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RESEARCH |
1 Institute of Zoology, Regent's Park, London NW1 4RY, UK2 Academic Unit of Reproductive and Developmental Medicine, The University of Sheffield, Level 4, The Jessop Wing, Tree Root Walk, Sheffield S10 2SF, UK3 The Royal Veterinary College, Royal College Street, London NW1 0TU, UK
Correspondence should be addressed to W V Holt; Email: bill.holt{at}ioz.ac.uk
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Abstract |
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 Top Abstract IntroductionResultsDiscussionMaterials and MethodsDeclaration of interestAcknowledgementsReferences |
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Introduction |
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TopAbstractIntroductionResultsDiscussionMaterials and MethodsDeclaration of interestAcknowledgementsReferences |
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Co-culture of spermatozoa with OEC has repeatedly been shown to prolong sperm viability in some species, including pigs. In this laboratory, we have consistently demonstrated the enhancement of boar sperm viability by a soluble fraction of oviductal apical plasma membrane (sAPM) proteins (Elliott et al. 2001, Fazeli et al. 2003, Holt et al. 2005), derived from APM vesicles, as originally described by Smith & Nothnick (1997). In bioassays involving incubation of boar spermatozoa with sAPM for 24 h at 39 °C, sperm viability was enhanced in a dose-dependent manner (Fazeli et al. 2003). Moreover, the enhancement effect was abolished if sAPM proteins had been denatured (Fazeli et al. 2003). These results confirmed that one or more proteins within the sAPM contribute to the enhancement of sperm viability.
However, sAPM represents a complex mixture of proteins, any of which might be active at low concentration. This might explain why a relatively high concentration of sAPM (200 µg/ml) is necessary in order to demonstrate the viability enhancing effect. In a previous publication (Holt et al. 2005), we described in detail the preparation method for sAPM and demonstrated that we could distinguish oviduct epithelium surface-exposed proteins by the use of an in situ biotinylation technique. In the current study, we have again used in situ biotinylated oviductal sAPM preparation and have tracked the binding of this protein preparation to the surface of co-incubated spermatozoa. We have also studied the sperm binding topology in some detail using confocal microscopy and used proteomic analyses to identify heat shock 70 kDa protein 8 (HSPA8 previously known as HSPA10, HSC71, HSC70 and HSP73) as a probable significant functional component of the sAPM. Ultimately, using bovine recombinant HSPA8 protein, we replicated the sperm-survival enhancement effect of sAPM, not only in boar spermatozoa but also in bull spermatozoa.
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Results |
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TopAbstractIntroductionResultsDiscussionMaterials and MethodsDeclaration of interestAcknowledgementsReferences |
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Examination of live images of the sperm–bead interactions revealed that unexpectedly small numbers of spermatozoa became bound to the bead surfaces. Even those that became bound tended to be released again after a few seconds. This lack of firm binding was not caused by any shortage of spermatozoa in the surrounding media and may therefore indicate that some degree of sperm selection was involved. Visual estimates nevertheless showed that the number of spermatozoa associated with sAPM-treated beads (regardless of whether the sAPM had been biotinylated ex-situ or in-situ) was about fivefold greater than became bound to untreated beads (five sperm/bead for ex-situ labelled sAPM; 4.4 sperm/bead for in situ labelled sAPM; control values were 0.75 and 0.82 sperm/bead respectively: P=0.02). As it was not possible to examine the whole of the bead surface by video microscopy, some sperm-bead preparations (made using live spermatozoa labelled with SYBR14) were fixed and examined by confocal microscopy to check the uniformity and low level of binding. Slices through an sAPM-treated bead and a control bead are shown in Fig. 1A and B respectively. The confocal microscopy confirmed that relatively few spermatozoa were bound to the bead surfaces.
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Identification of in situ biotinylated oviductal epithelial proteins that bind to spermatozoa
Immunoblotting of the combined solubilized sperm membrane protein fraction with sperm surface-bound biotin-labelled sAPM demonstrated specific binding of a protein fraction of 
70 kDa. In order to identify this protein(s), the corresponding protein band from a series of replicate SDS-PAGE gels was excised for proteomic analysis.
Figure 2A presents a PAGE gel showing that while many proteins were contained within the in situ biotinylated sAPM fraction, after incubation of the sAPM with spermatozoa only one band (MWt 70 kDa) was strongly detectable on the blot (Fig. 2B, Lane 1). Lane 2 in this figure shows that when spermatozoa were incubated with APM (i.e. non-solubilized biotinylated APM), they bound not only this protein band, but also another with a molecular weight of about 97 kDa (Fig. 2B, lane 2).
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This outcome was pursued further by repeating the bioassays, but using lower concentrations of HSPA8 (0, 0.25, 0.5, 2 and 8 µg/ml). As an additional comparison, the spermatozoa were also incubated in the presence of 200 µg/ml sAPM to evaluate the effectiveness of HSPA8 against the original oviductal preparation. The beneficial effects of HSPA8 were again evident at 2 µg/ml (P=0.0037; F1/84=8.89), as in the earlier experiment but to a lesser extent than both 0.25 and 0.5 µg/ml (P=0.001) for the comparison between TLP and 0.25 µg/ml and P<0.0001 for the specific comparison between TLP and both concentrations together). 8 µg/ml had no beneficial effect on maintaining sperm survival and it was concluded that the optimal concentration must be lower than 2 µg/ml (Fig. 3B).
A further series of bioassays was therefore undertaken using an even lower range of HSPA8 concentrations (0.1, 0.5 and 4 µg/ml); and sAPM (200 µg/ml) treatment was also included (Fig. 3C). Beneficial effects on sperm survival over a 24 h period were evident with HSPA8 at 0.1 µg/ml (P=0.005) and 0.5 µg/ml (P=0.0001). In this experiment, there was no beneficial effect observed with HSPA8 at 4 µg/ml. As 0.5 µg/ml HSPA8 had the greatest beneficial effect on sperm viability (Fig. 3A and C), it was concluded that this must be the optimal concentration of HSPA8.
In vitro fertilization assays
Spermatozoa were briefly (10 min) exposed to 0.5 µg/ml recombinant bovine HSPA8 protein before being diluted (1: 200) into the final fertilization droplet. A control treatment with no protein exposure was also set up. The summarised IVF results are shown in Table 2. Exposure to HSPA8 did not enhance the fertilization rate compared with the control, but it significantly enhanced the extent of monospermy (control versus HSPA8; 58.6 vs 88.9%; P<0.05). Exposing spermatozoa to HSPA8 also appeared to increase the rate of embryonic development so that by 60 h of incubation a markedly higher proportion of embryos had reached or passed the three or four cell stage (control versus HSPA8; 37.9 vs 74.1% and 3.5 and 22.2% respectively; P<0.05).
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When bull spermatozoa were incubated in the presence of HSPA8 for 48 h at 37 °C, 50% remained viable. However, the inclusion of HSPA8 in the TLP medium increased the rate of sperm survival in a dose dependent manner up to an optimal value at 1 µg/ml (TLP control versus 1 µg/ml HSPA8; P=0.0013). Although, increasing the dose of HSPA8 further (to 4 µg/ml) still resulted in a beneficial effect on sperm survival (P=0.006) compared with TLP alone, the higher dose was less effective at maintaining sperm survival. When spermatozoa were incubated in media containing tubulin as a control protein treatment, there was no significant difference in outcome with respect to the TLP control. These results are summarised in Fig. 4.
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Discussion |
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TopAbstractIntroductionResultsDiscussionMaterials and MethodsDeclaration of interestAcknowledgementsReferences |
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In this study, we have demonstrated that the HSPA8 is present within a soluble protein subset prepared from the porcine oviductal epithelium, sAPM, and that recombinant bovine HSPA8 enhances the in vitro survival of both boar and bull spermatozoa. Thus, we conclude that the HSPA8 is likely to be a biologically active sAPM component responsible for improved in vitro sperm survival that we have observed and reported previously (Elliott et al. 2001). Our investigative strategy involved two complementary approaches; one was the direct proteomic identification of biotinylated sAPM proteins that became bound to the sperm surface. This approach was informative because the sAPM had been prepared from in situ biotinylated OEC; therefore the biotin-labelled proteins must have originated on or near the oviductal cell surface. The second approach aimed to use the proteomic information by incubating the sAPM with anti-HSPA8 in an effort to inactivate HSPA8, the selected protein of interest. The investigative strategy was then taken a step further by testing the biological activity of commercially available bovine recombinant HSPA8 in sperm survival bioassays. This protein was shown to be capable of reproducing the sperm-enhancing properties of sAPM, but at considerably lower concentrations.
As a prelude to these studies we also showed, by confocal microscopy, that when spermatozoa and fluorescent-labelled sAPM proteins are co-incubated, the sAPM proteins bind predominantly to the apical region of the sperm head. This together with evidence of boar sperm binding to sAPM coated beads supports the findings of our earlier work whereby a small number of surface labelled sAPM proteins were found to associate with spermatozoa during incubation (Satake et al. 2006). The majority of boar spermatozoa (63.1%) bound FITC-labelled sAPM proteins at the apical region of the sperm head; this is the region that associates with OEC in vivo (Rodriguez-Martinez 2007) and suggests that the sAPM-sperm interaction mimics the natural situation. This also correlates with binding patterns and interactions seen in other studies (Pollard et al. 1991, Pacey et al. 1995, Satake et al. 2006). Binding over the anterior acrosome also coincides with the distribution of sulphated glycolipids reported by Gadella et al. (1995); interestingly, the sulphated glycolipids are known to bind specifically to members of the HSP70 family, especially HSPA8, through specific binding sites on the protein molecule (Boulanger et al. 1995, Lingwood et al. 2001, Mamelak et al. 2001). If this binding interaction is indeed functionally significant, it would explain the dose-dependent sperm–protein interaction observed here because the sperm surface-receptors would have to interact stoichiometrically with the exogenous protein. Since the anterior acrosomal sulphated glycolipid pattern reported by Gadella et al. (1995) undergoes a significant shift towards the equatorial segment during capacitation, we interpret our acrosomal patterns as indicating that most spermatozoa in our samples are uncapacitated; this would be expected as they had only been exposed to capacitation conditions for a very short period. This result would also agree with previous studies indicating that uncapacitated boar spermatozoa preferentially associate with OEC (Fazeli et al. 1999). That other spermatozoa either displayed extensive sAPM binding or no binding at all is therefore likely to be a reflection of sperm heterogeneity in the ejaculate; this is a well known property of boar ejaculates even though the specific reasons for the heterogeneity remain unclear. It is likely, however, that the differences correspond to inter-sperm differences in ability to colonise the oviduct.
Our attempt to mimic the sperm-oviduct interaction more precisely by incubating sAPM-coated beads with spermatozoa were of interest in that a surprisingly low number of spermatozoa became attached to each bead; although, this number was very low, it was nevertheless higher than the control beads that lacked surface-bound sAPM. There are a number of possible reasons for this, one plausible possibility being that the spermatozoa were only able to bind transiently to the bead surfaces, thus preventing the accumulation of spermatozoa. We did not, however, assess the effective density of sAPM bound to the bead surfaces, and a second possibility would be that the sAPM had been insufficiently coupled to the bead surface.
Further sperm/sAPM binding experiments enabled a subset of sperm-associating proteins to be identified using in situ biotinylated sAPM, followed by proteomic analysis of a significant 70 kDa sperm-binding protein band. Of the 19 proteins identified from the significant 70 kDa sperm-binding protein band, five were heat shock proteins and we chose to analyse one of these, namely HSPA8, in more detail. A western blot of sAPM proteins probed with anti-HSPA8 confirmed its presence in sAPM.
sAPM was unable to enhance sperm viability if it had been pre-treated with anti-HSPA8. We interpreted this result as showing that HSPA8 could be a member of the sAPM protein milieu participating in the protection of sperm viability in vitro. Incubations (bioassays) of spermatozoa with serial dilutions of the commercial bovine recombinant HSPA8 confirmed this view by clearly demonstrating its effectiveness in enhancing sperm survival in vitro at concentrations that were two orders of magnitude lower than the correspondingly bioactive sAPM concentration. It is of considerable interest that when the effectiveness of HSPA8 was tested with bull spermatozoa, it was again was seen to enhance sperm survival at low concentrations and in a dose-dependent manner.
In this study, the HSPA8 protein was detected in sAPM via the direct proteomic analysis of the sperm-binding protein band and by western blotting with an antibody (anti-HSPA8). To our knowledge, this is the first report suggesting membrane bound or extracellularly located HSPA8 may have a role in the protection of spermatozoa in the oviduct, but the associated literature is consistent with the evidence we present here. HSPA8 is a constitutive member of the heat shock protein 70 family that includes a number of HSP70 variants. Some are stress-inducible and others are constitutive with involvement in housekeeping functions (for reviews, see Tavaria et al. 1996, Daugaard et al. 2007). HSPA8 has been reported to be involved in a multitude of the housekeeping chaperone functions including folding of nascent polypeptides, protein translocation across membranes, chaperone-mediated autophagy, prevention of protein aggregation under stress conditions and disassembly of clathrin coated vesicles (Daugaard et al. 2007). Thus, HSPA8 is considered as an essential housekeeping gene and it has been reported that HSPA8 knockout-mice cannot be created due to the essential role of HSPA8 in cell survival.
When we undertook a parallel series of experiments using FITC-labelled HSPA8, we were unable to demonstrate the occurrence of any clear binding patterns to spermatozoa (data not shown). Logically, we conducted these experiments using HSP8A concentrations that matched the biologically significant range (1–4 µg/ml). It could be argued that the concentration of FITC-labelled HSP8A protein was too low to be detectable on spermatozoa. However, it is also possible that HSP8A binds transiently to the functional sperm surface, exerting its effects instead by plasma membrane-mediated signalling mechanisms that probably operate via exceedingly low protein concentrations. This explanation would also be consistent with the large discrepancy between the concentration of sAPM and HSP8A needed to elicit improved sperm survival.
The reduction of polyspermic fertilization and improved rate of embryonic development in vitro by briefly exposing spermatozoa to HSPA8 before interaction with oocytes is of considerable interest but difficult to explain. The results agree with a previous and larger IVF study, using the same conditions, in which spermatozoa were exposed to sAPM for 10 min before fertilization (Satake et al. 2007). In that study, the overall fertilization rate was significantly enhanced from 78 to 86% (n=1235 oocytes) when spermatozoa were pre-incubated with sAPM, and in the group of fertilized oocytes, polyspermic fertilization was also significantly reduced (47 vs 21%). Given that HSPA8 is a component of sAPM, it may also have been one of the agents responsible for producing these earlier results. However, the possibility that the HSPA8 protein acts in concert with proteins that are either already present in the sperm plasma membrane or within the oviductal epithelium in vivo cannot be ruled out.
Like the study reported here, experiments by Boilard and colleagues (Boilard et al. 2001) identified three chaperone proteins from cow oviducts that bound to bull spermatozoa; two of these were identified as Heat shock 60 kDa protein 1 (chaperonin; HSPD1 previously known as HSP60) and HSPA5 (Boilard et al. 2004). Interestingly, HSPA5 is also present in our sperm-binding 70 kDa sAPM band. Subsequent studies of these proteins have also confirmed their presence in the human female reproductive tract (Lachance et al. 2005). These proteins were found to elevate intracellular calcium concentrations in spermatozoa; (Lachance et al. 2007), thus confirming that the heat shock proteins are able to act upon spermatozoa via plasma membrane mediated cell-signalling mechanisms. This provides supportive evidence that HSPA8 may act via a similar route. In their study, Lachance et al. (2007) were unable to detect any viability enhancing effects of HSPD1 and HSPA5 on human spermatozoa, although given that their duration of incubation was only 4 h, any effects may have been difficult to detect.
Heat shock proteins of similar size and function to those identified here as components of the oviductal epithelium have also been identified as components of spermatozoa themselves. They are known to be synthesized during spermatogenesis (Allen et al. 1988, Maekawa et al. 1989) and they adopt specific localizations within sperm e.g. within their plasma membrane (Boilard et al. 2004, Mitchell et al. 2007). While endogenous heat shock proteins clearly have roles in sperm function, data in the present study clearly show that exogenous heat shock proteins, such as HSPA8 can also have an important effect on sperm function.
Our data showing an important role for HSPA8 in the maintenance of boar and bull sperm survival in vitro implies that the mechanism of action could be highly conserved across species. Consequently, the protein could be a useful additive to sperm extenders used for artificial insemination for more than one species. Indeed, inter-species variability in effectiveness is a major difficulty for the development of sperm extenders in mammals, but we speculate that such a highly conserved protein as HSPA8 may be useful in overcoming this problem.
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Materials and Methods |
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TopAbstractIntroductionResultsDiscussionMaterials and MethodsDeclaration of interestAcknowledgementsReferences |
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Apical plasma membrane preparations
Unlabelled fractions
The OEC from the reproductive tissue of freshly slaughtered sows were harvested manually and the sAPM fractions were isolated and solubilized as described previously (Holt et al. 2005). Briefly, epithelial cells were scraped from the lumena of oviducts and sequentially and alternately treated with 10 mM MgCl-2 and differential centrifugation to release the APMs. Soluble proteins were extracted from the membranes with 1 M NaCl and high-speed centrifugation. The supernatant was separated from the pellet and concentrated using Microcon 3 000 Dalton cut-off centrifugal filter devices (Millipore UK Ltd, Watford, UK). The concentrated sAPM protein fraction was washed twice with TLP and stored at ?80 °C. Protein concentration was measured using the Bradford (1976) method (kit supplied by Bio-Rad).
Biotin labelled sAPM fractions
Prior to epithelial cell collection and sAPM preparation the oviductal lumena were labelled with biotin ester Sulpho-NHS-LC-biotin (Pierce). This involved filling the oviducts with the prepared biotin ester (0.5 mg/ml) and incubating them for 30 min. The excess biotin was quenched by flushing with 50 mM NH4Cl. sAPM proteins were prepared using the same method as that for the unlabelled fractions.
Sperm preparation
Viable boar sperm samples were separated from the storage diluent, usually BTS, by washing through a Percoll–saline concentration gradient (Holt & Harrison 2002). Semen aliquots (7 ml) were layered over 2 ml 35% Percoll on 2 ml 70% Percoll and centrifuged for 15 min at 200 g followed by 15 min at 900 g. The supernatant was aspirated to leave 0.4 ml containing the sperm pellet.
Bull semen was supplied by Genus Freezing Unit, (Ruthin, Wales), in 1:5 dilution of Eqcellsire (IMV L'Aigle, France) and cooled to room temperature prior to overnight dispatch to our laboratory. A total of eight ejaculates were used in the current study. The samples were centrifuged for 10 min at 600 g and the supernatant removed. The sperm pellet was washed once and then diluted in TALP to give a sperm concentration of 100x106/ml (final concentration 50x106 sperm/ml).
Evaluation of sperm viability
Sperm viability (as defined by plasma membrane integrity) was assessed using SYBR-14 (200 nM) and ETHD-1 (2 µM) in PBS supplemented with 0.01% (v/v) formalin. Spermatozoa were incubated in this solution for 15 min at 39 °C in 5% CO2 and at least 200 cells were counted and classified as live (plasma membrane-intact; green) or dead (plasma membrane damaged; red). The data were expressed as mean viability index±S.E.M. Viability index was defined as the ratio of % viable spermatozoa at time=24 h/% viable spermatozoa at time=0 h.
Evaluation of interactions between in situ biotinylated sAPM and spermatozoa
Samples of in situ biotinylated sAPM were adjusted to 3 mg/ml in 200 µl Immobilized Neutravidin coated agarose beads in PBS (Pierce). The samples were incubated for 1 h on a small shaker at 600 rpm (room temperature). The bead/sAPM samples were centrifuged to remove unbound protein and washed in 5x1 ml PBS for 2 min each at 2500 g. A sample of beads without added sAPM was also washed. The final pellet of beads was resuspended in 100 µl TLP. Boar semen was washed in a Percoll–saline concentration gradient as described above; 2 µl aliquants of washed spermatozoa were added to each of three tubes containing 25 µl each of 1) TLP only, 2) beads without sAPM and 3) beads with sAPM; these were incubated for 20 min at 39 °C in 5% CO2. Clean electropositive slides (VWR, Lutterworth, UK) were prepared by warming to 37 °C. After 20 min an aliquot (10 µl) of the incubated sample was mounted on a slide beneath a coverslip. The coverslips were raised >65 µm above the slide surface using small amounts of silicon grease mixed with 65 µm diameter glass beads. The sperm-bead interactions were recorded on video tape for observation; two observers made blind and independent estimates of the number of spermatozoa bound to beads.
Microscopic localization of sAPM proteins on the surface of spermatozoa
sAPM proteins were labelled with FITC using a ProtOn Fluorescein labelling kit (Vector Laboratories Inc., Burlingame, CA, USA) following the manufacturer's instructions. Briefly, 100 µl sAPM (7.0 mg/ml) was supplemented with 2 µl ProtOn FITC reagent and incubated for 30 min at room temperature. The reaction was stopped with 2 µl ethanolamine (1 M) and incubated for 5 min at room temperature. The sample was passed through the salt extraction column provided (by centrifugation at 500 g for 1 min) to remove excess fluorescein. The protein concentration of the sample was then assessed (Bradford 1976).
The labelled protein (2.4 mg/ml) was diluted in TLP (1:10 v/v). 2 µl Percoll–saline washed spermatozoa was added to 100 µl aliquots of this labelled protein solution. The samples were incubated at 39 °C for 10 min prior to the addition of 1 µl PI for a further 3 min. These samples were centrifuged for 3 min at 3000 g: the supernatant was removed and the samples resuspended in 80 µl TLP. Aliquots were examined by confocal microscopy (Zeiss LSM 510, Oberkochen Germany). At least ten images of spermatozoa from each boar were recorded. Sperm/sAPM localization was assessed subjectively as to whether there was ‘no binding’, ‘binding to the acrosomal region’ or ‘binding to the head and mid-piece region’. Approximately 60 plasma membrane intact spermatozoa were evaluated from each of eight boar ejaculates.
In vitro fertilization technique
Sow ovaries were obtained from a local abattoir (A & G Barber, Purleigh, Essex, UK); these were placed in 0.9% saline solution supplemented with 100 IU/ml penicillin-G and 100 µg/ml streptomycin sulphate solution (pen/strep; Invitrogen Life Technologies) and maintained in a thermos flask at 25–30 °C for transportation to the laboratory. On arrival the ovaries were washed in lukewarm water then rinsed in warm saline solution with pen/strep supplements.
Follicular fluid from 3–6 mm diameter ovarian follicles were aspirated using an 18.5 gauge needle attached to a 10 ml disposable syringe containing 2 ml collection medium 1 (TCM 199 supplemented with 5 mM NaHCO3, 15 mM Hepes (Na+ salt or free acid), 0.05 g/l kanamycin sulphate, 0.4% (w/v) BSA fraction V and 0.04 g/l heparin). The aspirated fluid was then filtered through a 70 µm cell strainer to harvest the oocytes. The oocytes were washed and collected into pre-warmed collection medium 2 (TCM 199 (with Hank's salts and sodium bicarbonate) supplemented with 0.1% (w/v) polyvinyl alcohol, 3.05 mM D-glucose, 0.91 mM sodium pyruvate and pen-strep solution). Cumulus–oocyte complexes (COCs) were collected under a stereomicroscope; oocytes had at least two complete layers of cumulus cells and evenly granulated ooplasm. The selected COCs were washed in collection medium 2 and transferred in groups of 50 COCs into pre-equilibrated 500 ml wells of IVM medium (TCM 199 (with Earle's salts, L-glutamine and sodium bicarbonate) and supplemented with 0.1% polyvinyl alcohol, 3.05 mM D-glucose, 0.91 mM sodium pyruvate, 0.57 µM cysteine and 10 ng/ml epidermal growth factor and pen-strep solution (Abeydeera et al. 2000) and pre-equilibrated). Before oocyte transfer 0.5 µg/ml LH and 0.5 µg/ml FSH were added to the 500 µl maturation droplets. The preparations were incubated for 48 h.
After IVM incubation 0.1% (w/v) hyaluronidase was added for 30 min to the maturation droplets to loosen the cumulus cells. COCs were then washed in pre-equilibrated mTBM (modified Tris-buffered medium (mTBM (Abeydeera & Day 1997): 113.1 mM NaCl, 3 mM KCl, 20 mM Tris-base, 11 mM D-glucose and 5 mM sodium pyruvate supplemented with 5 mM NaHCO3 and 0.4% (w/v) BSA). After equilibration of the medium, 10 mM CaCl2 was added to avoid precipitation). Groups of 30–40 COCs were placed in pre-equilibrated 98 µl droplets of mTBM covered in mineral oil. Approximately 3x106 ml of Percoll-washed spermatozoa were incubated for 10 min in 1 ml pre-equilibrated mTBM supplemented with, or left without, 0.5 µg/ml bovine recombinant HSPA8. 2 µl sperm suspension were added to the IVF droplets, to give a final concentration of 6x104 spermatozoa/ml. The oocytes and spermatozoa were co-incubated for 6 h.
Following sperm–oocyte co-incubation, the presumptive zygotes were collected into 1 ml pre-equilibrated NCSU23 medium (North Carolina State University-23 medium: 108.73 mM NaCl, 4.78 mM KCl, 1.19 mM K2PO4, 1.19 mM MgSO4.7H2O, 1 mM L-glutamine, 5.55 mM D-glucose, 7 mM taurine, 5 mM hypotaurine, 25.07 mM NaHCO3 and 1.71 mM CaCl2 supplemented with 0.4% (w/v) BSA and pen-strep solution) in a 5 ml glass tube and vortexed for 2 min to remove loosely bound spermatozoa and cumulus cells. The presumptive zygotes were then washed twice in fresh pre-equilibrated 40 µl droplets of NCSU23 and transferred to 20 µl of the same medium in groups of 20–25. They were cultured for 48 h and then fixed in 2.5% paraformaldehyde fixative with 0.5% triton X-100 for 30 min at 39 °C. The embryos were washed (x 3) in warm PBS and stained with 10 µg/ml PI for 10 min before being mounted in mounting medium (Vectashield, Vector Laboratories). They were examined by confocal microscopy (Zeiss LSM 510, Germany) to identify fertilization status. The oocytes/embryos were classified as 1) unfertilized oocytes (the presence of metaphase plates or female pronuclei), 2) fertilized, with male and female pronuclei, 3) one-cell embryos, 4) cleaved embryos with two or more cells 5) embryos with more than four cells.
Identification of the oviductal epithelial proteins that bound to spermatozoa
Biotinylated APM and sAPM (prepared ex situ and in situ) was incubated with spermatozoa (150 µg/ml and 1x108 spermatozoa/ml) at 39 °C and 5% CO2 in air for 30 min. After incubation the sperm suspensions were concentrated by microcentrifugation and then washed three or four times (>1:100 dilution per wash) by microcentrifugation at 5000 g for 4 min for removal of APM and sAPM. To prepare a suspension of biotinylated proteins bound to sperm membranes, the washed pellets were resuspended in SDS sample buffer (50 mmol tris/l pH 6.8 and 0.5% SDS w/v), incubated at room temperature for 90 min and then centrifuged at 14 000 g for 10 min. The supernatant (soluble fraction, comprising sperm membrane proteins and surface-bound biotinylated proteins) was aspirated for evaluation by one-dimensional PAGE and immunoblotting to detect biotinylated proteins.
A biotin-positive band in the immunoblot of sperm proteins corresponded to a 70 kDa band in the PAGE gel preparation. This band was therefore excised and analyzed further. To prepare sufficient protein for proteomic analysis sAPM proteins were separated using 16 single-dimensional PAGE lanes and the appropriate band in each was extracted for analysis. Specifically, this was achieved as follows. Protein (15 µg) was loaded into each of eight wells in two 10-well gels. The two outer wells of each gel were loaded with Kaleidoscope standards for identification of molecular weight. Electrophoresis was performed, using the discontinuous buffer system (Laemmli 1970) for 45 min at 190 volts after which the gels were fixed (40% methanol v/v and 7% glacial acetic acid) for 1 h and stained overnight in Brilliant Blue G-Colloidal Concentrate. The gels were destained in 10% acetic acid in 25% methanol (v/v) for 30 s and rinsed in 25% methanol before being placed carefully onto clean glass plates. Using a new sterile blade, the prominent band (Fig. 2A arrow) corresponding to the 70 kDa in situ sAPM biotinylated protein that bound to spermatozoa (Fig. 2B arrows) was excised from each of the 16 separate sAPM lanes. Each piece was cut into 1 mm2 pieces and placed in 1 ml dH-2O in 1.8 ml centrifuge tubes for further proteomic processing.
Proteomic analysis
Gel pieces were washed in 25% (v/v) methanol, and then incubated for 1 h at 37 °C in Coomassie destain solution consisting of 40% (v/v) acetonitrile (ACN: VWR International Ltd., Leicester, UK) in 200 mM ammonium bicarbonate. The destain solution was removed, and gel pieces were incubated with ACN for 15 min at room temperature. ACN was subsequently removed, and the gel pieces were dried in a vacuum centrifuge. Dried gel pieces were stored at 4 °C until required. Proteins were digested with 20 ng/µl sequencing grade modified trypsin (Promega) in 50 mM ammonium bicarbonate at 37 °C for 12 h. The supernatant from the trypsin digest was transferred to a siliconized microcentrifuge tube. Peptides were sequentially extracted three times by incubation with peptide extraction solution consisting of 25 mM ammonium bicarbonate (10 min at room temperature), 5% formic acid (15 min at 37 °C), and ACN (15 min at 37 °C). Each extraction was followed by centrifugation and removal of supernatants. The original supernatant and the supernatants from the three sequential extractions were combined and dried in a vacuum centrifuge for 4–6 h. The dried peptides were dissolved in 7 µl 0.1% (v/v) formic acid in 3% (v/v) ACN in water. Samples were centrifuged for 5 min at 12 000 g, and the supernatants were subjected to liquid chromatography electrospray ionization tandem mass spectrometry (LC-ESI-MS/MS).
Liquid chromatographic separations of the tryptic digests were performed using a reverse phase CapLC system (Waters, Manchester, UK). Peptides were desalted by a PepMap C18 microguard column (300 µm internal diameterx1 mm; LC-Dionex, Leeds, UK) and were then transferred to the analytical column (PepMap C18; 75 µm internal diameterx15-cm column (LC-Dionex). The peptides were eluted in a 60 min gradient. The compositions of the hydrophilic and hydrophobic solvents were 5% ACN, 0.1% formic acid and 95% ACN, 0.1% formic acid. The column eluent was sprayed directly into the nano-ESI source of a Q-TOF microcolumn (Waters). An initial MS scan was performed, and selection of ions for CID was automated by Mass Lynx software (Waters). CID selection criteria were set for 2+ and 3+ ions within the range of 400–2000 m/z above 10 ion counts.
Spectra were searched against the mass spectrometry data base in a sequence query search using MASCOT 2.2.03 software (www.matrixscience.com). The taxonomy was limited to filter for only mammalian matches, and trypsin was used as the cleavage enzyme with one missed cleavage site allowed. The peptide tolerance was set to 1.2 kDa, and the MS/MS tolerance was set to 0.6 kDa. Carbamidomethyl modifications of cysteine and oxidized methionine were set as variable modifications. Matches were considered valid if MS/MS data for multiple unique peptides per protein were identified, each with ion scores above the threshold of statistical significance (values generated by MASCOT). Manual examination of MS/MS data was performed for single peptide matches. In these cases, a continuous stretch of peptide sequence covered by either the y- or b-ion series was required for the protein match to be considered valid.
Western blot analysis of sAPM
The proteomic analysis indicated that spermatozoa avidly bound a series of heat shock proteins. Of these, we chose to examine HSPA8 in more detail. First, for independent confirmation that HSPA8 was indeed present in sAPM a western blot were performed. sAPM proteins were separated by SDS-PAGE (10% gel), blotted on to nitrocellulose and incubated in blocking buffer for 1 h. Anti-HSPA8 (SPA-815), at the manufacturer's recommended dilution was added to the blot and incubated for 1 h at room temperature. The blot was washed 6 x10 min in tris-buffered saline (Pierce) supplemented with tween-20 (TTBS) followed by the addition of the goat anti-rat secondary antibody conjugated with alkaline phosphatase (AP; ab7098, Abcam), at the manufacturer's recommended dilution for a further hour. The blot was washed 6 x10 min in TTBS and, finally, incubated for 3 min in Lumi-phos for AP (Pierce) detection. The blot was exposed to X-ray film for periods between 5 and 60 s.
Sperm viability assays
Boar sperm viability in the presence of antibody-treated sAPM
Antibody-treated sAPM (200 µg/ml) was prepared by incubating sAPM with 1 µg/ml anti-HSPA8 (Ab1427) in TLP for 2 h on a rotary shaker at room temperature. Then, semen from eight boars was individually washed with Percoll–saline as previously described and each sample (in TALP) was adjusted to give a final concentration of 25x106 spermatozoa/ml. 15 µl droplets of sperm suspension and antibody-treated sAPM (with untreated sAPM and TLP as control treatments) were mixed 1:1 in 20 mm diameter wells in four well Multi-dish plates (Sigma Aldrich) and then covered with mineral oil. They were incubated at 39 °C in 5% CO2 for 24 h. The viability of spermatozoa was assessed at zero hours and after 24 h.
Dose response effect of HSPA8 protein on boar sperm viability
Boar spermatozoa were prepared for the bioassay as previously described. Semen samples from seven boars were used for series 1 and eight boars for series 2. Recombinant bovine HSPA8 (Stressgen; SPP-751) was diluted in TLP to give final concentrations of 16, 8, 4 and 2 µg/ml (series 1) and 8, 2, 0.5 and 0.25 µg/ml (series 2) when incubated with equal volumes of spermatozoa. Control samples were incubated in TLP only or TLP + sAPM (200 µg/ml). The samples were incubated at 39 °C in 5% CO2 for 24 h. Sperm viability was examined as described earlier.
Effect of HSPA8 protein on bull sperm viability
Serial dilutions of bovine recombinant HSPA8 and a control protein 
tubulin were prepared in TALP to give final concentrations of 4, 1, 0.5 and 0.1 µg/ml. Spermatozoa were added to droplets of TALP, TALP + tubulin (Abcam plc) or TALP + recombinant HSPA8 in equal volumes of 25 µl each. The 50 µl droplets were covered with mineral oil and incubated at 37 °C in a 5% CO2 incubator for 48 h. The viability of samples was assessed at zero hours and then at 48 h following the same procedure as that for boar spermatozoa.
Statistical analyses
Percentage and Viability Index data were log transformed and treatment effects were examined using analyses of variance (Statistica for Windows V7; Statsoft UK, Letchworth, UK). Individual contrasts between treatments were examined using orthogonal polynomial coefficients. Treatment effects in the IVF experiment were analyzed using Fisher's exact 
2-tests. Sperm – bead binding effects were assessed using the non-parametric Mann–Whitney U test.
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TopAbstractIntroductionResultsDiscussionMaterials and MethodsDeclaration of interestAcknowledgementsReferences |
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TopAbstractIntroductionResultsDiscussionMaterials and MethodsDeclaration of interestAcknowledgementsReferences |
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Received July 9, 2008
First decision August 20, 2008
Revised manuscript received September 27, 2008
Accepted November 7, 2008
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