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BOR - Papers in Press, published online ahead of print January 25, 2006.
Biol Reprod 2006, 10.1095/biolreprod.105.048587
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BIOLOGY OF REPRODUCTION 74, 923–930 (2006)
DOI: 10.1095/biolreprod.105.048587
© 2006 by the Society for the Study of Reproduction, Inc.

Research Article

Epididymal SPAM1 Is a Marker for Sperm Maturation in the Mouse1

Hong Chen , Genevieve Griffiths , Deni S. Galileo , and Patricia A. Martin-DeLeon 2 

Department of Biological Sciences, University of Delaware, Newark, Delaware 19716

ABSTRACT

Sperm adhesion molecule 1 (SPAM1), is a glycosyl phoshatidylinositol-linked sperm membrane protein that is dually expressed in testis and epididymis. Epididymal SPAM1 is secreted in all three regions of the epididymis in all mammalian species studied, including humans. It shares the same molecular mass and neutral hyaluronidase activity as the testicular and sperm isoforms that are responsible for the penetration of the cumulus during fertilization. Using wild-type (W/T) sperm and those from mice homozygous for either a null (Spam1–/–) or mutant Spam1 allele, which results in decreased mRNA and protein, we demonstrate that sperm binding of epididymal SPAM1 occurs in vitro after exposure to W/T sperm-free epididymal luminal fluid (ELF). Binding or adsorption that occurred after incubation at room temperature or 32°C was detected immunocytochemically and confirmed quantitatively using flow cytometry. The localization of SPAM1 on the plasma membrane of Spam1-null sperm mimicked that seen in the W/T. The remarkable increase in binding on W/T caudal sperm indicates that they are not fully saturated with SPAM1 during storage, and suggests that uptake of epididymal SPAM1 in vivo augments testicular SPAM1. Spam1-null sperm exposed to W/T ELF for 45–60 min during in vitro capacitation to allow epididymal SPAM1 binding showed a highly significant (P < 0.001) increase in cumulus penetration after 6–7 h compared to those incubated in ELF from null males. Similarly, the number of cumulus-free oocytes was also highly significantly greater (P < 0.001) than that for sperm capacitated in W/T SPAM1-antibody-inhibited ELF. Because epididymal SPAM1 uptake significantly increases cumulus penetration, we conclude that it is a marker of sperm maturation.

epididymal luminal fluid, epididymis, GPI anchor, in vitro fertilization, male reproductive tract, secretory protein, Spam1-null sperm, sperm capacitation, sperm hyaluronidase activity, sperm maturation

INTRODUCTION

Sperm maturation in mammals involves an intimate association or interaction between sperm and the epididymal epithelium. Whereas interactions with the proximal epididymis are essential for the acquisition of the fertilizing ability of sperm [1–3], the distal epididymis is thought to function primarily in temperature-regulated sperm storage [4]. Under the control of androgens, the epididymis synthesizes and secretes proteins, some of which become associated with the sperm plasma membrane [5–7]. Evidence suggests that certain epididymal glycosidases are adsorbed or incorporated into the sperm plasma membrane, where they are later involved in the steps of fertilization [8–10].

Sperm adhesion molecule 1 (SPAM1), the major testicular hyaluronidase, is among the glycosyl phosphatidylinositol (GPI)-linked glycoproteins secreted by the epididymal epithelium [11–13]. Originally thought to be testis-specific [14–16], SPAM1 has now been shown to be synthesized in the epithelium in all three regions of the epididymis, and is conserved in at least five mammalian species, as recently reviewed [17–19]. Based on its widespread expression in the epididymis, it is likely to play a role in both sperm maturation and storage. In the bull and mouse, it has been shown to be secreted in the epididymal luminal fluid (ELF) [11–13]. Consistent with the presence of SPAM1 in the ELF is the finding that it is secreted in conditioned media of cultured murine epididymal epithelial cells [11]. Importantly, it has been demonstrated that the secreted form of SPAM1 maintains an intact GPI anchor and is found in both the soluble and insoluble (epididymosomes) fractions of the ELF [12].

As a GPI-linked glycosidase, epididymal SPAM1 may be secreted for the modification of the sperm plasma membrane. In its capacity as a glycosidase, epididymal SPAM1 may be loosely adsorbed on the sperm surface. Unlike epididymal proteins such as CD52 [7] that are not expressed in the testis, the modification of sperm by proteins that are dually expressed in testis and epididymis may be the augmentation or replacement of the existing testicular isoforms via the lipid anchor. Replacement of the testicular isoform by an epididymal isoform is seen for Clusterin, which, like SPAM1, is expressed in both the testis and the epididymis [20, 21]. It should be noted that testicular and epididymal isoforms of SPAM1 share the same molecular mass (67 kDa) [12]. However, in 2-D gel electrophoresis, testicular SPAM1 has a broader isoelectric point range, 6.6 to 9.0, compared to epididymal isoforms (7.3 to 9.0). Also, epididymal and sperm SPAM1 share identical N-linked carbohydrate moieties that are not present in testicular SPAM1 [12].

The goal of this investigation was to determine in the murine model whether epididymal SPAM1 1) binds to sperm in vitro, 2) augments testicular SPAM1, and 3) whether its binding impacts sperm function via cumulus penetration. The challenge in detecting sperm binding of epididymal SPAM1 on sperm is distinguishing it from testicular SPAM1. To circumvent this problem, we have used Spam1-null (–/–) mice, making it possible to observe epididymal SPAM1 binding in vitro without the confounding presence of the testicular isoform. Although SPAM1 plays multiple essential roles in fertilization [22], Spam1-null mice have been shown to be fertile with reduced cumulus penetration capabilities [23]. This lack of a major effect on fertility in Spam1-null mice has been attributed to the presence of related rodent-specific reproductive hyaluronidases, HYAL5 and HYALP1, which have recently been shown to have redundant and overlapping functions with SPAM1 [24]. This redundancy is not surprising, considering the essential roles played by SPAM1.

Additionally, we have taken advantage of an available murine Spam1 mutation that is known to result in a decreased level of the steady-state RNA as well as the protein [16, 25]. This mutation, which is carried on a Robertsonian (Rb) translocation, Rb(6.16)24Lub, leads to subfertile sperm that are not fully saturated with SPAM1 because they have only 70% of the wild-type (W/T) level [25]. Thus, they are likely to accommodate exogenous SPAM1 on their surface and can therefore be used for uptake of epididymal SPAM1 in in vitro binding assays. Our results show that epididymal SPAM1 binds to sperm in vitro and augments testicular SPAM1 in both mutant and W/T ICR mice. Furthermore, in vitro binding of epididymal SPAM1 significantly increases the ability of Spam1-null sperm to penetrate the cumulus cells surrounding the oocytes in an in vitro fertilizing assay.

MATERIALS AND METHODS

Animals and Reagents

The studies were approved by the Animal Care Committee at the University of Delaware and conform to the Guide for the Care and Use of Laboratory Animals, published by the National Institutes of Health (publication 85–23, revised 1985). Spam1–/– mice were obtained from the laboratory of Dr. Tadashi Baba (University of Tsukuba, Tsukuba Science City, Ibaraki, Japan), where they were generated [23]. We confirmed their Spam1-deficiency by PCR genotyping using primers described by Baba et al. [23], Northern analysis of testicular RNA, and by immunocytochemistry. Sexually mature 3- to 6-mo-old Rb(6.16)24Lub Robertsonian translocation-bearing mice were purchased from The Jackson Laboratory and bred in our colony, and outbred ICR W/T (Spam1+/+) mice were obtained from Harlan Sprague-Dawley Inc. The Rb(6.16)24Lub mice were previously shown to have a gross genomic alteration at the Spam1 locus and 11 point mutations scattered in the 5' and 3' UTRs and the coding region, where one leads to the replacement of a conserved residue [25]. Consequently, the testicular Spam1 mRNA level is 25–30% of the congenic W/T [16] and the sperm have only 70% of the W/T level of SPAM1 [25]. All chemicals were purchased from Sigma Chemical Company or Fisher Scientific Company unless otherwise specified.

Collection of Luminal Fluid and Sperm

Luminal fluid. In each experiment luminal contents were collected from all three epididymal regions pooled from three 3- to 6-mo-old ICR W/T or Spam1–/– mice, using a method similar to that used by Zanich et al. [26], by slicing the whole tissue after removing excess blood and fat. The tissue was placed in 2 ml PBS containing 1 mM PMSF, a protease inhibitor, and the sperm allowed to swim out for 10 min after slicing the tissue and after 5 min of gentle agitation. The released sperm and fluid were removed from the tissue fragments in the dish and the supernatant centrifuged at 1000 x g for 5 min to remove the sperm. The resulting luminal fluid was further clarified by centrifugation at 3500 x g for 20 min to pellet cellular fragments, and was shown to be sperm-free after microscopic examination.

Sperm. To collect sperm from ICR and Rb(6.16) Spam1 mutants, cauda epididymides were finely sliced in PBS containing 1 mM PMSF and gently shaken. Sperm were allowed to disperse and the suspension separated from the tissue fragments by gravity settling. The suspension was then centrifuged at 1000 x g for 3 min to pellet the sperm without breaking their membranes. The pellets were washed twice with PBS containing 1 mM PMSF. In the case of Spam–/– mice, sperm were obtained from all three regions of the epididymides and pooled, and only caudal sperm were collected from W/T and Rb(6.16) mice.

In Vitro Binding of Epididymal SPAM1

Binding was assayed after sperm from the three strains of mice were coincubated individually with sperm-free dilute luminal fluid (LF; protein concentration 1.0 mg/ml) at room temperature (RT) or 32°C for 1 h, after washing sperm in PBS containing 1 mM PMSF. The control consisted of sperm that were incubated only in PBS and 0.1% (w/v) BSA, after washing in PBS containing PMSF. During coincubation, the dish was gently shaken to prevent the cells from settling at the bottom.

Following coincubation, sperm were washed to remove unbound protein and then processed for immunochemical detection of bound epididymal SPAM1, live or fixed. Fixation was done in 1.5% paraformaldehyde for 1 h at RT. After washing and blocking in 2% BSA in PBS, sperm were treated using a rabbit antipeptide mouse SPAM1 antiserum generated from a C-terminal 15-mer (#381- 395) oligopeptide (custom-made by Zymed; diluted 1:400). This antiserum was shown to be specific for SPAM1, with peptide blocking showing that the signal is specific [27]. The secondary antibody was fluorescein isothiocyanate (FITC)-conjugated goat anti-rabbit IgG (diluted 1:320).

Flow Cytometry and Immunofluorescence

Flow cytometric analysis. Flow cytometry was performed to quantify the amount of SPAM1 on the sperm surface. After several washes of the cells, fluorescence was measured (in samples of 104 sperm) using a FACScalibur (Becton-Dickinson) flow cytometer with a Cell Quest software package. The FACScalibur instrument uses an argon laser at 488 nm with detectors for FITC. Prior to preparing for cytometric analysis, an aliquot of the Spam1-null sperm suspension was used for indirect immunofluorescence of bound SPAM1.

Indirect immunofluorescence. Caput, corpus, and caudal sperm were studied from Spam1-null males. Sperm recovered from the three regions of the epididymal ducts were fixed and processed as above after exposure to LF or PBS with BSA, in the case of the controls. The sperm were then mounted on slides in {rho}-phenylenediamine antifade with 1.5 µg/ml of 4',6-diamidino-2-phenylindole (DAPI) for standard fluorescence microscopy, or mounted without the DAPI staining for confocal microscopy. The DAPI-stained specimens were examined using a Zeiss Axioskop (Carl Zeiss) with the appropriate FITC filter set, and images were obtained with the use of a charge-coupled device-cooled camera and IPLab software.

Cumulus Penetration of Spam1-Null Sperm after In Vitro Exposure to Epididymal LF Untreated or Treated with SPAM1 Antibodies

Egg preparation. For each experiment four 6- to 8-wk-old superovulated ICR females were used. Superovulation was achieved by intraperitoneal injections of 7.5 I.U. eCG and hCG given 48 h apart. Females were sacrificed 15–16 h after HCG and their eggs collected from the oviductal ampullae in prewarmed human tubal fluid (HTF; Specialty Media) under mineral oil and incubated at 37°C in 90% N2, 5% CO2, and 5% O2.

ELF and Caudal Sperm Preparation

In each experiment, two Spam1-null and two W/T ICR 3- to 4-mo-old males were used. One hour before egg retrieval, males were sacrificed and caudal epididymides were removed and minced in 800 µl of HTF in a 35-mm petri dish. The supernatants were collected from each of the two pairs of minced caudal epididymides after 10 min when sperm had swum out, and centrifuged at 300 x g for 5 min to ensure that sperm membrane would not be damaged. The resulting supernatant was centrifuged at 16100 x g for 10 min twice to obtain sperm-free ELF as confirmed by microscopic examination. Total protein concentration in each sperm-free ELF sample was determined using a biocinchoninic acid kit (Pierce) using different concentrations of BSA as standards.

SPAM1 Uptake In Vitro During Capacitation

To determine the impact of in vitro binding of epididymal SPAM1 on cumulus penetration, caudal sperm were capacitated by incubation for 45 min in a 0.2-ml drop of ELF (1.0–2.0 mg/ml protein) in HTF at 37°C under 90% N2, 5% CO2, and 5% O2. There were four groups: Group 1 (G1) was a positive control consisting of W/T caudal epididymal sperm in W/T ELF; group 2 (G2) was Spam1-null caudal epididymal sperm in W/T ELF with preimmune rabbit serum (1:100); group 3 (G3) was Spam1-null caudal epididymal sperm in W/T ELF with anti-SPAM1 rabbit antiserum (1:100) for 30 min; and group 4 (G4) was a negative control, Spam1-null caudal epididymal sperm in Spam1-null ELF. (This anti-SPAM1 antiserum is an antipeptide antibody which has been shown to be specific for SPAM1 [25] in a number of studies, including assays using peptide inhibition.) At the end of capacitation, an aliquot of the Spam1-null sperm from G2 that was incubated with W/T LF with preimmune serum was processed for immunocytochemistry to determine whether binding had occurred.

In vitro fertilization assay. Three experiments were performed. The capacitated caudal epididymal sperm were counted and then used to inseminate cumulus-intact eggs at a final concentration of 3.0 x 105 sperm/ml in a 0.2-ml drop of HTF medium. The eggs and sperm were then incubated for 6–7 h at 37°C under 5% CO2 in 90% N2 and 5% O2.

Observation of the stages of cumulus dispersal. The progress of cumulus cell removal was categorized in four stages, based on the appearance of the cumulus masses after microscopic observation. Stage 1 had eggs with tightly packed cumulus cells (such that the eggs were fully occluded); stage 2 had cumulus masses in which the eggs could be visualized with loosely associated cumulus cells; stage 3 had eggs in which most of the cumulus cells had been dispersed from the masses; and stage 4 had eggs with complete dispersal of the cumulus cells. Eggs were monitored during incubation after the first hour of insemination and the stages recorded. Subsequently, eggs were observed and stages recorded every 2 h up to 7 h. An additional control experiment was conducted to determine whether the very small volume of ELF added along with the sperm could by itself have a significant impact on penetration of the cumulus. Thus, cumulus dispersal in the presence of HTF only was compared to that in sperm-free W/T LF collected in HTF for up to 6 h.

Statistical Analysis

Fisher exact test was used to compare the efficiency of cumulus penetration when null sperm were capacitated in HTF containing W/T LF in which preimmune rabbit serum was present to those from 1) W/T LF containing the anti-SPAM1 antipeptide rabbit antiserum, and 2) null LF.

RESULTS

When pooled caput, corpus, and cauda sperm from Spam1-null mice were incubated at room temperature in W/T LF for 1 h and processed for the immunochemical detection of SPAM1, binding of epididymal SPAM1 was detected. Epididymal SPAM1 was found to be uniformly distributed on the acrosomal region of the head and on the midpiece of the tail, as seen in W/T caput mouse sperm [28] (Fig. 1. IB and IIB). It was also seen on the anterior head (in a crescent shape over the acrosome) and the midpiece, which are patterns seen in corpus and cauda W/T sperm [28] (Fig. 1, I, C and D, and IIC). The staining was particulate or patchy, indicating movement of epididymal SPAM1 on the plasma membrane, as occurs in W/T sperm [26, 28]. Thus, the distributions of epididymal SPAM1 on null sperm mimic all the patterns for WT sperm. Control samples that were incubated with PBS and those in which the antibody was blocked by peptide inhibition showed only background fluorescence (Fig. 1, IA and IIA).


Figure 1
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  		FIG. 1. I) Localization of in vitro binding of epididymal SPAM1 on Spam1-null sperm using confocal microscopy. Control null sperm incubated in PBS are shown in A. For treated sperm, the staining patterns for caput are seen in the two B panels, and for corpus and cauda in C and D, with the arrow showing regionalization seen in mature W/T sperm. Asterisks show epididymal SPAM1 on the midpiece of the tail. II) Localization of epididymal SPAM1 on null sperm after binding and imaging with the standard fluorescence microscope. After immunocytochemistry, cells are counterstained with DAPI (blue) to show nuclear DNA. A shows a sperm incubated in PBS + BSA; the green staining (FITC) in B for a sperm incubated in W/T ELF shows the typical uniform distribution seen in caput sperm; and C shows a corpus or caudal sperm with regionalization of the stain on the anterior head and the midpiece. In C, asterisks show heads without epididymal SPAM1. After binding, the protein is seen on the anterior head and midpiece (arrows labeled A and B). Original magnification I x630, II x1000.

To confirm and quantify the binding detected on Spam1-null sperm using immunocytochemistry, flow cytometry was performed on 30000 sperm incubated in ELF and PBS + BSA (treated and control). An arithmetic histogram displays a unimodal distribution for the control, with a mean of 11.95. However, the treated groups showed higher fluorescence (Fig. 2, B and C; right shift of the peak) with means of 25.53 and bimodal sperm populations. The heterogeneity of the populations incubated in ELF, which was not seen in those incubated in PBS + BSA (Fig. 2A), suggests that there are subsets of sperm exposed at room temperature with and without epididymal SPAM1. Repeat experiments (Fig 2, B and C) showed similar results, providing direct evidence that epididymal SPAM1 can be acquired by sperm in vitro from dilute unfractionated ELF.


Figure 2
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  		FIG. 2. Flow cytometric analysis of Spam1-null sperm after binding of epididymal SPAM1 at room temperature and staining with SPAM1 antipeptide/FITC. Control sperm incubated in PBS are seen in A and show a unimodal curve, whereas B and C show independent samples of sperm incubated in ELF in which the protein concentration is 1.0 mg/ml. For these two samples, there is increased fluorescence (right shift of peaks), and the distribution is heterogeneous.

To ask whether sperm with SPAM1 originating in the testis are also capable of binding epididymal SPAM1 in vitro, experiments were performed with Rb(6.16)-bearing caudal sperm at 32°C. Flow cytometric analysis of 50000 cells showed an increase in fluorescence intensity in sperm incubated with W/T LF, for which the mean was 559.24, compared to those incubated in PBS containing BSA, for which the mean was 362.25 (Fig. 3A). Importantly, uptake at physiological temperature showed a more robust increase in the intensity of the binding, and the distribution was normal rather than heterogeneous, as seen for null sperm at RT. To test whether W/T caudal sperm had reached maximum saturation levels of SPAM1, populations of 50000 W/T ICR caudal sperm were analyzed after exposure, at physiological temperature, to either PBS containing BSA as a control or W/T LF. The results also showed a marked increased in the fluorescent signal on sperm incubated in W/T LF, for which the mean was 555.66, compared to 297.56 seen in the control (Fig. 3B).


Figure 3
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  		FIG. 3. The results of flow cytometric analysis of caudal sperm from mice bearing the Rb(6.16) Robertsonian translocation (A) and from W/T ICR males (B) immunostained for SPAM1 after incubation at 32°C in W/T LF (1–1.5 mg/ml) or BSA (control) for 1.5 h. Dotted lines indicate control sperm, and dark lines show sperm incubated in ELF. Sperm exposed to ELF showed increased fluorescence intensities as compared to the BSA control, indicating that they had not reached maximum SPAM1 saturation levels in the cauda epididymis.

To determine if the binding of epididymal SPAM1 in vitro has an impact on the ability of sperm to penetrate the cumulus-oocyte complex, freshly superovulated oocytes were divided into four groups and inseminated with sperm that were capacitated under different conditions. Capacitated sperm processed for immunocytochemistry after exposure to medium containing LF from Spam1+/+ males showed binding of SPAM1 similar to that seen in LF diluted with PBS (data not shown). Only clutches of eggs that were normal in appearance were analyzed. In 3 experiments, approximately 431 oocytes in the four groups were monitored to determine the progress in the removal of cumulus cells. Clutches exposed to HTF containing W/T LF in equivalent amounts to that used in the test group, and those exposed to HTF alone, failed to produce oocytes at stage 4 after all periods of observations, and had similar proportions of stage 3 oocytes up to 6 h after insemination. This indicates that the penetration of the cumulus is essentially a result of the hyaluronidase activity from sperm that have bound SPAM1 and not from activity in the very small volumes of LF accompanying the sperm.

The numbers of oocytes analyzed in the four groups (G1, G2, G3, and G4) were 81, 129, 134, and 137, respectively. Figure 4 shows the progress in the rate of cumulus penetration at the different stages for each of the four groups over time. The rates were different for the groups, with the highest rate of progress seen for G1, which is the positive control W/T sperm. There was a marked difference in the progress made by oocytes inseminated with null sperm that were exposed to W/T LF containing the preimmune rabbit serum (G2) and those inseminated with sperm that were capacitated in W/T LF with the anti-SPAM1 rabbit antiserum (G3). In the latter group, approximately 20% of the oocytes were at stage 1 at 5 h after the initiation of incubation, whereas this number was 0% for sperm capacitated in preimmune serum. On the other hand, G3 was similar to G4 in the number of oocytes in stage 1 at this time (Fig. 4).


Figure 4
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  		FIG. 4. The progression of penetration of the cumulus-oocyte complexes shows rates that are different for the four groups. G1, which is the positive W/T control, W/T sperm capacitated in W/T ELF, had a progression rate that was similar to G2, which had null sperm capacitated in W/T ELF. The latter was markedly different from G3 and G4, which both had oocytes in stage 1 and the majority in stage 2 after 5 h incubation. The antibody inhibition in G3 appeared to have stalled the progression compared to G4, as the level of stage 4 oocytes remained virtually unchanged between 1 and 5 h.

Figure 5 shows the percentages of cumulus-free oocytes (stage 4) at 1 and 6–7 h for the four groups. It demonstrates that at 1 h postinsemination, 17.3%, 18.6%, 6.7%, and 10.9% of the oocytes in G1, G2, G3, and G4, respectively, are cumulus-free, as compared to 79%, 75.2%, 30.6%, and 43% at 6–7 h. Thus, null sperm in W/T ELF showed a similar penetrating ability to that of W/T sperm in W/T ELF. Using Fisher's exact test, the rate of complete dispersal of cumulus cells for null sperm in G2 compared to those in G3 at 1 h postinsemination is highly significantly different (P < 0.01). At 6–7 h postinsemination, the difference between these groups is even more highly significant (P < 0.001). Also at 6–7 h, the difference between the percentage of cumulus-free cells for G2 and G4 is highly significant (P < 0.001). Although for both the 1 and 6–7 h postinsemination periods, G4 had higher percentages of cumulus-free oocytes than G3, the difference at 1 h was not significant (P > 0.05), and at 6–7 h, it was only marginally significant (P < 0.05). The difference between these groups could be because of steric hindrance of the antibody affecting other hyaluronidases on the sperm surface.


Figure 5
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  		FIG. 5. Complete dispersal of cells from the cumulus masses in oocytes incubated for 1 to 6–7 h with W/T or null sperm exposed during in vitro capacitation to ELF from W/T or null mice. In G1, W/T sperm were capacitated in W/T LF; G2 is null sperm in W/T ELF in the presence of preimmune rabbit serum; G3 is null sperm in W/T ELF in the presence of SPAM1 antipeptide rabbit antiserum; and G4 is null sperm in null ELF. Highly significant differences are seen for G2 compared with G3 at 6–7 h, ##P < 0.0001; G2 compared with G4 at 6–7 h, ***P < 0.0001; G2 compared with G3 at 1 h, **P = 0.0047, P < 0.01; and G3 compared with G4 at 6–7 h, *P = 0.0437, P < 0.05. There were no significant differences for G3 and G4 at 1h, P = 0.2857, P > 0.05, and for G2 and G4 at 1 h, P = 0.0851, P > 0.05.

DISCUSSION

SPAM1 is a widely conserved mammalian sperm membrane protein [29] known to play multiple essential roles in fertilization, including cumulus penetration via hyaluronidase activity, zona binding, and the signaling involved in acrosomal exocytosis [22, 30]. Best known for its hyaluronidase activity, SPAM1 possesses both acidic and neutral activity. These are respectively found in the soluble form of the sperm protein that is released after the acrosome reaction [31] and the insoluble form that is present on the sperm plasma membrane [31]. Activity of the insoluble protein at neutral pH distinguishes SPAM1 from other hyaluronidase family members and is responsible for the dispersion of cumulus cells surrounding the oocyte [32, 33]. Acidic hyaluronidase activity in the soluble protein, which results from cleavage of the carboxy terminus [31, 34] and which in primates has a molecular mass of 53 kDa instead of 64 kDa (seen for the membrane insoluble form [31]) may be involved in the penetration of the zona and the perivitelline space, both of which contain hyaluronic acid [35–37].

Like sperm SPAM1, epididymal SPAM1 also exists in a soluble and an insoluble or epididymosomal form; however, both of these forms have the same molecular mass in the mouse [12]. This indicates that the murine epididymal soluble form is unlikely to result from proteolytic cleavage, as is the testicular soluble form [31, 34]. Further, epididymal SPAM1 has neutral hyaluronidase activity [11, 17, 18, 27] similar to testicular and sperm SPAM1, but lacks the acidic activity seen after the acrosome reaction [27]. It is noteworthy that in rats, in which epididymal SPAM1 undergoes endoproteolytic cleavage into two subunits, unlike the testicular isoform, the subunits are of similar sizes to the sperm-bound forms [18]. These observations, which reveal overlapping features for epididymal and sperm SPAM1, suggest a common source for at least a fraction of both of these isoforms.

Our results in the present study using both immunocytochemistry and flow cytometry provide proof of principle that Spam1-null sperm are able to acquire epididymal SPAM1 after in vitro exposure to dilute W/T ELF. This demonstration that epididymal SPAM1 binds to sperm in vitro suggests that binding occurs in vivo. The binding of epididymal SPAM1 to sperm is consistent with the finding that the protein is released with its lipid anchor intact and that it exists in the form of epididymosomes [12], vesicles that are similar to prostasomes and are known to serve as vehicles for the transfer of epididymal proteins [7, 38, 39]. It is known that several mammalian GPI-linked molecules can transfer spontaneously to plasma membranes in vitro and in vivo [40], and that both the soluble and insoluble forms can bind to cells [39]. As might be expected, because of increased membrane fluidity with increasing temperature, transfer was more pronounced for the Rb(6.16) and the W/T sperm at 32°C compared to the null sperm exposed at RT.

Immunocytochemistry showed that Spam1-null caput and caudal sperm that bind epididymal SPAM1 at RT displayed the protein over the acrosomes on the anterior head, the posterior head, or the midpiece of the flagella. In some sperm, there was a uniform distribution on the head. These localizations are typical of mature and immature W/T mouse sperm, respectively [28]. It should be noted that mouse SPAM1 has recently been shown to be a lipid raft-associated protein and that under noncapacitating conditions there is stability of the raft microdomains to which GPI-linked proteins are attached [41]. Thus localizations of epididymal SPAM1 binding on null sperm similar to the W/T pattern are consistent with uptake at 23°C (RT) or under noncapacitating conditions. Also, the protein staining after uptake was patchy or particulate, indicating its movement on the plasma membrane, as occurs in W/T sperm [26, 28]. Therefore, the localization and disposition of the protein after uptake mimic the in vivo situation and suggest that epididymal SPAM1 contributes to sperm phenotype. More importantly, exogenously-introduced GPI-anchored molecules are known to become functional in the target cell once they have acquired a distribution similar to that of endogenous GPI-anchored proteins [39].

It was important to determine whether sperm with testicular SPAM1 and, more importantly, W/T sperm are capable of binding epididymal SPAM1. Our results show that caudal sperm from both Rb(6.16)-bearing mice, in which testicular and sperm SPAM1 are respectively 43% and 70% of that in W/T [25, 42], and W/T mice are able to significantly bind SPAM1 in vitro at 32°C, as reflected by flow cytometric analysis. The increase in fluorescence intensity after binding of epididymal SPAM1 to W/T sperm suggests that, unlike Clusterin, for which the epididymal form of the protein primarily replaces the testicular form [21], epididymal SPAM1 augments the testicular isoform. However, this augmentation does not exclude the possibility of replacement of preexisting testicular SPAM1 on the sperm surface.

Our finding for W/T sperm also suggests that these sperm do not reach maximal saturation levels of SPAM1 in the cauda. This is likely to be because of the tight packing of the sperm in this organ. Our previous observations from immunohistochemical studies of sections of the rat cauda reveal a difference in the intensity of SPAM1 (2B1) staining, depending on the location of the sperm [18]. Tightly packed in the cauda, sperm positioned near the epithelial lining of the lumen shared the same intensity of staining as the stereocilia of the epithelial cells, and were far more immunopositive than those in the interior of the lumen [18]. In the in vitro assay performed in the present study, in which there were dilute suspensions of sperm, there would be greater accessibility of the sperm membrane, and this facilitates binding and accounts for the increase in the intensity of SPAM1 staining in the group incubated in ELF, compared to those incubated in PBS + BSA. It should be noted that during storage in vivo, thorough mixing of caudal sperm with the epididymal secretion occurs by peristaltic contractions over time and might be required for maximum levels of saturation of SPAM1 on the sperm surface.

In vitro binding of epididymal SPAM1 by caudal sperm is supported by a number of studies providing indirect evidence for sperm acquisition in vivo [19]. An improvement in the fertilizing abilities of mouse sperm bearing the Rb(6.16) or Rb(6.15) translocation carrying Spam1 mutations was seen when sperm from heterozygotes were physiologically aged by sexual rest [42–45]. This improvement was reflected in changes in the transmission ratio distortions seen in male carriers of these translocations [43–45]. A more drastic improvement was seen in males carrying the Rb(6.16) translocation when sperm were subjected to prolonged storage in the cauda after surgical ligation of the corpus epididymis: the distortion disappeared and sperm with the mutation and W/T genotypes were able to fertilize in comparable frequencies [46]. Studies on sperm from W/T males also support acquisition of SPAM1 from the ELF, because caudal mouse sperm were shown to have a >4-fold increase of SPAM1 as well as hyaluronidase activity compared to caput sperm [47]. Other species such as stallions [48] and bulls [13] also demonstrate a progressive increase in sperm SPAM1 during epididymal transit.

In the present study, in vitro binding of epididymal SPAM1 during capacitation was shown to significantly increase the ability of Spam1-null sperm to disperse the cumulus-oocyte complexes, compared to those in null ELF or SPAM1-antibody-inhibited W/T ELF. In W/T ELF, null sperm and W/T sperm had similar cumulus-penetrating abilities. This suggests that the acquisition of epididymal SPAM1 is associated with an increase in neutral SPAM1 hyaluronidase activity that is responsible for cumulus penetration [22]. It must be noted that this increase in enzyme activity is likely to be attributed not only to SPAM1 but also to the hyaluronidase activity of HYALP1 and HYAL5. These two rodent-specific functional hyaluronidases encoded by closely linked family members of Spam1, Hyalp1 and Hyal5, were shown to be GPI-linked proteins that have overlapping functions with SPAM1, including epididymal expression [24], and Hyal5 was thought to be responsible for the fertilizing ability of Spam1-null sperm [23]. The significant reduction of cumulus penetration in the presence of SPAM1-antibody-inhibited W/T ELF in this study suggests that epididymal SPAM1 is likely to play a predominant role in mouse epididymal hyaluronidase activity in vitro. It should be noted that the SPAM1 antibody used for the inhibition was made from a unique sequence in the C-terminus and should not have directly inhibited the HYALP1 and HYAL5 activity, although steric hindrance of these and other surface proteins cannot be ruled out. It should also be noted that by and large null sperm incubated in SPAM1-antibody-inhibited ELF (G3) and Spam1-null ELF (G4) gave results that were not markedly different. In summary, epididymal SPAM1 is a secretory epididymal protein that can be acquired on the surface of mouse sperm and that increases their fertilizing competence. It is thus a marker of sperm maturation.

Under in vitro conditions, it is known that CD59, another GPI-linked protein, is incorporated into U937 monocytic cells at 37°C after 2 h exposure when the protein equilibrates and becomes redistributed on the detergent-resistant membrane complexes and induces calcium signaling [39]. In the present study, biological activity of bound epididymal SPAM1 provides evidence for its correct integration in the sperm plasma membrane. During the 6–7 h incubation of gametes in this study, it is likely that redistribution and equilibration of epididymal SPAM1 would ensure that it becomes positioned in the appropriate location to perform at least the first step in fertilization. Interestingly, Sleight et al. [41] recently showed that membrane changes during capacitation initiate signaling events. In this respect, it is possible that binding of epididymal SPAM1 would not only increase cumulus penetration, but could also increase signal transduction and intracellular calcium, in which both human and mouse SPAM1 have been shown to be involved [28, 49]. Thus, studies are underway to determine the impact that in vitro binding of epididymal SPAM1 has on other steps in the fertilization process and on overall sperm fertilizing ability. Additionally, we are also investigating the role of the insoluble epididymosomes and the soluble fraction of the ELF in the transfer of epididymal SPAM1 to the sperm surface.

ACKNOWLEDGMENTS

We are grateful to Dr. Tadashi Baba for supplying us the Spam1-null mice.

FOOTNOTES

1 Supported by National Institutes of Health grant RO1 HD38373 to P.A.M-D. Back

2 Correspondence: Patricia A. Martin-DeLeon, Department of Biological Sciences, University of Delaware, Newark, DE 19716. FAX: 302 831 2281; pdeleon{at}udel.edu Back

Received: 20 October 2005.

First decision: 21 November 2005.

Accepted: 23 January 2006.

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