Mol. Hum. Reprod. Advance Access originally published online on June 13, 2006
Molecular Human Reproduction 2006 12(7):461-468; doi:10.1093/molehr/gal050
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HE1/NPC2 status in human reproductive tract and ejaculated spermatozoa: consequence of vasectomy
1Centre de Recherche en Biologie de la Reproduction and Département d’Obstétrique-Gynécologie, 2Département de Chirurgie, Faculté de Médecine, Université Laval, Ste-Foy, Québec, Canada and 3UMR 6175 Physiologie de la Reproduction et des Comportements INRA-CNRS-Un. de Tours-Haras, Nouzilly, France
4 To whom correspondence should be addressed at: Unité d’Ontogénie-Reproduction, Centre de Recherche, Centre Hospitalier de l’Université Laval, 2705 Boulevard Laurier, Ste-Foy, Québec, Canada G1V 4G2. E-mail: robert.sullivan{at}crchul.ulaval.ca
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Abstract |
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We have previously demonstrated that the amount of HE1/NPC2 mRNA and protein expressed in the human epididymis is decreased under vasectomy. In this study, western blot analyses showed that many vasovasostomized men are characterized by high HE1/NPC2 levels in spermatozoa when compared with fertile donors. HE1/NPC2 association with sperm from vasovasostomized men was not related to low motility per se as spermatozoa from asthenospermic men have HE1/NPC2 levels similar to those in normal fertile semen samples. Spermatozoa from vasovasostomized men with high amount of HE1/NPC2 are characterized by higher concentration of cholesterol and more lipid raft domains. HE1/NPC2 is secreted in different glycoforms by different tissues of human male reproductive tract. These forms are due to variation in N-glycosylation, and only the deglycosylated form is associated with spermatozoa from some vasovasostomized men. Compared with normal men, seminal plasma of vasectomized men is characterized by a major decrease in immunodetectable HE1/NPC2 without change in the glycosylation pattern. Following surgical vasectomy reversal, seminal plasma HE1/NPC2 was found in similar amounts to the ones characterizing normal men. Considering the potential role of HE1/NPC2 in cholesterol transport during sperm maturation, unusual high levels of this protein associated with spermatozoa of vasovasostomized men may reflect epididymal sequelae occurring when the vas deferens is obstructed.
Key words: cholesterol/epididymis/HE1/NPC2/raft/sperm maturation/spermatozoa/vasectomy/vasovasostomy
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Introduction |
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Vasectomy is the third most common contraceptive method. Worldwide,
4 million vasectomies are performed each year. Although vasectomy rates plateaued in the last decade, the demand for surgical vasectomy reversal (vasovasostomy) is increasing. Following vasovasostomy, the pregnancy rate is much lower than the surgical success of reanastomosis (Belker et al., 1991
; Shannon, 1994; Kolettis et al., 2003). The pregnancy rate decreases with the increase of time between vasectomy and its reversal (Belker et al., 1991). The discrepancy between the surgical success of vasovasostomy and the pregnancy outcome has, however, been recently challenged (Silber and Grotjan, 2004). Many factors may be responsible for the discrepancy between the surgical success of vas deferens reanastomosis and the pregnancy outcome, such as age of the female partner, epididymal obstruction by granuloma, sperm antibodies and idiopathic infertility (Belker et al., 1991; Nieschlag et al., 1997).
Leaving the testis, the mammalian spermatozoa have to transit along the epididymis before reaching the vas deferens. The epididymis is a long, single convoluted tubule classically divided into three segments: the caput, the section connected to the testis, the corpus and the cauda epididymidis. Although the distal cauda serves as a sperm reservoir, the caput and corpus segments are involved in the acquisition of sperm fertilizing ability. Each segment expresses a unique set of genes that results in a pattern of protein secretion that varies from one segment to the other (Kirchhoff, 1999; Rodriguez et al., 2001; Johnston et al., 2005). These intraluminal epididymal proteins sequentially modify the sperm cells to generate fully functional male gametes. Taken together, this process has been named epididymal sperm maturation (Sullivan, 1999; Cuasnicu et al., 2002; Gatti et al., 2004).
The consequences of vasectomy on epididymal physiology are poorly documented, especially in humans. Vasectomy has been shown to selectively affect the expression of a cystein-rich secretory protein-1 (CRISP-1) in the rat caput epididymidis (Turner et al., 1999) and of HE2-like mRNA in the corpus segment of the cynomolgus monkey (Lamontagne et al., 2001). At least in rats, the consequences of vasectomy on epididymal protein secretion are not reversible following vasovasostomy (Turner et al., 2000). Vasectomy disturbs gene expression also in the human epididymis. We showed that vasectomy modifies the pattern of the expression of P34H (Legare et al., 2001), a protein expressed in the human corpus epididymidis (Legare et al., 1999), added to the sperm surface during maturation (Boue et al., 1996) and involved in zona pellucida binding (Boue et al., 1994). The absence of P34H from spermatozoa of men presenting with idiopathic infertility is associated with the inability of these gametes to interact with the zona pellucida (Boue and Sullivan, 1996). Similarly, a significant proportion of vasovasostomized men produce spermatozoa lacking this epididymal sperm protein (Guillemette et al., 1999). Although the expression of P34H in the epididymis is relocated under vasectomy, HE1/NPC2, a gene expressed all along the human epididymis, is down-regulated under vasectomy (Legare et al., 2004). Considering that HE1/NPC2 is a modulator of cholesterol transport (Garver and Heidenreich, 2002; Vanier and Millat, 2004), the objective of this study was to investigate the consequences of the low epididymal amount of this protein on spermatozoa from vasovasostomized men. We characterized the regional modifications of the glycosylation status of HE1/NPC2 in the reproductive tract of normal men as well as in the seminal plasma of vasectomized men.
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Materials and methods |
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Ethic consent
These studies were conducted with the approval of the ethic committee for research on human subjects of the Université Laval.
Biological material
Semen samples from donors were considered fertile according to the WHO 1999 criteria: a minimum of 2 ml of semen volume, a sperm concentration of at least 20 x 106/ml and a viability and motility >50% (WHO, 1999). Semen samples from vasovasostomized and asthenozoospermic men were obtained from the clinical andrology laboratory of our institution. The time period between vasectomy and vasovasostomy varied from 2 to 19 years. Samples were obtained 2–11 months after vasovasostomy. Asthenozoospermy was defined by a percentage of motility <40 (WHO, 1999). Samples were obtained by masturbation, and spermogram parameters were determined using computer-assisted sperm analysis. After 30–120 min of liquefaction at room temperature, spermatozoa were pelletted and washed twice by centrifugation at 800 g in Dulbecco’s phosphate-buffered saline (D-PBS; Gibco-BRL, Burlington, Ontario, Canada). Seminal plasma and pellets of spermatozoa were frozen at –80°C until use.
Human epididymides were obtained through our local organ transplantation programme. Donors were of 47 and 48 years of age with no known medical pathologies that could affect reproductive function. Tissues were collected under optimal conditions while artificial circulation was maintained to preserve organs assigned for transplantation. Epididymides were immediately sent on ice and transported to the laboratory and dissected into caput, corpus and cauda epididymidis, as previously described (Legare et al., 1999).
Segments of the vasa deferentia were obtained from healthy patients of proven fertility who were undergoing vasectomy or vasovasostomy. The vasectomy was performed under local anaesthesia by high bilateral incisions in the scrotum. Once the vas was identified, a short segment was removed. Ligation of both ends was performed with metallic clips after cauterization of the lumen. The vasovasostomy was performed under regional anaesthesia by two scrotal incisions. The scarred portion of vas was removed. Small segments of both abdominal and testicular ends were excised. These vas deferens segments were pooled for HE1/NPC2 determinations without making distinction between abdominal and testicular segments. A modified one-layer anastomosis was performed under the microscope with interrupted 9-0 nylon sutures. Prostate and seminal vesicle tissues were obtained from patients who were undergoing prostatectomy. This surgical procedure was performed by laparoscopy under general anaesthesia.
Protein preparation
Spermatozoa
Sperm pellets (107) were resuspended in sodium dodecyl sulphate–polyacrylamide gel electrophoresis (SDS–PAGE) sample buffer (Laemmli, 1970) prepared without reducing reagent. After 10 min, spermatozoa were pelletted by centrifugation and the protein supernatants recovered and reduced by the addition of 2% ß-mercaptoethanol followed by 5 min of heating in a boiling water bath.
Tissues
Tissues were homogenized with a Polytron (InterSciences, Markham, Ontario, Canada) in a homogenization buffer [10 mM EDTA, 1 mM phenylmethylsulphonyl fluoride (PMSF), 1% SDS in sterile water]. Extracts were then centrifuged at 3000 g for 15 min at 4°C. Supernatants were precipitated with MEOH/CHCl3, and proteins were resuspended in a sample buffer and submitted to SDS–PAGE (Laemmli, 1970). Protein concentrations were determined by amido black staining of dot blots (Chapdelaine et al., 2001).
Seminal plasma
Seminal plasma samples were centrifuged twice at 3000 g for 10 min to eliminate spermatozoa and other cellular constituents. Supernatants were precipitated with MEOH/CHCl3, and proteins were resuspended in a sample buffer and submitted to SDS–PAGE (Laemmli, 1970). Protein concentrations were determined by amido black staining of dot blots (Chapdelaine et al., 2001).
Deglycosylation of N-linked carbohydrates
HE1/NPC2 from corpus epididymidis and that from seminal plasma were deglycosylated using endoglycosidase F/N-glycosidase F (PNGase; Sigma, Oakville, Ontario, Canada). Epididymal and seminal plasma proteins (100 ug) were incubated for 3 h at 37°C in 50 mM phosphate buffer (pH 7), 0.75% Triton X-100 and 0.1% SDS in the presence of 1% 2-mercaptoethanol. Control samples were incubated under the same conditions without N-glycosidase. The deglycosylated proteins were then analysed by SDS–PAGE and western blotting.
Antibodies
Rabbit polyclonal antibody was produced against HE1/NPC2 purified from ram epididymal fluid (Fouchecourt et al., 2000; Gatti et al., 2005) and used at 1/2000 (v/v). Rabbit polyclonal antibody against prostatic phosphatase acid (PAP) purified from human seminal plasma was used at 1/1000 (v/v). This antiserum was a generous gift of Dr R.R. Tremblay and was used as a marker of prostatic protein secretions. Mouse monoclonal antibody against ß-actin was purchased from Sigma and used at 1/20 000 (v/v). Goat anti-rabbit immunoglobulin G (IgG) conjugated to horseradish peroxidase (HRP) was purchased from Jackson Laboratory (Missisauga, Ontario, Canada) and used at 1/10 000 (v/v). Goat anti-mouse IgG conjugated to HRP was purchased from BIO/CAN Scientific (Missisauga, Ontario, Canada) and used at 1/2000 (v/v).
Western blot analysis
Proteins were separated on 12% SDS–PAGE and transferred to nitrocellulose membranes (Towbin et al., 1979). After saturation with 1% bovine serum albumin (BSA) or 5% milk in PBS with 0.1% Tween 20, membranes were incubated with an anti-HE1/NPC2, an anti-PAP or an anti-ß-actin. Goat anti-rabbit or anti-mouse IgG conjugated to HRP was used for chemiluminescent detection of proteins [enhanced chemiluminescence (ECL) reagent; Amersham, Baie d’Urfée, Quebec, Canada]. HE1/NPC2, PAP and ß-actin signals were quantified by densitometry in the linear range of film exposure and expressed as arbitrary units (AU).
Cholesterol measurements
Cholesterol from 4 x 107 spermatozoa was extracted with chloroform/methanol (v/v 1:2) by Folch’s method (Folch et al., 1957). Total cholesterol content was quantified by the Liberman-Burchard method. Liberman-Burchard reagent (sulphuric acid and acetic anhydride) reacts with sterol to produce a characteristic green colour whose absorbance is determined by spectrophotometry at 640 nm.
Ganglioside M1 determination
About 107 spermatozoa were lysed in 200 µl of ice-cold TNE buffer [10 mM Tris (pH 7.5), 150 mM NaCl, 5 mM EDTA] containing 1% Triton X-100 for 30 min on ice. Cell lysates were centrifuged at 12 000 g for 15 min at 4°C. To detect the ganglioside M1 (GM1) raft marker (Lagerholm et al., 2005), we dotted 50 µl of supernatants on a polyvinylidene difluoride membrane, which was then incubated with CTB-HRP (HRP-conjugated cholera toxin B subunit; Sigma) and subjected to chemiluminescent detection using an ECL kit (Amersham). GM1 was quantified by densitometry and expressed as AU.
Statistical analysis
Statistical analyses were performed by analysis of variance using super ANOVA software (ABACUS Concepts, Berkeley, CA, USA). Results were compared by Fisher’s test. Differences were considered to be significant at P < 0.05.
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Results |
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Effect of vasectomy on HE1/NPC2 associated with spermatozoa
The amount of HE1/NPC2 associated with a constant number of ejaculated spermatozoa showed a great inter-individual variability in both normal and vasovasectomized samples (Figure 1). On the basis of our data, we arbitrarily defined a threshold of 30 units (AU) of HE1/NPC2 to distinguish sperm samples with low and high amounts of HE1/NPC2. In sperm protein extracts from fertile men, HE1/NPC2 was detectable in high amount (>30 AU) in only 18% (5/27) of the samples. In samples from vasovasostomized men, high HE1/NPC2 values were more frequent, i.e. in 62% (18/29) samples tested (Figure 1). The high value was mainly due to an increase of the amount of the 21–23 kDa HE1 doublet associated with spermatozoa of the vasovasostomized men (as shown in the Figure 1 inset).
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No correlation could be found between the level of HE1/NPC2 in spermatozoa from vasovasostomized men and the period of time between vasectomy and surgical vasectomy reversal, neither with the time period between post-surgical vasectomy reversal and semen collection (data not shown).
Relationship between vasovasectomy and HE1/NPC2 associated with spermatozoa and motility
The percentage of motility also showed a great variability in semen samples obtained from vasovasostomized men. Interestingly, in samples characterized by normal motility (>50%), the amount of HE1/NPC2 was 20 HE1/NPC2 AU, a value significantly lower (P < 0.05) than the 62 AU found in samples with low motility (<50%) (Figure 2). To clarify this association between HE1/NPC2 and motility, we analysed the level of HE1/NPC2 associated with spermatozoa of asthenozoospermic men (Figure 3). We found no difference in HE1/NPC2 quantities between asthenozoospermic and fertile semen samples (24 and 25 AU, respectively), which is significantly lower than that in vasovasostomized samples (52 AU). This suggested no direct association between HE1/NPC2 levels and motility.
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Association of high levels of HE1/NPC2 on cholesterol and spermatozoa membrane raft
Considering that HE1/NPC2 is a cholesterol transporter, cholesterol content was determined in spermatozoa from vasovasostomized men and compared with normal semen samples. This determination was performed on vasovasostomized sperm protein samples characterized by low and high levels of HE1/NPC2. In samples from vasovasostomized men with high HE1/NPC2 levels, the amounts of cholesterol associated with spermatozoa was 50% higher than those measured in fertile samples, whereas no differences were found in vasovasostomized samples with low levels of HE1/NPC2 (Figure 4). As high concentrations of membrane cholesterol is found in lipid rafts, it was relevant to determine whether cholesterol concentration found in samples from vasovasostomized men was associated with an increase in raft domains using GM1 as a marker. Higher amounts of GM1 were associated with spermatozoa of vasovasostomized men when compared with fertile samples (Figure 5).
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HE1/NPC2 glycoforms in human reproductive tract
To understand the origin of HE1/NPC2 associated with cholesterol-rich spermatozoa from vasovasostomized men, we investigated the regional modifications of the glycosylation status of HE1/NPC2 in the reproductive tract of normal men by western blotting. In epididymal tissues, HE1/NPC2 was detected as two distinct immunoreactive protein forms on 1D gel electrophoresis: a form at 26 kDa and a doublet at 21–23 kDa. Whereas the 21–23-kDa form was detectable in caput, corpus and cauda epididymidis, the 26-kDa form was significantly detectable only in cauda epididymidis (Figure 6A). In the vas deferens, two forms of HE1/NPC2 were found, a major form at 26 kDa and a doublet at 21–23 kDa. In prostate homogenate, HE1/NPC2 was detected as two forms, 28 and 26 kDa, whereas only the 28-kDa form was present in seminal vesicles tissues. Both 26- and 21–23-kDa forms were detected in seminal plasma, whereas only the doublet at 21–23 kDa was associated with spermatozoa from some vasovasostomized men. The quantity of these different glycoforms in the different tissues was measured, and the results are shown in Figure 6B.
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These different glycoforms of HE1/NPC2 detected by western blotting resulted from N-glycosylation. In fact, when seminal plasma or corpus epididymidis was treated by PNGase and then submitted to HE1/NPC2 immunodetection, the higher molecular weight (MW) form of HE1/NPC2 was highly diminished and the 21–23-kDa doublet increased in intensity (Figure 7).
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Vasectomy effect on HE1/NPC2 glycoforms in seminal plasma and vas deferens
Compared with normal men, seminal plasma of vasectomized men was characterized by a major decrease in the quantity of HE1/NPC2 immunodetectable without change in the protein glycosylation pattern. Approximately 40% less HE1/NPC2 was detected in seminal plasma in samples from vasectomized men. Following surgical vasectomy reversal, HE1/NPC2 levels were comparable with the ones characterizing normal men (Figure 8A). This contrasts with PAP known to be secreted by the prostate, which was present in similar quantities in the seminal plasma of normal, vasectomized and vasovasostomized men (Figure 8B).
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In agreement with our previous finding, we also observed by western blot analysis of the vas deferens homogenates from vasectomized men that HE1/NPC2 was strongly decreased as compared with tissues from normal men (Figure 9).
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Discussion |
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First described as an epididymal-specific cDNA (Krull et al., 1993
), HE1/NPC2 mRNA has been detected in different reproductive tissues including vas deferens, seminal vesicles and prostate (Kirchhoff et al., 1996). HE1/NPC2 transcript is detectable as a single band on northern blots of RNA extracted from all male reproductive tissues investigated (Kirchhoff et al., 1996). It has been previously shown that HE1/NPC2 associates with human epididymal spermatozoa, and its association with ejaculated spermatozoa has, however, been considered questioned (Kirchhoff et al., 1996). These observations have, of course, been obtained using fertile semen samples. In our study, significant HE1/NPC2 association with ejaculated spermatozoa is detectable by western blot in only 18% of semen samples from normal men. HE1/NPC2 association with spermatozoa is much more easily observed when using spermatozoa from vasovasostomized men. Interestingly, HE1/NPC2 not only binds to spermatozoa from vasovasostomized men but also binds mostly to those with high cholesterol content (Figure 4).
HE1/NPC2 was identified as a major secretory protein of the human epididymis by substrative cDNA libraries (Kirchhoff et al., 1996). It has also been characterized in stallion (Uhlenbruck et al., 1993; Fouchecourt et al., 2000), boar (Uhlenbruck et al., 1993; Okamura et al., 1999), chimpanzee (Frohlich and Young, 1996), dog (Ellerbrock et al., 1994), bull (Uhlenbruck et al., 1993) and rat (Kappler-Hanno and Kirchhoff, 2003). HE1/NPC2 cDNA sequence is well conserved among species, suggesting an important function. Mutations in HE1/NPC2 gene are responsible for an autosomal recessive disorder named Niemann-Pick Type C (NPC) disease (Steinberg et al., 1994; Naureckiene et al., 2000). These rare pathologies are late endosomal/lysosomal storage disorders to which neurodegeneration and organomegaly are associated (Garver and Heidenreich, 2002). Although HE1/NPC2 function in the epididymis remains to be defined, it facilitates cholesterol transport within the lumen of late endosome/lysosomes (Garver and Heidenreich, 2002; Vanier and Millat, 2004). In fact, HE1/NPC2 structure reveals a cholesterol-binding pocket (Friedland et al., 2003; Ko et al., 2003; Vanier and Millat, 2004), and HE1/NPC2 purified from porcine epididymal fluid binds cholesterol with a stoichiometry of 1:1 with a Kd = 2.3 uM (Okamura et al., 1999). Therefore, spermatozoa with higher cholesterol content should bind more efficiently HE1/NPC2 through its cholesterol-binding pocket, which is in agreement with our results.
During the epididymal transit, spermatozoa undergo major modifications including cholesterol/phospholipids molar ratio modifications (Jones, 1998). Considering that HE1/NPC2 binds cholesterol, it is hypothesized that this epididymal protein could be involved in the decrease in sperm plasma membrane cholesterol/phospholipids ratio occurring during the epididymal transit in humans (Haidl and Opper, 1997). Under vasectomy, HE1/NPC2 expression in the human epididymis is selectively down-regulated (Legare et al., 2004). In the absence of HE1/NPC2, spermatozoa may not be properly processed during the epididymal transit. This could explain why spermatozoa from some vasovasostomized men are characterized by higher cholesterol content. Knowing that membrane remodelling of maturing spermatozoa is essential for flagellar motility acquisition, it is not surprising that poorly motile ejaculated spermatozoa of vasovasostomized men bind more HE1/NPC2 than spermatozoa of other individual for which epididymal function was less affected by vasectomy.
Lipid rafts are cholesterol- and sphingolipid-enriched microdomains present in the plasma membrane of most mammalian cells investigated (Brown and London, 2000; Edidin, 2003). Rafts are enriched in glycosylphosphatidylinositol-anchored proteins and transmembrane proteins and are involved in cell signal transduction (Simons and Ikonen, 1997). The major increase in GM1 content in spermatozoa from vasovasostomized men is in agreement with the increase in cholesterol content in these ejaculated spermatozoa. This reflects major modification of sperm raft domains following vasectomy. Rafts being involved in membrane signalling, this can have major consequences in sperm physiology and in the sequence of events during the fertilization process (Cross, 2004). Raft domain increases in spermatozoa from vasovasostomized men suggest that the alterations of their plasma membrane are more complex than a simple modification in cholesterol content. Comprehension of the differences between spermatozoa from normal and vasovasostomized men can provide information regarding the biochemical modifications undergone by the male gamete during the epididymal transit.
Most men who undergo vasectomy reversal have fathered before sterilization by vas deferens ligation. Following vasovasostomy, even though they recover spermogram values comparable with those characterizing their fertile period, some of them remain infertile. Thus, some epididymal sequelae may not be completely reversible as is had been shown for CRISP-1 expression in the rat epididymis (Turner et al., 2000), and this is reflected by the discrepancy between the surgical success of vasectomy and the fertility recovery. The consequences of vasectomy include HE1/NPC2 down-regulation that affects the normal sperm maturation process. In a subpopulation of vasovasostomized men, spermatozoa will thus have an increase in cholesterol with a decrease in motility.
HE1/NPC2 present in seminal plasma may originate from the accessory sex glands (seminal vesicles and the prostate), the vas deferens and the epididymis. Considering that HE1/NPC2 concentration in seminal plasma is significantly reduced under vasectomy, the epididymides contribute to approximately half of the HE1/NPC2 concentration present in seminal plasma of normal men. Even though HE1/NPC2 is encoded by a single mRNA, different forms of HE1/NPC2 can be detected along the male reproductive tract as well as in the seminal plasma. These forms result mainly from N-glycosylation. Although two forms of HE1/NPC2 are present in the seminal plasma, only the deglycosylation form of lower MW is associated with spermatozoa of a subpopulation of vasovasostomized men. Considering the different pattern of HE1/NPC2 glycoforms detected in different reproductive tissues in humans, we hypothesized that the deglycosylated form of HE1/NPC2 associated with these spermatozoa originates from the epididymis and/or the vas deferens.
We also hypothesize that at ejaculation in vasovasostomized men, HE1/NPC2 secreted along the reproductive tract will bind to improperly mature spermatozoa characterized by high cholesterol content. This would explain why, in most vasovasostomized men, HE1/NPC2 is detectable in sperm pellet in contrast to normal men or vasovasostomized men with less pronounced epididymal damage (Figure 10). We have tried without success to add purified HE1/NPC2 to spermatozoa preloaded with cholesterol under different in vitro conditions. This probably reflects the fact that sperm cholesterol efflux is not the only factor disturbed during epididymal maturation of spermatozoa in some vasovasostomized men and that binding on HE1/NPC2 depends of complex biochemical factors.
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In conclusion, in humans, vasectomy causes sequelae to the epididymis that are not necessarily reversed following vasovasostomy. A better understanding of the biochemical parameters of the spermatozoa ejaculated following these procedures can help us to better understand the function of epididymis in men and to predict fertility recovery following vasectomy reversal.
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Acknowledgements |
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Dr R.R. Tremblay from Université Laval is thanked for generous gift of the anti-PAP antiserum and Dr P. Manjunath from Université de Montréal for providing us with biological material. We thank Dr Pierre Leclerc, Mr Gilles Frenette and Mrs Julie Girouard for technical help and for the stimulating discussion and advices. We also thank the technicians of the clinical andrology laboratory of our institution for their collaboration. The cost of open access was funded by a CIHR grant.
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Submitted on April 3, 2006; accepted on May 10, 2006.
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