sperm was defined by the manufacturer as appropriate for the human species: A-grade sperm (rapidly progressive with velocity ≥ 35 µm/sec at 37 C), B-grade (slow/sluggish progressive with velocity ≥ 10 µm/sec but <35 µm/sec), C-grade (nonprogressive motility with velocity < 10 µm/sec), and D-grade (immobile).

Statistics

Results shown represent mean ± SEM. Statistical analysis was performed by ANOVA with a post hoc analysis by the least significant difference t test. Differences were considered significant and highly significant for P values of <0.05 and <0.01, respectively.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Expression of opioid receptors in human sperm

We detected the presence of {kappa}- and µ-opioid transcripts but not {delta}-opioid transcripts in human spermatozoa using RT-PCR. The expected 352-bp fragment for {kappa} and the 440-bp fragment for µ were detected in samples of human cerebral cortex used as a positive control and in sperm. The 222-bp fragment corresponding to the {delta}-opioid receptor amplicon was observed in human cerebral cortex but not in human sperm (Fig. 1AGo). To assure that the mRNA species represented specific receptor mRNAs, we sequenced the RT-PCR products and confirmed its identity with the known receptor sequences (data not shown). Real-time quantitative PCR further corroborated these findings. Thus, although {kappa}- and µ-receptors were detected, no {delta}-receptor mRNA was present in sperm (Fig. 1BGo). Retrotranscriptase negative controls were performed to assure the absence of genomic DNA (data not shown).


Figure 1
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  		FIG. 1. A, Ethidium-bromide-stained 2% agarose electrophoresis gels of the RT-PCR products for {delta}-, {kappa}-, and µ-opioid receptors and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) in human sperm (Sp) and cerebral cortex (Cx). DELTA, amplified fragment using primers specific for the human {delta}-opioid receptor (222-bp band); KAPPA, amplified fragment using primers specific for the human {kappa}-opioid receptor (352-bp band); MU, amplified fragment using primers specific for the human µ-opioid receptor (440-bp band). GAPDH was used as endogenous control. A representative RT-PCR experiment is shown; n = 5. B, Cycles of amplification (Ct ± SEM) for {delta}-, {kappa}-, and µ-opioid receptors assessed by real-time quantitative PCR using specific primers for human {delta}-, {kappa}-, and µ-opioid receptor transcripts.

 
Expression of {delta}-, {kappa}-, and µ-proteins in human sperm

Figure 2Go shows representative Western blots using plasma membranes from human sperm and human cerebral cortex (positive control). The anti-{delta} polyclonal antiserum labeled a band at 50 kDa in sperm protein extracts. In the cortex, the approximately 50-kDa band was also observed together with other bands of approximately 70 and 35 kDa (Fig. 2AGo). The anti-{kappa} polyclonal antiserum recognized four bands of about 100, 65, 56, and 36.5 kDa in sperm; a 65- to 56-kDa band was also observed in the cortex (Fig. 2BGo). Finally, the anti-µ polyclonal antiserum labeled bands at approximately 70 and 50 kDa in sperm, whereas in cerebral cortex the approximately 50-kDa band appears predominantly (Fig. 2CGo). When primary anti-{delta}, -{kappa}, or -µ antisera were omitted before secondary antibody addition, no bands appeared in the blots (data not shown).


Figure 2
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  		FIG. 2. Western blotting analysis of {delta}-opioid (A), {kappa}-opioid (B), and µ-opioid (C) receptors in human sperm (Sp) and cerebral cortex (Cx). The molecular mass markers (kDa) are indicated on the left. Western blots representative of those obtained with five normozoospermic donors are shown.

 
Localization of {delta}-, {kappa}-, and µ-opioid receptors in human sperm

Immunocytochemistry revealed that the {delta}-opioid receptor was present in the plasma membrane at the front part of the sperm head (over the acrosomal region), in the middle region, and uniformly distributed along the tail (Fig. 3AGo). Considerable differences in staining were not observed when the plasma membrane was permeabilized (Fig. 3BGo). In approximately 20% of the cells, prolonged staining at the beginning of the postacrosomal region in the head was observed (data not shown). {kappa}-Opioid receptor immunostaining was found in the plasma membrane in the sperm head, in the middle region, and in the tail (Fig. 3CGo), but when the plasma membrane was permeabilized, we found intense immunostaining on the neck of the sperm, and in approximately 17% of these cells, immunostaining at the equatorial/postacrosomal region was also observed (Fig. 3DGo), as well as in the middle region and the tail. Finally, µ-opioid receptor labeling was observed in the plasma membrane of the sperm head with more intensity at the equatorial/postacrosomal region, in part of the middle piece, and in the tail, both in nonpermeabilized (Fig. 3EGo) and permeabilized (Fig. 3FGo) cells. When primary anti-DOR, -KOR, or -MOR antibody was omitted before secondary antibody addition, the fluorescent staining pattern was not evident (Fig. 3HGo).


Figure 3
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  		FIG. 3. Immunofluorescence analysis of opioid receptors in human sperm cells under nonpermeabilized (A, C, and E) or permeabilized (B, D, and F) conditions. The distribution of {delta}-opioid receptors (A and B), {kappa}-opioid receptors (C and D), and µ-opioid receptors (E and F) is shown. H, Negative control treated with Hoechst 33342 and secondary antibody; G, its phase-contrast image; n = 3. Arrows indicate the different labeling after permeabilization (C and D) or the variability of staining in one sample (D). Representative photomicrographs are shown. Scale bar, 10 µm.

 
Effects on sperm motility of opioid agonists and antagonists

Motility effects of the {delta}-receptor agonist (DPDPE) and antagonist (naltrindole). As shown in Table 1Go, the {delta}-receptor antagonist naltrindole (10–4 M) decreased the percentage of sperm A plus B grades immediately after its addition to the sample (naltrindole 25.7 ± 5.9% vs. control 62.9 ± 4.1%; P < 0.01), without affecting sperm viability (data not shown). This decrease of sperm motility caused by naltrindole was weakened over time and was not significant after 3.5 h of incubation (naltrindole 37.4 ± 7.3% vs. control 50.2 ± 5.6%). The addition of lower doses of naltrindole did not significantly decrease sperm motility. The addition of different doses of the DPDPE agonist did not significantly affect sperm motility either.


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  		TABLE 1. Dose-response effects of {delta}-agonist (DPDPE) and antagonist (naltrindole) in sperm motility

 
Motility effects of the {kappa}-receptor agonist (U50488) and antagonist (nor-binaltorphimine). The {kappa}-receptor agonist U50488 and antagonist nor-binaltorphimine did not produce significant changes in the motility of spermatozoa during the course of 3 h of incubation (Table 2Go).


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  		TABLE 2. Dose-response effects of {kappa}-agonist (U50488) and antagonist (nor-binaltorphimine) in sperm motility

 
Motility effects of the µ-receptor agonist (morphine) and antagonist (naloxone). The addition of the µ-agonist morphine or antagonist naloxone to the incubation medium did not change the percentage of sperm A plus B grades in the sample, neither instantly nor after 1 h of incubation. However, samples exposed to 10–7 M morphine for 3 h had less sperm A plus B grades compared with those incubated with vehicle (45.8 ± 5.9 vs. 29.6 ± 9%; P < 0.05; see Table 3Go.) This decrease in motility was abolished by preincubation with naloxone (50.3 ± 5.3 vs. 29.6 ± 9%; P < 0.05; see Table 3Go.). Higher doses of morphine or incubation with naloxone alone did not further alter the motility of sperm in the sample (Table 3Go).


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  		TABLE 3. Dose-response effects of µ-agonist (morphine) and antagonist (naloxone) in sperm motility

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Opioid receptor expression has been reported in a wide variety of tissues in addition to the CNS. However, apart from the description of the µ-opioid receptor in horse spermatozoa (11), the existence of different types of opioid receptors in male gametes has not yet been reported. The present study demonstrates for the first time that human spermatozoa express {delta}-, {kappa}-, and µ-opioid receptors.

RT-PCR revealed the presence of {kappa}- and µ-opioid receptor mRNA in human spermatozoa. However, although the presence of {delta}-opioid receptor immunoreactivity was observed, there was no evidence of the expression of the mRNA for this opioid receptor in these mature cells. On the one hand, it is known that mature sperm do not perform transcription and translation processes (17) because in the final stages of spermatogenesis, the spermatozoa lose most of the cytoplasm (including most of the mRNA), with the exception of a thin layer (21). Hence, the presence of RNA would be limited to the nucleus/perinucleus or mitochondria, as has been shown by in situ hybridization (22, 23, 24). On the other hand, selective RNA degradation has been reported in human semen, in which specific RNA populations appear to be protected from such damage, indicating the existence of a stable population of RNAs (25). It is thus likely that spermatozoa do not retain {delta}-opioid receptor mRNA on the grounds that the gamete would have no further need for it (21), thus explaining its absence in mature spermatozoa but the presence of the receptor protein that would have been expressed during spermatogenesis. Moreover, this would suggest that {kappa} and µ mRNAs may have a role as selected messages in early zygote development, as has been reported for other sperm transcripts (26, 27). In any event, the function and utility of sperm mRNAs is still a controversial issue that continues to generate considerable debate (21).

This study includes the first Western blot analysis of {delta}-, {kappa}-, and µ-opioid receptors in human spermatozoa. With regard to the {delta}-opioid receptor, our observations coincide with previous results in the literature. Thus, recent studies have reported a predominant 46-kDa species, although {delta}-receptor immunoreactivity after SDS-PAGE appeared at different molecular masses (28). Persson et al. (29) found three bands of about 72, 48, and 36 kDa, suggesting that the 48-kDa band could represent the full-length rat {delta}-opioid receptor.

On the other hand, the 65- to 56-kDa bands observed in human sperm using an anti-KOR antibody, which also appeared in the human cerebral cortex, have been described in some studies using rat and frog brain and human placenta (30, 31, 32). Estimates from the KOR1 cDNA clone predicted a protein with a molecular mass of about 43 kDa, and it has been suggested that this mass difference was most likely the result of posttranslational modifications (33). The 100-kDa band has been proposed to be a dimer (33, 34).

Finally, we localized µ-opioid receptor in human sperm at 70 and around 50 kDa. Albrizio et al. (11) detected two similar nonglycosylated bands at 65 and 50 kDa. The theoretical molecular mass of the µ-receptor is 49.8 kDa, but as we have observed, bands with higher molecular masses also appear, presumably because of posttranslational modifications of the receptor (35).

Fluorescent immunomicroscopy of nonpermeabilized spermatozoa also revealed the presence of {delta}-, {kappa}-, and µ-opioid receptors on the plasma membrane of sperm cells. The {delta}-opioid receptor staining over the acrosomal region of sperm cells coincide with the localization of the precursor of [Met5] enkephalin, ligand of {delta}-opioid receptor (7). The differences in the {kappa}-opioid receptor staining pattern between nonpermeabilized and permeabilized spermatozoa indicate that this receptor could be present in internal structures (36). The pattern of µ-opioid receptor immunostaining was similar to that previously reported in mammalian sperm (11). The presence of receptors in the equatorial segment could imply their involvement in the fertilization process because this segment is supposed to have a crucial role in sperm/oocyte fusion (37). Different immunostaining patterns observed in cells that have undergone an identical immunostaining procedure would suggest that the membranes of these cells may be in a different functional state because, during the activation of the sperm cell, which can take place spontaneously (36), the sperm plasma membrane undergoes reorganization to achieve the ability to fertilize the oocyte (17).

We incubated sperm cells with opioid agonists and antagonists to examine the function of each opioid receptor. Thus, incubation of human spermatozoa with the µ-opioid receptor agonist morphine increased the number of immobile sperm; this action was reversed by naloxone. This result is consistent with that reported for cooled equine spermatozoa (38) and with the belief that decreased motility is the main sperm pathology of heroin (µ-agonist) addicts (12). Therefore, the low semen quality and male infertility found in opiate addicts seem to be a result not only of their effects in the hypothalamus (39, 40) and pituitary gland (4) but also of their direct action on sperm cells. This dual effect may explain why sperm pathologies can be found in heroin addicts with both normal and abnormal serum gonadotropin levels (12).

On the other hand, although different doses of the {delta}-opioid agonist DPDPE had no clear effect on sperm cell motility, incubation of spermatozoa with the {delta}-antagonist naltrindole produced a decrease in sperm motility. Previous studies also revealed an apparently contradictory effect of enkephalin (an opioid peptide with higher affinity for {delta}- than for µ-opioid receptor) on sperm function. Thus, high doses of enkephalin decreased sperm motility (1, 41), whereas lower doses of the pentapeptide were necessary to maintain sperm motility (9). Our results may explain this paradox. Thus, the inhibitory effect of the {delta}-antagonist naltrindole reported in the present work could be a result of the displacement of endogenous enkephalin from the {delta}-opioid receptor, which could be necessary to maintain motility. However, the inhibitory action of higher doses of enkephalins reported by others (41) may be a result of their effect on µ-opioid receptors.

It was intriguing to find that morphine at 0.1 µM had an inhibitory effect on sperm motility, whereas at higher concentrations (1 and 10 µM), morphine seems to be without effect. However, it is possible that at higher concentrations, morphine binds µ-opioid receptors (dissociation constant Kd of ~23 nM) and {delta}-opioid receptors (Kd of ~390 nM) (42), exerting a {delta}-effect that, as we have seen, could maintain sperm motility.

Our results may also explain the apparently contradictory effect of the different doses of the opioid antagonist naloxone on sperm motility. Albrizio et al. (11) reported that progressive motility was significantly reduced after incubation with high doses of naloxone, whereas it increased significantly after low doses of naloxone. Despite its relative lack of specificity, naloxone has more affinity for µ- than for {delta}-opioid receptors. Therefore, low naloxone doses, by antagonizing the µ-opioid receptor, can increase motility because activation of these receptors leads to an antimotility effect. However, higher naloxone doses may also displace {delta}-opioid ligands and, as we observed for naltrindole, may cause a loss of sperm motility.

The {kappa}-agonist and antagonist did not significantly affect the progressive motility of human sperm cells. However, the presence of this type of opioid receptor in different parts of the spermatozoa suggests that the {kappa}-opioid receptor may play other roles in sperm physiology. In fact, the distinct distribution of the three types of opioid receptors, together with the fact that different doses of opioid peptides and antagonists have distinct actions on the motility of spermatozoa, suggest that each receptor type has indeed specific functions because the highly polarized structure and function of spermatozoa requires the compartmentalization of particular metabolic and signaling pathways to specific regions (43).

Our findings corroborate the notion that endogenous opioid peptides may control reproductive function at multiple sites (1). Thus, the opioid system may operate as a multimessenger system in the CNS (40), the pituitary gland (4), and the testis (44), exerting a direct action on the spermatozoa.

In conclusion, we report for first time the presence of functional {delta}-, {kappa}-, and µ-opioid receptors in human sperm membranes. The {delta}- and µ-opioid receptors participate in distinct manners in regulating sperm motility, whereas the function of the {kappa}-receptor remains to be characterized. These findings open up novel avenues of research of the opioid system as a biochemical tool for the diagnosis and treatment of male infertility.


   Acknowledgments
 
We thank Ricardo Andrade (University of the Basque Country) and Asier Ruiz (University of the Basque Country) for their important contribution to this study. Likewise, we express their thanks to Quattro Translations (http://www.euskalnet.net/acts) for having improved the English of this paper.


   Footnotes
 
This work was supported by the University of the Basque Country (UPV/EHU, 00081.327-EA-4512/1998). E.A. holds a fellowship from the University of the Basque Country.

Disclosure statement: The authors have nothing to declare.

First Published Online September 19, 2006

Abbreviations: CNS, Central nervous system; DOR, {delta}-opioid receptor; KOR, {kappa}-opioid receptor; MOR, µ-opioid receptor; TNC, tyrode-modified noncapacitating medium.

Received March 17, 2006.

Accepted September 11, 2006.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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