| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
ARTICLES |
a Instituto de Histología y Embriología, Facultad de Ciencias Médicas, Universidad Nacional de Cuyo, Mendoza, Argentina
 |
ABSTRACT |
|---|
 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
epididymis, sperm, sperm maturation
|
INTRODUCTION |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
Numerous studies have demonstrated the presence of MPRs in the male reproductive tract of different mammalian species. For example, Brown and Farquhar [14] observed that CI-MPR is distributed in the trans-Golgi network of the epididymal principal cells in rats. Likewise, O'Brien et al. [15] found that in mice, pachytene spermatocytes and round spermatids predominantly synthesize CD-MPR and lower levels of CI-MPR. In contrast, cultured Sertoli cells synthesize higher levels of CI-MPR. Furthermore, Sertoli cells secrete glycoproteins containing mannose-6-phosphate residues, which are then endocytosed by spermatogenic cells [16]. This mechanism may be related to the regulation of spermatogenesis [17]. Both CI-MPR and CD-MPR have also been found on the sperm surface of rat cauda epididymis [18], although their role in the gametes remains unclear.
It is well known that mammalian spermatozoa undergo post-testicular modifications in the epididymis and in the female genital tract, thereby acquiring their fertilizing capacity. During exposure to the epididymal microenvironment the spermatozoa interact with some components of the lumen and, as a consequence, the gamete surface undergoes modifications [19–23].
We wondered if the changes in distribution of MPRs may reflect those changes that spermatozoa undergo during epididymal transit, and if they are related to the maturation process of gametes. To test this possibility, we examined by indirect inmunofluorescence (IIF) and transmission electron microscopy (TEM) the distribution of both CD-MPR and CI-MPR in testicular spermatozoa and those gametes obtained from different regions of epididymis.
|
MATERIALS AND METHODS |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
Rabbit polyclonal antisera directed against human CD-MPR were generously provided by Dr. Annette Hille-Rehfeld, Zentrum Für Biochemie und Molekulare Zellbiologie, Göttingen, Germany. Mouse polyclonal antibodies to bovine CI-MPR were developed in BALB/cd mice in our laboratory. FITC-conjugated anti-mouse (F-8771) or anti-rabbit (F-0382) immunoglobulin G (IgG) were purchased from Sigma Chemical Company (St. Louis, MO). Colloidal gold (10 nm) conjugated anti-mouse or anti-rabbit IgGs were also purchased from Sigma, as were trypsin (from porcine pancreas, T-4799), digitonin, Triton X-100, biotin-conjugated anti-mouse (B-9904) and anti-rabbit (B-8895) IgG, and ExtrAvidin peroxidase. All the reagents for electrophoresis were purchased from Bio-Rad (Hercules, CA).
Animals
Adult male Sprague-Dawley rats (90–120 days old) were housed under standard conditions (food and water ad libitum at 20–22°C and a light cycle of 12L:12D). They were killed by inhalation of ether and decapitated according to the rules reported in the National Institutes of Health guide of the National Research Council, and the epididymides were removed.
Preparation of Spermatozoa
The epididymides were separated into three segments: caput, corpus, and cauda, and each was minced in 2 ml of PBS at 37°C. After swirling for 10 min and letting the fragments settle, the supernatant fractions containing spermatozoa were aspirated, placed in 15-ml Falcon tubes, centrifuged at 500 x g for 7 min at room temperature, and the pellets containing spermatozoa were washed once with PBS. The spermatozoa were finally resuspended in small volumes of PBS, fixed with 2% p-formaldehyde (PAF; in PBS) for 10 min, placed on slides, and processed for immunofluorescence. To obtain spermatozoa from rete testis, the rats were ligated between testis and epididymis and maintained alive for 4 days. The spermatozoa that accumulated in rete testes were obtained by puncture and aspiration. The subsequent procedures were similar to those for epididymal spermatozoa.
Treatment of Spermatozoa
One million spermatozoa extracted from either rete testis or different regions of epididymis were incubated with 1 mg/ml trypsin at 37°C for 15 min in 200 µl PBS. The incubation was stopped by adding of 2 mg/ml trypsin inhibitor, and the spermatozoa were sedimented and washed once with 2 ml PBS. For high-strength treatment, two million spermatozoa were incubated with 0.3 M NaCl as described by Belmonte et al. [24]. They were then processed for immunofluorescence. Other pools of spermatozoa were either treated with 0.05% digitonin (30 min at 37°C) or 0.5% Triton X-100 (5 min at room temperature) [25] after fixation of the cells with 2% PAF and then processed for immunofluorescence.
In Vitro Sperm Capacitation
For capacitation, spermatozoa were collected from cauda epididymis by puncture of the ductuli and the first drop was collected in a 15-ml Falcon tube. A 1.5-ml aliquot of capacitating medium [26] was added and the tubes were incubated at 37°C for 5 min with 5% CO2. The spermatozoa that swam up were collected from the top of the tubes, placed in tissue culture wells (16 mm, Sigma) and diluted to one million cells/ml with fresh capacitating medium overlaid with mineral oil. After incubation at 37°C for 5 h in an atmosphere of 5% CO2, the spermatozoa were collected and fixed with 2% PAF for 10 min. The spermatozoa were washed three times with PBS containing 4 mg/ml BSA (Fraction V, Sigma), placed on polylysine-coated slides, and air-dried. The slides were then processed for IIF.
The percentages of motile sperm were determined on prewarmed slides and observed by light microscopy. To assess viability, we measured the incorporation of eosin Y (0.1% in saline solution) by the spermatozoa.
Incubation with Ionophore A23187
After incubating the spermatozoa for 4.5 h in capacitating medium, the ionophore was added to a final concentration of 5 µM and newly incubated for 15 min. Spermatozoa were collected and fixed with 2% PAF for 10 min, washed with PBS containing 4 mg/ml BSA, and then placed on polylysine-coated slides and processed for IIF.
Immunofluorescence
The slides containing spermatozoa were blocked for 1 h at room temperature with 5% horse serum in PBS. They were then incubated in either rabbit polyclonal anti-CD-MPR or mouse anti-CI-MPR (diluted 1:50 and 1:100, respectively, in PBS containing 1% horse serum [PBS-HS]) overnight at 4°C. After three washes with PBS, the corresponding second antibody (FITC-conjugated anti-rabbit or anti-mouse IgG, 1:100 in PBS-HS) was added and incubated for 1 h at room temperature. The slides were washed three times with PBS and mounted with 0.1 mg/ml propylgalate and 50% glycerol in PBS. Slides were examined on a Nikon Optiphot microscope (Nikon, Tokyo, Japan). The percentage of stained spermatozoa was estimated by counting >100 cells with epifluorescence optics at a magnification of 300x.
Transmission Electron Microscopy
Spermatozoa obtained as described earlier were fixed for 10 min with PAF containing 0.1% glutaraldehyde, pelleted, washed twice with 1% glycine in PBS, blocked for 1 h at room temperature with 5% PBS-HS, and incubated with either anti-CD or anti-CI-MPR for 2 h at room temperature. The samples were then washed three times with PBS and incubated for 2 h with 10 nm colloidal gold particles covered with either anti-rabbit or anti-mouse IgG (each diluted 1:40 in PBS). After three washes with PBS the samples were processed for TEM. Spermatozoa were fixed according to the method of Mollenhauer et al. [27]; they were then washed with 50 mM phosphate buffer (pH 7.2) and postfixed overnight with 2% osmium tetroxide (OsO4). Spermatozoa were then washed with distilled water, dehydrated in increasing grades of ethanol and acetone, and finally embedded in Epon 812. Ultrathin sections were obtained with a Leica Ultracut R ultramicrotome and stained with 2% lead citrate followed by 2% uranyl acetate. Observations were made with a Siemmens Elmiskop I microscope (Siemmens, GA, Karlsruhe, Germany).
Gel Electrophoresis and Western Blotting
Sperm proteins were extracted by heating twice at 95°C for 5 min each in Laemmli sample buffer without a reducing agent. The mixtures were centrifuged at 12 000 x g for 10 min, the supernatants were supplemented with 10 mM dithiothreitol, and boiled for 3 min before loading.
The proteins were run on SDS-PAGE (8% for CD-MPR, 7% for CI-MPR) at 25 mA for 45 min. After that, they were electrotransferred to nitrocellulose membranes (pore size of 0.2 µm, Pierce, Rockford, IL) as described by Burnette [28]. Nonspecific binding sites were blocked overnight at 4°C with 0.05% Tween-20 in 20 mM PBS (buffer B) containing 3% low-fat milk. All the following steps were carried out at room temperature: Membranes were incubated with either anti-CD-MPR or anti-CI-MPR antibody for 2 h (each diluted 1:500 in buffer B), washed three times for 15 min with buffer B, and incubated with biotin-conjugated anti-rabbit IgG (1:5000 in buffer B) or anti-mouse IgG (1:5000 in buffer B) for 2 h. After five washes with buffer B, the membranes were incubated for 1 h with peroxidase-conjugated ExtrAvidin (1:2000 in buffer B). After washes, the protein band of MPR 46 or MPR 300 were detected by the enhanced chemiluminescence method according to the manufacturer's instructions (Amersham, Buchler, Germany).
Statistical Analysis
The percentage of immunostained spermatozoa were examined by ANOVA followed by Duncans test. A value of P < 0.02 was accepted as significant.
|
RESULTS |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
|
We observed that MPRs changed their localization on sperm surface as they passed through the epididymal duct. CI-MPR was initially distributed on the dorsal region of the head in spermatozoa obtained from rete testis. Sperm extracted from epididymis (caput, corpus, or cauda) showed additional immunoreactivity that was distributed over a region that corresponded to the equatorial segment of the head (Fig. 2, c, e, and g). In all cases, no labeling was observed on the postacrosomal region of spermatozoa (Fig. 2, a, c, e, and g; compared with a', c', e', and g'). The controls incubated with preimmune serum did not show fluorescence (Fig. 2, b, d, f, and h).
|
CD-MPR was distributed on the dorsal region of the head in sperm from rete testis, as well as caput and corpus, whereas immunoreactivity extended to the ventral region in caudal spermatozoa (Fig. 3). Additional labeling of CD-MPR was observed on the middle piece of corpus and cauda spermatozoa (Fig. 3, e and g). As with CI-MPR, no CD-MPR immunoreactivity was detected on the postacrosomal region (Fig. 3). The labeling observed on the dorsal region of the sperm head (for CI-MPR and CD-MPR) likely correspond to the acrosomal region because the staining disappeared when incubated with the ionophore A23187 under conditions in which the acrosome reaction occurs [25, 29] (Fig. 4, Table 1). In addition, CD-MPR labeling diminished drastically on the ventral side of the equatorial region after the acrosome reaction (Fig. 4).
|
|
|
In order to evaluate whether the regional differences observed in the distribution of the receptors were due to the existence of proteins that mask MPRs, spermatozoa were either extensively washed at high ionic strength (0.3 M NaCl) or were digested with trypsin, but no differences in patterns of staining were observed with respect to controls (data not shown). When spermatozoa were permeabilized with either Triton X-100 or digitonin, the distribution of CD-MPR and CI-MPR immunoreactivity did not change (data not shown). We confirmed with electron microscopy and colloidal gold that sperm remained intact and that both CI-MPR and CD-MPR were localized on the plasmalemma (Fig. 5, A and C; Fig. 6A). This localization was observed in spermatozoa from all regions studied. No colloidal gold particles were observed when preimmune serum was used (Fig. 5, B and D; Fig. 6B). In addition to regional differences in the pattern of distribution of CD-MPR and CI-MPR, we found that the percentages of sperm labeled with CI-MPR progressively increased from rete testis to cauda epididymis. The percentages of sperm immunostained for CD-MPR, however, increased from rete testis to caput epididymis, and they were maintained along the epididymis (Table 2).
|
|
|
|
DISCUSSION |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
We also observed that the percentages of cells that were immunoreactive for CI-MPR progressively increased from rete testis to cauda, whereas the percentages of immunostained spermatozoa for CD-MPR increased to a maximum when the gametes entered the epididymis.
We deduce that the observed changes in localization of both MPRs were the result of a redistribution, but not to an unmasking, given the failure of high ionic strength or trypsin treatment to induce these changes in spermatozoa.
The labeling observed on the dorsal region of the sperm head (for CI-MPR and CD-MPR) likely corresponds to the acrosomal region because the staining disappeared when cauda spermatozoa were incubated with the ionophore, A23187, under conditions in which the acrosome reaction occurs [25, 29]. It may also indicate that both receptors localize either in the gamete plasmalemma or the external, or both, but not the internal acrosome membrane. Likewise, the fact that no additional labeling was observed in gametes that had been permeabilized with Triton X-100 or digitonin may indicate the absence of MPRs in the internal acrosome membrane. Electron micrographs confirmed that MPRs reside at least in the sperm plasmalemma.
The redistribution of membrane proteins on the surface of cells is a prevalent feature of differentiation in a variety of cells, and two possible mechanisms for the relocalization of surface proteins have been postulated so far; passive diffusion or active translocation [30]. A temporal correlation between migration of proteins and proteolytic processing has also been described in spermatozoa [31]. Whether the redistribution of MPRs in spermatozoa occurs by one of these mechanisms remains to be studied.
Maturation of spermatozoa in epididymis involves remodeling of glycoproteins, lipids of the plasma membrane, or both, and redistribution of both to different domains of the plasmalemma. This remodeling includes modification and loss of pre-existing glycoproteins or addition of new glycoproteins from epididymal secretions, and exchange of lipids [32]. All these changes collectively contribute to sperm maturation in epididymis. The repositioning of MPRs may follow redistribution of lipids and other glycoproteins in the plasmalemma of spermatozoa as one step in the maturation process [33]. Since CD-MPR polymerizes to efficiently interact with ligands [34], it is possible that the redistribution may group MPRs in certain domains of the sperm membrane to improve their binding ability.
In somatic cells, both CI-MPR and the CD-MPR mediate the specific transport of acid hydrolases to lysosomes [1], but the presence of these receptors in sperm plasmalemma is an intriguing phenomenon because they are normally confined to intracellular compartments [1–14]. We have previously demonstrated that 
-mannosidase from epididymal fluid binds with high affinity and in saturable form to CI-MPR [24], and we have presented indirect evidence that suggests the existence of epididymal ligands for sperm CD-MPRs [35]. From those results, we could infer that spermatozoa use MPRs to transport enzymes, other glycoproteins, or both from the epididymal lumen to an extraepididymal milieu (e.g., the female genital tract). In support of this, the female reproductive tract provides an optimal pH for acid hydrolase activity.
The possibility that MPRs may interact with ligands that are present in the oocyte zona pellucida should not be discarded. An essential step in the process of mammalian fertilization is the recognition and binding of spermatozoa to the egg's zona pellucida. Although the complementary molecules involved in interaction of the opposite gametes are poorly characterized, a growing body of evidence suggests that carbohydrates mediate the interaction [36, 37]. If glycoproteins of the zona pellucida contain mannose-6-phosphate residues, a role for MPRs in the sperm-egg primary binding could be postulated.
Other roles for MPRs on sperm surface may, however, be considered. Because CI-MPR can also interact with IGF-II [8, 9] we could propose a function that is related to signal transduction in a relevant process, such as the acrosome reaction.
We have confirmed the existence of MPRs on sperm surface and suggested a possible relationship between their distribution and the maturation process of the gametes. From these results we postulate a novel role for MPRs, other than the selective transport of acid hydrolases to lysosomes.
|
ACKNOWLEDGMENTS |
|---|
|
FOOTNOTES |
|---|
1 Supported by grant PLI-323/98 from PLACIRH. 
2 Correspondence: Silvia A. Belmonte, Instituto de Histologia y Embriologia, Facultad de Ciencias Médicas, Universidad Nacional de Cuyo, Casilla de Correo 56, 5500 Mendoza, Argentina. FAX: 54 261 4494117;sbelmont{at}fmed2.uncu.edu.ar
Accepted: May 31, 2000.
Received: March 29, 2000.
|
REFERENCES |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
