Molecular Human Reproduction, Vol. 5, No. 9, 816-824,
September 1999
© 1999 European Society of Human Reproduction and Embryology
Regulation of sperm function |
Relationship between sperm motility and the processing and tyrosine phosphorylation of two human sperm fibrous sheath proteins, pro-hAKAP82 and hAKAP82
Center for Research on Reproduction and Women's Health, Department of Obstetrics and Gynecology, University of Pennsylvania School of Medicine, 421 Curie Blvd., Philadelphia, PA 19104, USA
Abstract
Sperm motility is regulated by the cAMP-dependent protein kinase (protein kinase-A)-mediated phosphorylation of a group of largely unidentified flagellar proteins. Human AKAP82 (hAKAP82) and its precursor protein, pro-hAKAP82, are members of the A-kinase anchor protein (AKAP) family. These proteins tether protein kinase-A to the fibrous sheath of human spermatozoa and presumably localize the activity of the kinase near specific targets in the sperm flagellum. In this way, pro-hAKAP82 and hAKAP82 may be involved in regulating sperm motility. Similar to its homologues in other species, pro-hAKAP82 is proteolytically processed to hAKAP82. However, the amount of processing of pro-hAKAP82 in human spermatozoa is less than the amount of processing of the precursor in other species. We postulated that this lower extent of processing may be related to lower percentages of human sperm motility. In addition, both pro-hAKAP82 and hAKAP82 are tyrosine phosphorylated in a capacitation-dependent manner. Since capacitation is associated with hyperactivated motility, we postulated that tyrosine phosphorylation of pro-hAKAP82/hAKAP82 is associated with changes in motility. However, using a combination of immunofluorescence and immunoblotting approaches, we found no evidence for an association between either processing or tyrosine phosphorylation of pro-hAKAP82/hAKAP82 and significant differences in motility in spermatozoa from normal men.
hAKAP82/human/protein processing/sperm motility/tyrosine phosphorylation
Introduction
Male factor infertility is a significant problem in humans. It is estimated that ~17% of all couples are infertile and that in 26–46% of these couples the infertility is, at least in part, due to the man (Lunenfeld and Insler, 1993
). In subfertile men, the percentage of motile spermatozoa and the percentage of spermatozoa with progressive motility are highly correlated with fertilizing ability (Aitken et al., 1982a,b, 1983; Holt et al., 1985) and it is generally accepted that spermatozoa with significant aberrations in motility are incapable of fertilization without extensive laboratory intervention (Boyle et al., 1992). In spite of its obvious clinical importance, little is known about the molecular mechanisms that regulate human sperm motility.
Cyclic-AMP (cAMP)-dependent phosphorylation of flagellar proteins is involved in the initiation and maintenance of sperm motility; however, most of the target proteins have not been identified (Tash and Means, 1982; Brokaw, 1987; Tash et al., 1988; Tash and Bracho, 1994). In mammalian spermatozoa, the major downstream target of cAMP is protein kinase-A (PK-A), making it likely that PK-A is involved in these phosphorylation reactions (Visconti et al., 1997). The question of how a soluble enzyme such as PK-A localizes to the sperm flagellum, a highly compartmentalized structure with little cytoplasm, has been answered by the identification of two major fibrous sheath proteins, AKAP82, and its precursor, pro-AKAP82, as members of the A-kinase anchor protein (AKAP) family in mouse, human, and bull spermatozoa (Carrera et al., 1994, 1996; Turner et al., 1998; Moss et al., 1999). AKAPs function to anchor PK-A via the type II regulatory (RII) subunit of the kinase to the cytoskeleton or to subcellular organelles (Scott and McCartney, 1994). In response to cAMP binding to the RII subunit of PK-A, the catalytic subunit of the kinase is released and becomes free to phosphorylate its substrates. In spermatozoa, AKAP82 anchors PK-A to the fibrous sheath, a cytoskeletal structure that surrounds the axoneme and outer dense fibres and is found only in the principal piece of the mammalian sperm flagellum (Lindemann et al., 1992). Thus, AKAP82 anchors PK-A in close proximity to its flagellar target proteins. Recently, there have been several reports of AKAPs, including an alternative splice variant of AKAP82 called Fsc1, which, like AKAP82, have been identified in the fibrous sheath/principal piece of mammalian spermatozoa (Mei et al., 1997; Miki and Eddy, 1998; Vijayaraghavan et al., 1999). Additionally, it has been determined that peptides which block the ability of RII to bind to AKAPs will arrest mammalian sperm motility (Vijayaraghavan et al., 1997). These data support the hypothesis that, by anchoring the activity of PK-A in the fibrous sheath, AKAPs play central roles in the regulation of normal sperm motility. In addition, the proteins which make up the human sperm fibrous sheath have been analysed by sodium dodecyl sulphate–polyacrylamide gel electrophoresis (SDS–PAGE) (Jassim et al., 1992; Kim et al., 1997) and the most prominent bands represent proteins with relative molecular weights similar to those of human AKAP82 (hAKAP82) and human pro-AKAP82 (pro-hAKAP82). Thus, in addition to their roles as AKAPs, it is likely that hAKAP82 and pro-hAKAP82 are major fibrous sheath structural proteins as well.
In the mouse, bull and human, pro-AKAP82 is processed into mature AKAP82 by proteolytic cleavage of the amino terminal `pro' domain (Johnson et al., 1997; Turner et al., 1998; Moss et al., 1999). Immunoblots of mouse, human and bull spermatozoa probed with an antibody that recognizes both the precursor and mature proteins demonstrate that the ratio of pro-AKAP82 to AKAP82 varies among species and is greatest in human spermatozoa. This finding indicates that processing of pro-AKAP82 in the human is less complete than in the mouse and bull (Turner et al., 1998). Additionally, immunofluorescence experiments using an antibody directed against the mouse homologue of pro-hAKAP82 (pro-mAKAP82) have shown that while pro-mAKAP82 localizes to the entire length of the principal piece in mouse testicular spermatozoa, it is found only in the proximal portion of the principal piece in mature cauda epididymal mouse spermatozoa. This finding, together with immunoblot experiments which did not detect mAKAP82 in testicular spermatozoa, suggest that mAKAP82 is made as a precursor in post-meiotic germ cells which then is processed into mAKAP82 in a distal to proximal direction along the length of the fibrous sheath during epididymal transit (Johnson et al., 1997). In contrast, in ejaculated (mature) human spermatozoa, pro-hAKAP82 is present along the entire length of the fibrous sheath, suggesting that either the precursor is processed less completely in the human than in the mouse and/or that processing occurs throughout the fibrous sheath rather than in a distal to proximal direction (Turner et al., 1998). Since human ejaculates typically contain a lower percentage of motile spermatozoa than bull ejaculates or mouse cauda epididymal sperm samples (Albert and Roussel, 1984; Mortimer, 1994; Barth, 1998), we postulated that inefficient processing of pro-hAKAP82 into hAKAP82 might be associated with populations of spermatozoa with poor motility. If this is true in human spermatozoa, then an increase in processing of pro-hAKAP82 into hAKAP82 in motile spermatozoa might be indicated by differences in distribution of the precursor in the fibrous sheath (i.e. the precursor might be found only in the proximal portion of the principal piece in spermatozoa with good motility). Additionally, populations of motile human spermatozoa would contain a lower pro-hAKAP82:hAKAP82 ratio when compared with populations of human spermatozoa with significantly reduced sperm motility.
Another difference between mouse and human AKAP82 is that, in the mouse, pro-mAKAP82 and mAKAP82 are phosphorylated on serine/threonine residues prior to capacitation and are not phosphorylated on tyrosine residues (Carrera et al., 1996; Johnson et al., 1997). However, although the serine/threonine phosphorylation status of pro-hAKAP82 and hAKAP82 is not known, both human proteins are tyrosine phosphorylated in a capacitation-dependent manner (Carrera et al., 1996). Additionally, not all `capacitated' human spermatozoa contain tyrosine phosphorylated pro-hAKAP82/hAKAP82 at a given point in time, consistent with the hypothesis that spermatozoa are recruited to capacitate in a stochastic fashion, thus increasing the likelihood that a given subset of spermatozoa will be capable of fertilizing an egg at the time of ovulation (Yanagimachi, 1994). Since capacitation is closely associated with a hyperactivated state of sperm motility (Yanagimachi, 1994; Suarez, 1996), our second hypothesis is that tyrosine phosphorylation of pro-hAKAP82 and/or hAKAP82 results in some undefined modification in the activity of the protein(s) during capacitation that may be associated preferentially with motile spermatozoa. The purpose of this paper is to evaluate the association of pro-hAKAP82 processing and pro-hAKAP82/hAKAP82 tyrosine phosphorylation status with the motility of spermatozoa from normal men.
Materials and methods
Immunological reagents
Anti-AKAP82 is a polyclonal rabbit antiserum raised against electrophoretically purified mAKAP82 (Carrera et al., 1994). Anti-AKAP82 has been used previously to detect pro-hAKAP82 and hAKAP82 by immunoblotting and immunofluorescence analysis of human spermatozoa (Carrera et al., 1996).
Anti-hpro is a polyclonal rabbit antiserum raised against a synthetic peptide corresponding to amino acids 131–145 of the pro domain of pro-hAKAP82 (NH2-CVGDTEGDYHRASSEN-COOH). Anti-hpro has been used previously to identify pro-hAKAP82 and the free human pro domain (hpro) by immunoblotting and immunofluorescence analysis of human spermatozoa (Turner et al., 1998). Anti-hpro does not recognize mature hAKAP82. Anti-phosphotyrosine is a mouse monoclonal antibody (clone 4G10) purchased from Upstate Biotechnology (Lake Placid, NY, USA).
Collection and analysis of semen samples
Samples of human semen were obtained by masturbation from healthy donors with good sperm motility (total sperm motility >75%, progressive motility >60%). Semen samples were allowed to liquefy for 60–70 min at ambient temperature. Following liquefaction, the volume of each ejaculate was measured using a disposable, warm pipette. To determine sperm concentration, and based on initial assessment of the semen, a small aliquot of each sample was diluted either 1:20 or 1:50 in phosphate-buffered saline (PBS) using a positive displacement pipette. The diluted sample was then mixed and 10 µl was loaded into each chamber of a haemocytometer. The total number of spermatozoa in the four corner squares of each chamber was counted using phase contrast microscopy at x200 magnification using a Zeiss Photomicroscope III. The total numbers for the two different chambers were averaged. The average was then multiplied by the dilution factor, divided by 4 and multiplied by 10 000 to determine the number of spermatozoa/ml of sample. The percentage of total motile spermatozoa was evaluated in each sample according to previously described methods (Kenney, 1975), by viewing a drop of raw semen on a prewarmed slide under a prewarmed coverslip with phase contrast microscopy at x400 magnification using a Zeiss Photomicroscope III. For these experiments, a motile spermatozoon was considered to be one with any visible flagellar movement. Sperm motility was assessed by two independent blinded observers and the two assessments were averaged. If the opinions of the two observers differed by more than 15 percentage points, then a third independent, blinded estimate was obtained, and the two assessments which were in closest agreement were averaged.
Swim-up assays and capacitation
A portion (two thirds) of each ejaculate was divided into two equal volumes in round-bottomed, polypropylene tubes (Falcon Brand; Fisher Scientific, Pittsburgh, PA, USA). Each of these fractions was overlaid with 600 µl of protein-free medium [human tubal fluid (HTF); Irvine Scientific, Santa Ana, CA, USA] warmed to 37°C. The samples were placed at a 45° angle in a 37°C, 5% CO2 (in air) incubator and spermatozoa were allowed to swim up into the HTF medium for 1 h. The top 500 µl of fluid were then removed from each fraction and pooled to form the swim-up (SU) sample. The remaining fluid from these two fractions was pooled to form the non-swim-up (NSU) sample. Sperm motility in each SU and NSU fraction was assessed by two independent observers, one of whom was blinded to the procedure, and the two assessments were averaged. As described previously, if the opinions of the two observers differed by over 15 percentage points then a third independent, blinded estimate was obtained and the two assessments which were in closest agreement were averaged. Spermatozoa from each sample were pelleted by centrifuging for 10 min at 375 g in a clinical centrifuge. The pellets were resuspended in 500 µl of capacitating medium [HTF containing 30 mg/ml bovine serum albumin (BSA)] warmed to 37°C. Samples were incubated at 37°C in 5% CO2 for 90 min and then centrifuged at 16 000 g for 3 min. The supernatant was removed and the sperm pellets were resuspended in PBS containing 200 mmol/l sodium vanadate (Sigma, St Louis, MO, USA). Volume, sperm concentration and total sperm numbers were determined for each fraction. Samples again were centrifuged for 3 min and the supernatant was removed down to ~20 µl. Samples were centrifuged for an additional 3 min and the remainder of the supernatant was removed.
Indirect immunofluorescence of spermatozoa
Indirect immunofluorescence was performed as previously described (Turner et al., 1998). SU and NSU sperm pellets were resuspended in PBS. Spermatozoa were permeabilized in solution in 0.1% (v/v) Triton X-100 in PBS for 15 min and washed once in PBS. Pellets were resuspended in 1 ml PBS and the cells transferred onto coverslips. After settling for 15 min, excess liquid was aspirated gently from the coverslip and the sperm were fixed by overlaying the coverslips with 4% (w/v) paraformaldehyde in PBS for 15 min at room temperature. Coverslips containing the sperm were transferred to individual wells of a plastic 6-well plate and washed three times by gently flooding the coverslips with PBS and then removing the PBS. Coverslips containing the spermatozoa then were submerged in methanol at –20°C for 2 min and washed three times in PBS as described above. Coverslips were transferred sperm-side up into a humid chamber and spermatozoa were blocked by overlaying the coverslips with 10% (v/v) filtered normal goat serum for 30 min at 37°C (if subsequently incubated in anti-hpro) or overnight at 4°C (if subsequently incubated in anti-phosphotyrosine). Following blocking, the goat serum was removed and the coverslips containing the spermatozoa were overlaid and incubated overnight at 4°C in anti-hpro diluted 1:10 (v/v) in 10% goat serum or for 1 h at 37°C in anti-phosphotyrosine diluted 1:100 (v/v) in 10% goat serum. After washing in PBS, coverslips containing spermatozoa were overlaid for 1 h at 37°C in the appropriate secondary antibody [anti-hpro: fluorescein isothiocyanate (FITC)-conjugated goat anti-rabbit immunoglobulin G (IgG), Jackson Immunoresearch Laboratories Inc, West Grove, PA, USA; anti-phosphotyrosine: FITC-conjugated anti-mouse IgG, Boehringer Mannheim, Indianapolis, IN, USA) diluted 1:100 (v/v) in 10% goat serum, washed again in PBS and mounted on slides with mounting media (Fluoromount-G; Southern Biotechnology Associates Inc, Birmingham, AL, USA). Control samples were incubated in preimmune serum (for anti-hpro) or in the secondary antibody only (for anti-phosphotyrosine). Slides were viewed with a Zeiss Photomicroscope III equipped with epifluorescence optics.
A total of 100 spermatozoa from each fraction were counted and scored as either labelled or not labelled with the respective antibody. Only those spermatozoa that stained throughout the length of the principal piece were counted as labelled. Photographs were taken with Kodak T-Max film, 3200 ASA. Paired sample and control photographs were exposed for the same amount of time.
Preparation of sperm proteins
To extract sperm proteins, SU and NSU sperm pellets were washed three times in PBS. Prior to the final centrifugation, the volume, sperm concentration, and total sperm numbers were determined for each sample. After the final wash, sperm pellets were dissolved in SDS sample buffer containing 40 mmol/l dithiothreitol (DTT) and boiled for 5 min. The samples were centrifuged at 16 000 g in an Eppendorf 5415C centrifuge for 3 min and the supernatant was saved. The amount of protein in each sample was determined by the Amido Black procedure (Schaffner and Weissman, 1973).
Electrophoresis and immunoblotting
Proteins from ejaculated human spermatozoa were separated under reducing conditions by SDS–PAGE on a 10% (w/v) polyacrylamide gel. Either equal numbers of sperm equivalents (2x105 spermatozoa) or equal amounts of protein (5 µg) were analysed in each lane. After electrophoresis, proteins were electrophoretically transferred to polyvinylidene fluoride (PVDF) membranes (ImmobilonTM-P Transfer Membranes; Millipore Corporation, Bedford, MA, USA). Following blocking in PBS containing 10% (v/v) fish gelatin, the blots were probed for 1 h with anti-AKAP82 [1:10,000 (v/v)] or anti-phosphotyrosine [1:10,000 (v/v)] in PBS containing 0.1% Tween 20 and 3% (w/v) BSA. The blots were washed 3x10 min with PBS containing 0.2% (v/v) Tween 20. Blots probed with anti-AKAP82 were then incubated for 1 h with donkey anti-rabbit IgG conjugated to fluorescein (Vistra Western blotting kit; Molecular Dynamics Inc, Sunnyvale, CA, USA) diluted 1:600 in PBS containing 0.2% (v/v) Tween 20 and 3% (w/v) BSA. These blots were washed 3x10 min in PBS containing 0.2% (v/v) Tween 20 and incubated for 1 h with an anti-fluorescein alkaline phosphatase-conjugated tertiary antibody fluorescein (Vistra Western blotting kit; Molecular Dynamics) diluted 1:2,500 (v/v) in PBS containing 0.2% (v/v) Tween 20. Blots were washed five times in PBS and overlaid with the fluorescent AttoPhos® reagent (Vistra Western blotting kit; Molecular Dynamics). Fluorescence was detected using a fluorescence imaging system (StormTM system; Molecular Dynamics). Protein bands were quantified using ImageQuantTM software (Molecular Dynamics).
Blots probed with anti-phosphotyrosine were incubated for 1 h with goat anti-mouse IgG conjugated to horseradish peroxidase (Bio-Rad Laboratories, Hercules, CA, USA) diluted 1:5000 in PBS containing 0.2% (v/v) Tween 20 and 3% (w/v) BSA. Blots were washed five times in PBS, overlaid with ECLTM detection reagents (Amersham Life Science, Arlington, IL, USA) and exposed to Reflection film (New England Nuclear, Boston, MA, USA). Protein bands were quantified using the public domain NIH Image software program (developed at the United States National Institutes of Health and available on the internet at http://rsb.info.nih.gov/nih-image/).
Statistical analysis
Based on the distribution of the data, the Wilcoxon signed rank test (the non-parametric equivalent of the paired t-test) was used to determine differences between groups. P < 0.01 was considered to be statistically significant.
Results
Sperm motility differed significantly in swim-up (SU) and non-swim-up (NSU) fractions
To identify associations between sperm motility and the processing and/or tyrosine phosphorylation of pro-hAKAP82 and hAKAP82, we used a swim-up assay to separate sperm ejaculates into fractions with significantly different motilities. SU fractions contained a significantly higher percentage of total motile spermatozoa than the NSU fractions (P < 0.01). Typically, >80% of spermatozoa in SU fractions were motile while <50% of spermatozoa in NSU fractions were motile (Table I). These separated sperm populations were used for further analyses.
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Distribution of pro-hAKAP82 and hpro was not different in populations of spermatozoa with different motility
In the mouse, pro-mAKAP82 is processed into mAKAP82 in a distal to proximal direction along the length of the fibrous sheath. As a result of this processing, pro-mAKAP82 and the free mouse pro domain are present only in the proximal portion of the principal piece in mature cauda epididymal mouse spermatozoa. In contrast, pro-hAKAP82 and the free human pro domain (hpro) are present along the entire length of the principal piece in human spermatozoa (Turner et al., 1998
). This difference in pro-AKAP82/hpro localization led us to postulate that a decrease in processing of pro-hAKAP82 into hAKAP82 might be reflected in a difference in distribution of the precursor protein and the free hpro domain in the fibrous sheath of highly motile versus less motile human spermatozoa. To test if this was the case, we performed immunofluorescent analysis using an antibody (anti-hpro) that specifically recognizes a region within the hpro domain of pro-hAKAP82. Thus, anti-hpro recognizes both pro-hAKAP82 and hpro, both of which are present in mature spermatozoa, but does not recognize hAKAP82 (Turner et al., 1998). In both SU and NSU sperm fractions, anti-hpro localized to the entire length of the principal piece in essentially all spermatozoa regardless of the motility of the sample (Figure 1). There was no significant difference in the percentage of labelled spermatozoa in the SU and NSU fractions (Table II). These findings indicated that there was no difference in the distribution of pro-hAKAP82 and/or hpro in individual spermatozoa with different motilities and provided initial evidence that processing of the precursor occurred in a similar fashion in all spermatozoa, regardless of motility.
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No difference in relative amounts of pro-hAKAP82 and hAKAP82 in different sperm populations
Since anti-hpro can not distinguish between pro-hAKAP82 and hpro in immunofluorescence experiments, we could not determine with certainty whether processing of pro-hAKAP82 was occurring at similar rates in SU and NSU spermatozoa or whether processing differed in the two populations but the free hpro domain was not degraded in the principal piece. To determine which of these two scenarios was the case, we used immunoblotting to quantify the relative amounts of pro-hAKAP82 and hAKAP82 in the SU and NSU sperm populations. We predicted that a reduction in processing would be indicated by a maintenance of high pro-hAKAP82 levels at the expense of hAKAP82 production. Immunoblot analyses were performed using anti-AKAP82, an antibody that recognizes both pro-hAKAP82 and hAKAP82 but not hpro (Carrera et al., 1996
; Turner et al., 1998). Immunoblots comparing proteins from SU and NSU sperm fractions probed with anti-AKAP82 showed no apparent differences in the relative intensities of the protein bands representing pro-hAKAP82 and hAKAP82 (Figure 2). To assess the quantities of the two proteins objectively, band intensities were assigned numerical values using image analysis software. The ratio of pro-hAKAP82 to hAKAP82 in paired SU and NSU samples then was calculated by dividing the pro-hAKAP82 value by the paired hAKAP82 value. No significant differences were detected in the ratio of pro-hAKAP82 to hAKAP82 in SU and NSU sperm fractions (Table III). These findings suggested that neither a decrease nor an increase in processing of pro-hAKAP82 into hAKAP82 was correlated with differences in motility of SU and NSU spermatozoa from normal men.
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Motility was not correlated with the percentage of capacitated spermatozoa which contained tyrosine phosphorylated proteins in the principal piece
Previous work in our laboratories showed that proteins in human spermatozoa are tyrosine phosphorylated in a capacitation-dependent manner and that the two major tyrosine phosphorylated proteins are pro-hAKAP82 and hAKAP82 (Carrera et al., 1996
). Additionally, immunofluorescence experiments demonstrated that the principal pieces of only a subpopulation of capacitated human spermatozoa immunoreact with an anti-phosphotyrosine antibody. This is consistent with the hypothesis that capacitation is a stochastic process and suggests that capacitation-dependent tyrosine phosphorylation of these flagellar proteins may be associated with a subset of spermatozoa, e.g., motile spermatozoa. To test whether tyrosine phosphorylation of flagellar proteins is correlated preferentially with motile spermatozoa, we first analysed SU and NSU sperm fractions by immunofluorescence using an anti-phosphotyrosine antibody. The percentage of spermatozoa labelling with anti-phosphotyrosine along the entire length of the principal piece was not significantly different between the SU (average of 47%) and NSU fractions (average of 43%, Figure 3, Table IV). These findings suggested that differences in tyrosine phosphorylation of sperm flagellar proteins are not associated with changes in motility in spermatozoa from normal men.
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Changes in the tyrosine phosphorylation status of pro-hAKAP82 and hAKAP82 were not associated with alterations in sperm motility
To determine specifically whether the tyrosine phosphorylation of pro-hAKAP82 and/or hAKAP82 was accompanied by an increase in sperm motility, immunoblots of capacitated spermatozoa from SU and NSU fractions were probed with the anti-phosphotyrosine antibody (Figure 4
). Bands at molecular mass ratio (Mr) 97 and 82 kDa represent tyrosine phosphorylated pro-hAKAP82 and hAKAP82 respectively (Carrera et al., 1996). Although we originally attempted to quantify these bands using the fluorescence imaging system and image analysis software, we were unable to obtain reliably distinct bands using the required tertiary and/or secondary antibodies. Instead, analysis of these immunoblots was completed using a horseradish peroxidase-conjugated secondary antibody and bands were detected by chemiluminescence using standard autoradiography film. Relative band intensities were analysed visually and no apparent differences in the amount of phosphorylated pro-hAKAP82 or hAKAP82 between the SU and NSU fractions were detected (Figure 4).
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To obtain a more objective measure of the amount of tyrosine phosphorylation of pro-hAKAP82/hAKAP82, bands representing tyrosine phosphorylated pro-hAKAP82/hAKAP82 were quantified using the NIH Image software program. Identical immunoblots then were prepared and incubated with the anti-AKAP82 antibody. Bands representing total pro-hAKAP82/hAKAP82 were quantified as described above using a fluorescence imaging system and image analysis software. The ratios of tyrosine phosphorylated pro-hAKAP82 (determined by the anti-phosphotyrosine antibody) to total pro-hAKAP82 (determined by the anti-AKAP82 antibody) and tyrosine phosphorylated hAKAP82 to total hAKAP82 were calculated. There was no significant difference in the ratio of tyrosine phosphorylated pro-hAKAP82 to total pro-hAKAP82 between SU and NSU sperm fractions (Table V
). Similarly, there was no significant difference in the ratio of tyrosine phosphorylated hAKAP82 to total hAKAP82 between SU and NSU sperm fractions. These findings suggested that alterations in the phosphorylation status of these proteins were not associated with changes in motility in spermatozoa from normal men.
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Discussion
Processing of pro-hAKAP82 into hAKAP82 is less complete than processing of either bull or mouse pro-AKAP82 into AKAP82 (Johnson et al., 1997; Turner et al., 1998; Moss et al., 1999). Since human ejaculates typically contain a lower percentage of motile spermatozoa than either bull ejaculates or mouse cauda epididymal spermatozoa, our first hypothesis was that inefficient processing of pro-hAKAP82 into hAKAP82 with the resultant maintenance of high levels of pro-hAKAP82 at the expense of hAKAP82, are correlated with populations of human spermatozoa with poor motility. However, our studies did not find evidence for an association between the degree of processing of pro-hAKAP82 and increases or decreases in motility in spermatozoa from normal men. This was evident by both the distribution of pro-hAKAP82 in individual SU and NSU spermatozoa as determined by immunofluorescence experiments and by the relative amounts of pro-hAKAP82 and hAKAP82 in populations of SU and NSU spermatozoa as determined by immunoblotting experiments.
If processing of pro-hAKAP82 is not involved in changes in sperm motility, then what is its role? In spermatozoa, several examples of proteolytic processing of precursor molecules have been reported, including processing of acrosomal matrix proteins, plasma membrane proteins and surface membrane proteins. These processing events have been shown to affect protein localization, assembly, and function (Anakwe et al., 1991; Hardy and Garbers, 1995; Tulsiani et al., 1995; Jones et al., 1996; Lum and Blobel, 1997; Olson et al., 1998). It is possible that pro-mAKAP82 may represent an assembly-incompetent form of the protein which is transported from the cell body to the flagellum (Johnson et al., 1997). Once the precursor has reached its site of assembly, proteolytic removal of the pro domain then allows for the addition of mAKAP82 into the fibrous sheath. Thus, one potential role for pro-hAKAP82 processing may be to regulate its assembly into the fibrous sheath. Alternatively, cleavage of the amino terminal pro region of pro-hAKAP82 could result in some functional change in the molecule. At this time, the most extensively studied function of AKAPs is their ability to target PK-A to specific subcellular locations by anchoring the RII subunit of the kinase. Gel overlay studies show that both pro-hAKAP82 and hAKAP82 are capable of binding RII in vitro (Carrera et al., 1996; Turner et al., 1998). However, these studies use denatured protein and so may not accurately represent the binding abilities of the proteins in vivo. Thus, it remains possible that the RII binding ability of pro-hAKAP82 may be altered in vivo by the removal of its amino terminus. Finally, some as yet unknown role of pro-hAKAP82 and hAKAP82 may be affected by processing. For example, multiple pathways may be co-ordinated by the binding of numerous signalling enzymes to a common AKAP (Coghlan et al., 1995; Klauck et al., 1996; Burton et al., 1997; Huang et al., 1997; Nauert et al., 1997). In this regard, it has been shown that the alternative splice variant of mAKAP82, Fsc1, is able to bind both the RI and RII subunits of PKA (Miki and Eddy, 1998). If hAKAP82, like other members of the AKAP family, acts as a scaffold for numerous signalling molecules, then its ability to bind these molecules could be affected by removal of the pro domain.
Since tyrosine phosphorylation of pro-hAKAP82/hAKAP82 was detected only in a subgroup of capacitated spermatozoa, our second hypothesis was that tyrosine phosphorylation of pro-hAKAP82/hAKAP82 would be associated with motile spermatozoa. In the studies reported here, we did not find evidence to support this hypothesis. Although statistical analysis of the ratio of tyrosine phosphorylated pro-hAKAP82 to total hAKAP82 showed no significant difference between the SU and NSU fractions, the P value was approaching the level of significance (P = 0.04, Table V
). The results were similar for the ratio of tyrosine phosphorylated hAKAP82 to total hAKAP82 (P = 0.08, Table V). The low sample numbers and high standard deviations are likely to be the reasons for the lack of significance. It is possible that, with a larger sample number, a significant difference would be identified. We intentionally chose a very stringent cut-off for significance due to the inherent variability of immunoblotting and immunofluorescence.
In this study, we did not employ any objective measure of capacitation status of the spermatozoa in our SU and NSU groups. Thus, although the two groups were subjected to identical, previously-reported capacitating conditions, it is possible that the NSU group may have contained a higher percentage of non-viable spermatozoa and so may have contained a lower absolute number of capacitated spermatozoa at the end of the incubation period than did the SU group. If this were the case, then any capacitation-associated differences in protein tyrosine phosphorylation between the NSU and SU sperm populations would have been amplified. Since we detected no significant difference in protein tyrosine phosphorylation between the two groups, any potential differences in capacitation status did not impact our results.
It is worth noting that the assays used in these studies are probably not sensitive enough to detect small differences in tyrosine phosphorylation, such as those that might be found if only select tyrosine residues within the protein(s) became phosphorylated or dephosphorylated. Alternatively, it is possible that some residues lose phosphate groups while others gain them. Such a balanced change in the overall phosphorylation status of the protein(s) also would not be detected using immunoblotting or immunofluorescence. Finally, if only a small subset of flagellar proteins (other than pro-hAKAP82 or hAKAP82) are tyrosine phosphorylated in association with differences in motility, then it is possible that immunofluorescence would not identify these differences. Thus, based solely on these studies, we can not definitively conclude whether or not tyrosine phosphorylation of flagellar proteins is associated with changes in sperm motility. In the future, it will be important to identify which tyrosine residues are phosphorylated in association with capacitation.
Tyrosine phosphorylation of mouse sperm proteins is regulated by the serine/threonine kinase PK-A (Visconti et al., 1997). Since pro-hAKAP82 and hAKAP82 are proteins that bind PK-A and are themselves tyrosine phosphorylated, it is likely that, in response to cAMP, the catalytic subunit of PK-A dissociates from the hAKAP82-anchored RII subunit and is positioned to activate local downstream tyrosine kinases. These activated tyrosine kinases then phosphorylate pro-hAKAP82 and hAKAP82. Post-translational modification of substrate proteins by phosphorylation is used in a variety of signalling pathways to regulate protein activity and examples of the effects of protein phosphorylation on other AKAPs have been reported. The phosphorylation status of the neuronal AKAP, AKAP79, may play a role in targeting of the protein to the cell membrane (Dell'Acqua et al., 1998). However, in the case of pro-mAKAP82, targeting to the fibrous sheath occurs prior to phosphorylation, making it unlikely that targeting is being directed by phosphorylation (Johnson et al., 1997). Similarly, tyrosine phosphorylation of pro-hAKAP82/hAKAP82 occurs during capacitation, well after targeting to and assembly in the fibrous sheath. Returning to the proven role of hAKAP82, that of anchoring the RII subunit of PK-A, it is possible that phosphorylation of pro-hAKAP82/hAKAP82 may affect the protein's ability to bind RII. Phosphorylation of the neuronal MAP-2, a microtubule-associated AKAP which also binds calmodulin, has been shown to reduce binding of MAP-2 to microtubules and decrease the rate of microtubule assembly (Murthy and Flavin, 1983; Lohmann et al., 1984). This raises the possibility that phosphorylation of pro-hAKAP82/hAKAP82 may affect some undetermined binding function of the AKAP, such as its ability to bind other signalling enzymes.
Because tyrosine phosphorylated proteins are present in the sperm flagellum, it has been postulated that these proteins would be related to sperm motility. In trout, the cAMP-dependent tyrosine phosphorylation of a 15 kDa protein has been closely linked to the onset of sperm motility (Hayashi et al., 1987); however similar associations have not yet been identified in other species. In fact, very few tyrosine phosphorylated mammalian flagellar proteins have been identified to date (Tash and Means, 1982; Brokaw, 1987; Tash et al., 1988; Tash and Bracho, 1994; Carrera et al., 1996). The identification and characterization of these proteins, such as pro-hAKAP82 and hAKAP82, are likely to contribute substantially to our understanding of the molecular mechanisms underlying human sperm motility. This knowledge could lead to improved clinical tests to identify defects in associated genes. While the data reported here do not identify an association of either phosphorylation or processing of pro-hAKAP82 and hAKAP82 with sperm motility, it must be kept in mind that our studies used spermatozoa from men with normal semen quality. Given this, it remains possible that an association between sperm motility and changes in phosphorylation status or processing of pro-hAKAP82/hAKAP82 may still be found in infertile, asthenozoospermic men. Spermatozoa from asthenozoospermic individuals may have more extreme differences in their ability to process and/or phosphorylate hAKAP82. In particular, spermatozoa from men with `dysplasia of the fibrous sheath' have severe fibrous sheath structural abnormalities and are immotile (Chemes et al., 1987). It is possible that defects in hAKAP82 are involved in this phenotype. In several cases, this pathology appears to be familial, making it likely that a genetic component is involved (Chemes et al., 1998). It will be important to determine if alterations in hAKAP82 are involved in this or in any other cases of naturally-occurring infertility in asthenozoospermic men. Further studies on the role of hAKAP82 in spermatozoa from both fertile and asthenozoospermic men are indicated as are studies focusing on the identification and characterization of other human sperm flagellar phosphoproteins.
Acknowledgments
Our thanks to Drs Kurt Barnhart, Laura Diaz-Cueto and Esther Noiles for their help with statistical analyses and to Drs Laura Diaz-Cueto, Linda Johnson, Gregory Kopf and Pablo Visconti for critical reading of this manuscript. This work was supported by NIH HD-01189 and HD-07305 (R.M.O.T.), NIH P01-HD06274 (G.L.G. and S.B.M.) and a University Foundation Research Grant (G.L.G.).
Notes
1 To whom correspondence should be addressed 
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