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J Biol Chem, Vol. 274, Issue 34, 24131-24136, August 20, 1999

Deletion of Type II Regulatory Subunit Delocalizes Protein Kinase A in Mouse Sperm without Affecting Motility or Fertilization^*

Kimberly A. Burton, Barbara Treash-Osio, Charles H. Muller^§, Elizabeth L. Dunphy, and G. Stanley McKnight^¶

From the Departments of  Pharmacology and ^§ Urology, University of Washington School of Medicine, Seattle, Washington 98195-7750

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Cyclic AMP stimulates sperm motility in a variety of mammalian species, but the molecular details of the intracellular signaling pathway responsible for this effect are unclear. The type II isoform of protein kinase A (PKA) is induced late in spermatogenesis and is thought to localize PKA to the flagellar apparatus where it binds cAMP and stimulates motility. A targeted disruption of the type II regulatory subunit (RII) gene allowed us to examine the role of PKA localization in sperm motility and fertility. In wild type sperm, PKA is found primarily in the detergent-resistant particulate fraction and localizes to the mitochondrial-containing midpiece and the principal piece. In mutant sperm, there is a compensatory increase in RI protein and a dramatic relocalization of PKA such that the majority of the holoenzyme now appears in the soluble fraction and colocalizes with the cytoplasmic droplet. Unexpectedly the RII mutant mice are fertile and have no significant changes in sperm motility. Our results demonstrate that the highly localized pattern of PKA seen in mature sperm is not essential for motility or fertilization.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Cyclic AMP analogues and phosphodiesterase inhibitors increase the motility of mature spermatozoa (1-4), and the phosphodiesterase inhibitor, pentoxyfylline, is routinely used to enhance the motility and acrosome reaction in sperm from infertile men (5). It has been proposed that elevated cAMP stimulates the phosphorylation of sperm proteins by protein kinase A (PKA)¹ leading to increased motility (2, 3, 6, 7). In support of this, PKA activity has been shown to rise during capacitation of motile sperm (8). However, other targets for cAMP action are possible. Cyclic nucleotide-gated ion channels are found along the flagellum of mammalian sperm (9, 10), and cAMP-regulated guanine nucleotide exchange factors are expressed in the testes (11). Either cyclic nucleotide-gated ion channels or cAMP-regulated guanine nucleotide exchange factors could conceivably mediate the stimulation of sperm motility by increases in cAMP levels.

In sperm, cAMP levels are elevated when adenylyl cyclase is activated by bicarbonate (12-14) or by calmodulin and Ca²⁺ (15, 16). The activation of the PKA holoenzyme occurs when cAMP binds to the regulatory subunit of PKA and causes the dissociation of the catalytic (C) subunit. The PKA holoenzyme is designated either type I or type II, depending on whether it contains RI or RII, and in mammalian sperm, both are expressed. In mouse postmeiotic spermatids RII predominates (17), with RII mRNA and protein being highly expressed in mature elongating spermatids (18, 19). Greater than 50% of the RII holoenzyme remains in the detergent-resistant tail fraction of mature sperm, suggesting that the majority of the RII holoenzyme is firmly attached to the flagellum (20). Although conflicting data exist on whether the RII subunit is present in the epididymal sperm head, both RII and C subunits are abundant in the midpiece and principal piece of the flagellum (21-23). These results suggest that type II PKA is the primary isoform of PKA in mature sperm and that it is tightly anchored to the particulate fraction of the sperm flagellum.

Recently it was found that PKA anchoring proteins (AKAPs) bind RII subunits with high affinity and that AKAPs also bind other signaling molecules such as calcineurin and protein kinase C (24). Multi-enzyme complexes tethered by AKAPs may position protein kinases and phosphatases near their organelle-bound substrates to promote the rapid and selective phosphorylation and dephosphorylation of target proteins. Several AKAPs have been characterized in mouse sperm including S-AKAP84 (also identified as D-AKAP1), which has been localized to the mitochondria of immature sperm but is lost during maturation (25), and AKAP82, which has been localized to the fibrous sheath of mature sperm in mouse (26). Recently, both S-AKAP84/D-AKAP1 and AKAP82 (also identified as the fibrous sheath component 1 (FSC1)) were shown to bind the RI subunit in addition to RII subunits (27, 28). The binding affinity of S-AKAP84/D-AKAP1 for RII is nearly 25-fold greater than for RI (27), suggesting that S-AKAP84/D-AKAP1 preferentially binds RII in cells expressing both RII and RI. In contrast, a region in AKAP82/FSC1 (domain B) was observed to bind RI exclusively (28), suggesting that in sperm, RII is bound to S-AKAP84/D-AKAP1 and AKAP82/FSC1, and RI is bound to AKAP82/FSC1. By using a synthetic peptide that mimics the amphipathic helix RII binding motif in AKAPs (29), the association between RII subunits and AKAPs can be disrupted (30). This synthetic peptide also blocks motility of bovine sperm (31). Together these findings have led to the proposal that PKA is anchored to AKAPs by RII in the flagellum and that this interaction is required for sperm motility.

Based upon these findings we hypothesized that gene-targeted disruption of the RII subunit of PKA would produce infertile male mice as a consequence of having sperm with compromised motility. However, unexpectedly the RII-deficient male mice are fertile, and both the curvilinear velocity and percent motility of mutant sperm are not significantly different from that of wild type sperm. Furthermore, we show that a compensatory increase in RI protein in mutant sperm correlates with a change in the fractionation and localization of PKA such that the majority of the holoenzyme is now found in the soluble fraction and cytoplasmic droplet. We conclude from these studies that anchored PKA is not essential for sperm motility.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Animals-- Wild type and RII mutant males were generated as described previously (32). Animals used in this study were 75, 87.5, or 97% C57BL/6 with the remaining genetic background as 129 Sv/J. Female C57BL/6 mice used in this study were purchased from Jackson Laboratories (Bar Harbor, ME).

Western Blot Analysis-- The cauda epididymides from adult wild type and mutant mice were removed, minced, and placed in phosphate-buffered saline (PBS). Sperm were allowed to swim out for 20 min at 37 °C. The sperm suspension was diluted in sample buffer to a final concentration of 63 mM Tris, pH 6.8, 2% sodium dodecyl sulfate, 5% glycerol, and 0.05% bromphenol blue and sonicated. DNA concentration was determined using Hoescht dye fluorescence and was used to adjust for equal sample loading on 10% polyacrylamide gels. Proteins were transferred to nitrocellulose membranes. These blots were blocked overnight with 5% bovine serum albumin and 0.1% Tween 20 and then probed with affinity purified polyclonal antibodies to RI, C, RII, or RII. After washing and incubation with horseradish peroxidase-conjugated secondary antibody, immunoreactivity was visualized with the Amersham Pharmacia Biotech ECL^TM system.

Cell Fractionation-- Cauda epididymal sperm were allowed to swim out in buffer A, which contained 12 mM KH₂PO₄, 58.5 mM NaCl, 4.8 mM KCl, 1 mM MgCl₂, 5 mM glucose, and 50 mM Tris-HCl, pH 7.4. Volumes were adjusted to provide equal concentrations (determined with a hemacytometer) of sperm from wild type and mutant mice. After gentle washing in 5 ml of cold 0.1 M PBS, cells were collected (600 × g for 10 min) and resuspended in buffer A containing protease inhibitors and 1% Triton X-100. The sperm were then frozen/thawed three times in liquid nitrogen and placed on ice for 30 min. These extracts were clarified (40,000 × g for 15 min), and the supernatant was taken as the soluble fraction. The pellet was washed once by dispersal and sedimentation as above and then resuspended in the same volume of fortified buffer A as used for the supernatant fraction (above). Equal volumes of these fractions from wild type and mutant sperm were loaded on 10% polyacrylamide gels, and PKA subunits were visualized as described above by Western blot.

Immunocytochemistry-- Sperm were recovered from the cauda epididymides as described above using PBS. Sperm were allowed to settle and attach on glass slides for 30 min then fixed in 4% paraformaldehyde and 0.05% gluteraldehyde in phosphate buffer overnight at 4 °C. Fixed sperm were washed and then blocked in 5% nonfat milk in PBS for 1 h at room temperature. Incubation with primary antibody diluted in 2% nonfat milk in PBS proceeded for 1.5 h at room temperature and then by 24 h at 4 °C. The polyclonal antibodies to RII and C were affinity purified and used at concentrations of 1:500 and 1:2000, respectively. A monoclonal antibody to RI (Transduction Laboratories) was used at a concentration of 10 µg/ml. Samples were washed, probed with a biotinylated secondary antibody, washed again, and then probed with avidin-fluorescein (1 h at 37 °C). After a final wash, a coverslip was mounted with Vectashield (Vector Labs, Inc.). Samples were viewed and photographed on a Nikon Microphot-FXA microscope.

Controls used to determine the level of nonspecific staining were subjected to the treatments described above but in the absence of primary antibody. Minimal staining was observed in these controls.

Density Gradient-- Cytoplasmic droplets were separated from sperm following a previously described protocol (33). Briefly, cauda epididymal sperm were layered on a discontinuous sucrose gradient composed of 1.5 ml of 1.0 M sucrose plus 1.0 ml of 0.25 M sucrose in PBS and then sedimented (1500 × g for 20 min). Cytoplasmic droplets were collected as a band in the 0.25 M sucrose layer while avoiding the 0.25 M/1.0 M interface and were subsequently centrifuged for 20 min at 27,000 × g. Sperm and cytoplasmic droplet pellets were initially resuspended in PBS (100 µl) and examined under the light microscope to determine the presence of sperm and droplets in each layer. Few sperm were detected in the cytoplasmic droplet pellet, but droplets were still observed attached to sperm in the sperm pellet. Both the sperm suspension and the cytoplasmic droplet suspension were then mixed with sample buffer (as described in Western analysis) to the same final volume. DNA quantification and Western analysis (as described above) were then performed on the samples. There was no detectable DNA in the cytoplasmic droplet sample. Equal amounts of sperm (as estimated by the DNA concentration) were loaded for wild type and mutant sperm samples. Equivalent volumes of cytoplasmic droplet and sperm samples were loaded.

Sperm Motility-- Males that were 6-8 months of age were used for this study. Two different protocols were used. The first used medium that supports capacitation and contains NaHCO₃ (25 mM), Ca²⁺ (1 mM), and bovine serum albumin (Fraction V, Sigma; 20 mg/ml) (34, 35). The motility of cauda epididymal sperm from males with proven fertility was assessed after 2 h of incubation in this medium. The other protocol examined the effects of cAMP analogues and used a medium that contained 135 mM NaCl, 5 mM KCl, 1 mM MgSO₄, 2 mM CaCl₂, 10 mM lactic acid, 1 mM sodium pyruvate, 30 mM Hepes, pH 7.4, and 20 mg/ml bovine serum albumin. After a 5-h incubation and a further 30 min in the presence or absence of either 1 mM Sp-cAMP or 1 mM Rp-cAMP (Research Biochemicals Inc.), sperm motility was examined.

For both protocols, quantitative sperm motility analysis on videotaped sperm samples was done by using an automated Hamilton-Thorn Motility analyzer as described (36). A slide chamber of 50-µm depth was used and motile sperm were tracked at 60 frames/s. The analysis was performed by an observer unaware of the sperm source. In the first study, 10 fields were recorded for 10 s each, and in the second study, 20 fields were recorded for 5 s each. Percent motility was determined on a minimum of 100 sperm from the recorded sperm samples. All sperm with moving flagella were counted as motile. The criteria for hyperactivation of sperm motility was defined as linearity (departure of the cell track from a straight line) less than 40%, lateral head displacement (average of sperm track width) greater than 6.5 µm, and track speed greater than 100 µm/s.

Fertility Assessment-- In this study, 10-15-week-old wild type and mutant males and 8-week-old C57BL/6 females were used. Two females were placed with each male. Females were checked daily for vaginal plugs as an indication that mating had occurred. Cohabitation continued either for 5 days at which point the females were removed for 2 days or until a plug was found. Matings continued until each male had successfully mated with at least four females. All mated females were euthanized 14 days after the discovery of the plug and the number of live and reabsorbed fetuses were counted. Pregnancy rate was calculated as the proportion of matings that produced pregnancy. Litter size was counted from implantations. The proportion of live fetuses was determined from the ratio of live fetuses to total implantations.

Assessment of Acrosomal Status-- The acrosomal status of spermatozoa was analyzed using a Pisum sativum lectin staining method (37). Sperm were incubated in the bicarbonate-free medium used for sperm motility measurements (above) for 5.5 h and then diluted 10-fold. Hoechst 33258 dye (bisbenzidine, Riedel-de Haen AG, Hanover, Germany; 1 µg/ml) was added to stain the nuclei of permeable ("dead") sperm. After 2 min of incubation the sperm were diluted 2-fold with fresh medium and collected (250 × g; 5 min). Cells were resuspended and washed by two more cycles of dilution and sedimentation. The cell pellet was resuspended in 50 µl of ice-cold ethanol and stored at 4 °C. Aliquots of the ethanol-permeabilized sperm were dried onto spots on 4-spot Teflon-coated slides (Cel-Line Associates) and hydrated with 10 mM Hepes buffer, pH 7.4, containing 150 mM NaCl, 0.1 mM CaCl₂, 0.01 mM MnCl₂, and fluorescein isothiocyanate-labeled P. sativum (Vector Laboratories, 25 µg/ml). After 20-30 min, the slides were washed in distilled water, mounted in buffered 90% glycerol solution (containing 0.05 M Tris, pH 8.5, and 1% 1,4-diazabicyclo-(2,2,2)octane; Sigma). Stained sperm were examined by epifluorescence microscopy under fluorescein isothiocyanate, UV, and phase contrast conditions on a Nikon Labophot-2 microscope. For each genotype, 100-200 sperm that had excluded the Hoechst dye were scored for acrosomal loss. Sperm with intact acrosomes were easily distinguished by the presence of a broad, uniform, brightly fluorescent arc corresponding to the anterior or dorsal portion of the acrosome. Sperm that had lost their acrosomes had only diffuse, less intense staining in the equatorial segment. Only sperm oriented so as to display the acrosomal crescent were assessed.

Statistical Analysis-- Unpaired t tests were performed when comparing between wild type and mutant groups.

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

The initial characterization of mice that contain a targeted deletion of the RII gene was described in a previous report (32). It was shown that the RII protein is absent in all tissues examined including the testis and that these mice are viable and healthy.

Compensation in RII Mutant Sperm-- To examine the effect of a genetic deletion of RII on the expression of PKA subunits in sperm, we performed a Western analysis on sperm samples from wild type and mutant mice. As shown in Fig. 1, the mutant sperm are completely deficient in RII protein. However, RI protein levels are elevated severalfold compared with wild type sperm. There is no apparent change in C protein content of mutant sperm. RII and RI protein were not detected in wild type or mutant sperm (data not shown). The compensatory increase in the expression of RI is most likely the result of its increased association with C subunit that is no longer bound to RII. We have observed a similar increase in RI subunit expression in the RII mutant mouse and determined that the RI subunit has an increased half-life presumably as a consequence of its sequestration in the holoenzyme complex (38). The lack of a difference in C subunit content of mutant and wild type sperm suggests that the association with RI prevents a change in C subunit levels. In summary, the loss of RII in mutant sperm results in a compensatory increase in RI with no change in C subunit levels.

View larger version (43K): [in this window] [in a new window]   Fig. 1.   Compensation by RI in RII mutant sperm. Western blot comparing levels of PKA subunits in wild type (+/+, n = 3) and RII mutant (/, n = 3) sperm using antibodies to RII, RI, and C. Equal amounts of sperm extract (10 µg of DNA) were loaded in each lane.

Particulate Association of PKA in Sperm-- The RI and RII isoforms differ in their affinity for various anchoring proteins (AKAPs). Therefore we anticipated that a shift from the expression of RII in wild type sperm to RI in mutant sperm would alter the intracellular localization of PKA. An examination of the partitioning of PKA subunits into the detergent-soluble and detergent-insoluble fraction (Fig. 2) confirms this prediction. In wild type sperm, RII and C protein are found in both the detergent-soluble and detergent-insoluble fractions. RI protein content in both fractions is much lower. With longer exposure times these immunoblots show that RI protein is present in equivalent amounts in both fractions of sperm (data not shown). By contrast, in mutant sperm RII protein is absent in both fractions, and RI and C proteins are detected primarily in the detergent-soluble fraction. These results suggest that in mutant sperm, PKA is no longer bound to the detergent-resistant structures but is found primarily in the soluble fraction.

View larger version (43K): [in this window] [in a new window]   Fig. 2.   Solubilization of C subunit in RII mutant sperm. A comparison of PKA subunit levels in detergent-soluble (Sup) and detergent-insoluble (Pellet) fractions of wild type (+/+) and RII mutant (/) sperm homogenized in buffer containing 1% Triton X-100 and centrifuged at 40,000 × g. Antibodies to RII, RI, and C were used on three separate experiments, and a representative figure is shown.

Immunolocalization of PKA Subunits in Mutant Sperm-- To confirm the differences in PKA distribution within mutant and wild type sperm, as indicated by cell fractionation, we examined the immunolocalization of PKA subunits in sperm (Fig. 3). As shown in Fig. 3a, RII immunoreactivity was found in the flagellum of wild type sperm. The staining was greater in the midpiece and in the distal portion of the principle piece. No RII immunoreactivity was detected in the mutant sperm (Fig. 3d). Fig. 3b shows that RI immunoreactivity was undetectable in wild type sperm. However, in mutant sperm (Fig. 3e), RI immunoreactivity was strong in the cytoplasmic droplet. C and RII were similarly distributed along the flagellum (with higher staining in the midpiece and in the end of the principal piece) in wild type sperm. Interestingly, in mutant sperm, C and RI were colocalized. C immunoreactivity was limited to the cytoplasmic droplet (Fig. 3f) for the vast majority of cells. In less than 10% of the mutant sperm, C staining was also faintly detectable in the midpiece (data not shown). Immunocytochemistry performed on wild type and mutant sperm in the absence of primary antibody produced samples with no staining (data not shown).

View larger version (31K): [in this window] [in a new window]   Fig. 3.   Immunolocalization of PKA subunits in sperm. Immunocytochemistry was performed on wild type (Wt, a-c) and RII mutant (d-f) sperm. Sperm were incubated with an antibody to either RII (a and d), RI (b and e), or C (c and f). Arrowhead, sperm head; arrow, cytoplasmic droplet.

The colocalization of RI and C subunit suggests that the type I PKA holoenzyme (RI₂C₂) is found primarily in the cytoplasmic droplet of mutant sperm. The density gradient studies in Fig. 4 support this interpretation. Cytoplasmic droplets were separated from whole sperm by a discontinuous sucrose gradient, and protein extracts from both were separated by SDS-polyacrylamide gel electrophoresis and probed for the presence of PKA subunits. It is important to note that the gradient separation is only partially successful with a considerable level of cytoplasmic droplet containing sperm still present in the whole sperm fraction but only a few sperm contaminating the droplet fraction. For wild type sperm, RII and C subunits were found primarily in the whole sperm fraction with only low levels appearing in the cytoplasmic droplet fraction. RI protein was not detected in either fraction. For mutant sperm, C and RI were present in both the cytoplasmic droplet and whole sperm fractions. In summary, by both immunocytochemical and cell fractionation studies, we find that PKA is no longer anchored to the flagellum but is redistributed to the cytoplasmic droplet.

View larger version (36K): [in this window] [in a new window]   Fig. 4.   C subunit in cytoplasmic droplets of RII mutant sperm. Western blot comparing levels of PKA subunits in whole sperm (Sperm) and cytoplasmic droplets (Droplets) separated by discontinuous sucrose gradient. Antibodies to RI and C were used on wild type (+/+) and mutant (/) sperm and droplets. A representative experiment is shown. Similar results were obtained in two other experiments.

Fertility and Sperm Motility in RII Mutant Mice-- The unchanged levels of C and enhanced expression of RI in sperm of mutant mice might allow PKA to fulfill some of its normal functions in sperm. However, the loss of anchoring of PKA to the flagellum suggested that deficits in motility (and therefore fertility) would occur. We examined this hypothesis by comparing the fertility of wild type and mutant males. These mice were mated with females until four plugged females were identified. On day 14 of gestation, plugged females were euthanized, and litter size was measured. Results are shown in Table I. There was no significant difference between wild type and mutant males in length of time to plug four females, pregnancy rate, litter size, or percentage of live fetuses.

View this table: [in this window] [in a new window]   Table I Male fertility in wild type and RII knockout mice

Despite the indication that mutant mice have normal fertility, we reasoned that more subtle defects in sperm function might be revealed by a comparison of motility parameters for wild type and mutant sperm. Sperm were incubated in vitro either in a medium that supports capacitation or in a simpler medium that was supplemented with permeant cAMP analogues. Fig. 5 (a and b) shows that in the capacitating medium, swimming speed and the proportion of motile sperm were not significantly different for mutant and control animals, and similar results were obtained when motility was examined in a simpler, bicarbonate-free medium (Fig. 5, c and d). The percentage of sperm that were hyperactivated was no different between wild type and mutant sperm in either medium (data not shown). The effects of both Sp-cAMP in stimulating and Rp-cAMP in inhibiting sperm motility were small or absent, respectively, indicating that once initiated, sperm motility is independent of PKA activity. In summary, these results indicate that mutant male mice are fertile and that mutant sperm motility is not significantly different from that of wild type.

View larger version (26K): [in this window] [in a new window]   Fig. 5.   A comparison of motility between RII mutant and wild type sperm. Motility analysis of sperm from wild type (+/+) and mutant (/) mice in media with (a and b) and without (c and d) NaHCO3. Curvilinear velocity (a and c) and the percentage of motility (b and d) in the absence and presence of 1 mM Sp-cAMP or 1 mM Rp-cAMP (c and d). Numbers of animals used in each experiment are shown.

Acrosome Reaction of RII Mutant Sperm-- Although most attention has been directed to the role of PKA in the control of sperm motility, some reports indicate that sperm cAMP content increases under conditions that promote spontaneous acrosome reaction (3). Therefore we also considered the possibility that alterations in the composition and localization of PKA in mutant sperm might alter this measure of sperm exocytotic function. However, we observed no difference in the proportion of wild type and mutant sperm that had undergone the acrosome reaction (wild type, 36 ± 4%; knockout, 32 ± 3%). These results indicate that RII-deficient sperm are not significantly different than wild type sperm when comparing the ability of the sperm to release acrosomal contents.

	DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Despite much study, the intracellular signaling pathways that govern sperm motility have not been defined. Considerable attention has focused on the cAMP system because it was shown that drugs that elevate cAMP (phosphodiesterase inhibitors) lead to increased motility. Until recently, the only pathway for cAMP action in sperm was assumed to be PKA, and this assumption was bolstered by the observed induction of the RII regulatory subunit late in spermatogenesis and the expression of several high affinity PKA anchoring proteins associated with either the mitochondria (S-AKAP84/D-AKAP1) or the fibrous sheath (AKAP82/FSC1). However, the recent demonstrations of a cyclic nucleotide-gated ion channel in sperm and of the cAMP-mediated guanine nucleotide exchange factors in testes provide additional pathways by which cAMP might act to stimulate motility. With these possibilities in mind, we have reinvestigated the potential role of PKA in stimulating sperm motility. Our approach was to produce a mutation in the major sperm regulatory subunit gene, RII, that eliminates its expression. This study shows that male mice lacking RII protein are fertile and that the motility of sperm from mutant mice is not impaired.

RI subunit levels are dramatically elevated in mutant sperm compared with levels in wild type sperm. This compensation by RI has been observed in other tissues of RII mutants (32) and RII mutants (39). The increase in RI levels in sperm from RII mutants most likely results from increased protein stabilization of RI in a holoenzyme complex that serves to protect the sperm from unregulated C subunit activity (38). As a consequence of the increased levels in RI subunit, C subunit is bound in the stable holoenzyme complex and its levels are therefore not altered in mutant sperm.

The PKA isozyme shift from a predominantly type II PKA, as seen in wild type sperm (17, 20), to a type I PKA in mutant sperm correlates with a change in the intracellular distribution of the C subunit. In wild type sperm, a major fraction of RII and C subunits is found in the Triton-insoluble pellet. By immunocytochemistry, these subunits are localized in the flagellum with highest levels in the midpiece. This pattern of RII and C staining has been observed by others (22, 23, 40). In mutant sperm, RI and C subunits are found primarily in the Triton-soluble fraction and, by immunocytochemistry and density gradient experiments, in the cytoplasmic droplet. The fractionation studies demonstrate that the detergent-soluble type I PKA in mutant sperm is not anchored to the flagellum and is, therefore, more readily released into the Triton-soluble fraction. Recent biochemical studies have shown that RI is capable of binding to a subset of the AKAPs identified in sperm including AKAP82/FSC1, which forms part of the fibrous sheath surrounding the flagellum (28), and SAKAP-84/D-AKAP1, which localizes to mitochondria (27) but is found only in immature sperm and is lost during maturation (25). The relative affinity of FSC1 for RI and RII has not been measured, but the apparent dissociation of PKA from the flagellum in RII mutant sperm suggests that the interaction of RI with FSC1 is not strong enough to localize the holoenzyme in vivo. S-AKAP84/D-AKAP1 binds to RI with about a 25-fold weaker affinity compared with RII, but this may be sufficient to localize PKA to mitochondria during the elongating spermatid stage. As the sperm mature, S-AKAP84/D-AKAP1 is lost, and we would not expect to see mitochondrial localization in mature sperm of an RI-containing PKA holoenzyme.

Although RI is not the major regulatory subunit expressed in mature sperm, it is present in low but detectable amounts in both the pellet and Triton-soluble fractions of wild type sperm. However, by immunocytochemistry, this subunit was not detected in wild type sperm. This result is not in agreement with previous reports in which RI was found throughout the entire sperm (8, 41) and suggests that our assay is not sensitive enough to detect small amounts of RI. This raises the possibility that small but undetectable amounts of RI may be associated with the flagellum of RII mutant sperm. If PKA exists as a holoenzyme in RII mutant sperm, then the C subunit should also be detected along its flagellum. However, in the majority of RII mutant sperm, C subunit was found in the cytoplasmic droplet, a localization that was dramatically different from that observed in wild type sperm flagellum. Although C and RI subunits were not detected along the flagellum in RII mutant sperm, it is possible that a small amount of PKA is associated with the flagellum and stimulates sperm motility.

Our demonstration that the majority of PKA is not anchored to the flagellum in RII mutant sperm with no deleterious effects on sperm motility does not support the findings of Vijayarghavan et al. (31), who showed that the motility of bovine and primate sperm was inhibited by a cell-permeant peptide (stearated Ht31) that blocks the association of PKA with AKAPs and presumably prevents the phosphorylation of key PKA substrates. This discrepancy suggests either a species difference in the dependence of sperm motility on anchored PKA or a nonspecific effect of stearated Ht31. Indeed, if stearated Ht31 blocked the ability of PKA to phosphorylate those substrate proteins required for motility, then it would be predicted that stearated PKI, an inhibitor of PKA activity, would likewise block motility. However, in this same study, stearated PKI did not block motility, which leads one to question the specificity of these stearated peptides.

RII knockout mice are fertile. Although the sample size in this fertility study is too small to detect small differences in pregnancy rate (42), this study, as well as ongoing successful breeding of the mutant mice for 3 years, demonstrates that the RII protein is not required for successful reproduction. This result is supported by our findings that there is no significant deficit in sperm motility or in the ability of mutant sperm to undergo the acrosome reaction. It is conceivable, however, that subtle deficits in fertilization capability are present in RII mutant sperm that would be revealed by a sperm competition assay, as was shown with acrosin knockout mouse sperm (43), but this awaits further study.

Our findings clearly indicate that RII is not essential for sperm motility and fertilization. In addition the loss of anchored PKA along the flagellum, as a result of the compensation by RI, does not observably affect normal sperm function. The presence of PKA primarily in the cytoplasmic droplet of motile mutant sperm raises the possibility that PKA is not required for motility of mature sperm and that other targets of cAMP action are mediating its effects. It remains to be determined, however, whether PKA is anchored in earlier stages of spermatogenesis in the RII mutant by the interaction of RI with AKAPs that are expressed in immature sperm, such as S-AKAP84/D-AKAP1, and whether this localization is essential for the maturation of sperm.

	ACKNOWLEDGEMENT

We thank Donner Babcock for helpful suggestions on the experiments and critical comments on the manuscript.

	FOOTNOTES

^* This work was supported by the NICHD, National Institutes of Health through Cooperative Agreement U54-HD12629 as part of the Specialized Cooperative Centers Program in Reproduction Research. This work was also supported by National Institutes of Health Grant 5 F32 HD08034-03 (to K. A. B.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

^¶ To whom correspondence should be addressed: Dept. of Pharmacology, University of Washington School of Medicine, Box 357750, Seattle, WA 98195-7750. Tel.: 206-616-4237; Fax: 206-616-4230; E-mail: mcknight@u.washington.edu.

	ABBREVIATIONS

The abbreviations used are: PKA, cAMP-dependent protein kinase; AKAP, A-kinase anchoring protein; C subunit, catalytic subunit; FSC1, fibrous sheath component 1; PBS, phosphate-buffered saline.

	REFERENCES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

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				K. S. Nadella, G. N. Jones, A. Trimboli, C. A. Stratakis, G. Leone, and L. S. Kirschner Targeted Deletion of Prkar1a Reveals a Role for Protein Kinase A in Mesenchymal-to-Epithelial Transition Cancer Res., April 15, 2008; 68(8): 2671 - 2677. [Abstract] [Full Text] [PDF]

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				K. K. Stein, J. C. Go, P. Primakoff, and D. G. Myles Defects in Secretory Pathway Trafficking During Sperm Development in Adam2 Knockout Mice Biol Reprod, November 1, 2005; 73(5): 1032 - 1038. [Abstract] [Full Text] [PDF]

				R. V. Schillace, S. F. Andrews, S. G. Galligan, K. A. Burton, H. J. Starks, H. G. A. Bouwer, G. S. McKnight, M. P. Davey, and D. W. Carr The Role of Protein Kinase A Anchoring via the RII{alpha} Regulatory Subunit in the Murine Immune System J. Immunol., June 1, 2005; 174(11): 6847 - 6853. [Abstract] [Full Text] [PDF]

				I. Bossis and C. A. Stratakis Minireview: PRKAR1A: Normal and Abnormal Functions Endocrinology, December 1, 2004; 145(12): 5452 - 5458. [Abstract] [Full Text] [PDF]

				M. A. Nolan, D. F. Babcock, G. Wennemuth, W. Brown, K. A. Burton, and G. S. McKnight Sperm-specific protein kinase A catalytic subunit C{alpha}2 orchestrates cAMP signaling for male fertility PNAS, September 14, 2004; 101(37): 13483 - 13488. [Abstract] [Full Text] [PDF]

				M. Luconi, V. Carloni, F. Marra, P. Ferruzzi, G. Forti, and E. Baldi Increased phosphorylation of AKAP by inhibition of phosphatidylinositol 3-kinase enhances human sperm motility through tail recruitment of protein kinase A J. Cell Sci., March 1, 2004; 117(7): 1235 - 1246. [Abstract] [Full Text] [PDF]

				K. TASKEN and E. M. AANDAHL Localized Effects of cAMP Mediated by Distinct Routes of Protein Kinase A Physiol Rev, January 1, 2004; 84(1): 137 - 167. [Abstract] [Full Text] [PDF]

				R. M. Turner Tales From the Tail: What Do We Really Know About Sperm Motility? J Androl, November 1, 2003; 24(6): 790 - 803. [Full Text] [PDF]

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				M. D. Anway, N. Ravindranath, M. Dym, and M. D. Griswold Identification of a Murine Testis Complementary DNA Encoding a Homolog to Human A-Kinase Anchoring Protein-Associated Sperm Protein Biol Reprod, June 1, 2002; 66(6): 1755 - 1761. [Abstract] [Full Text] [PDF]

				B. S. Skalhegg, Y. Huang, T. Su, R. L. Idzerda, G. S. McKnight, and K. A. Burton Mutation of the C{alpha} Subunit of PKA Leads to Growth Retardation and Sperm Dysfunction Mol. Endocrinol., March 1, 2002; 16(3): 630 - 639. [Abstract] [Full Text] [PDF]

				S. M. Jackson and C. A. Berg An A-kinase anchoring protein is required for Protein kinase A regulatory subunit localization and morphology of actin structures during oogenesis in Drosophila Development, January 10, 2002; 129(19): 4423 - 4433. [Abstract] [Full Text] [PDF]

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				J. T. San Agustin and G. B. Witman Differential Expression of the Cs and C{{alpha}}1 Isoforms of the Catalytic Subunit of Cyclic 3',5'-Adenosine Monophosphate-Dependent Protein Kinase Testicular Cells Biol Reprod, July 1, 2001; 65(1): 151 - 164. [Abstract] [Full Text] [PDF]

				M. Belyamani, E. A. Gangolli, and R. L. Idzerda Reproductive Function in Protein Kinase Inhibitor-Deficient Mice Mol. Cell. Biol., June 15, 2001; 21(12): 3959 - 3963. [Abstract] [Full Text]

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				J.-L. Desseyn, K. A. Burton, and G. S. McKnight Expression of a nonmyristylated variant of the catalytic subunit of protein kinase A during male germ-cell development PNAS, June 6, 2000; 97(12): 6433 - 6438. [Abstract] [Full Text] [PDF]

				J. Pan and W. J. Snell Regulated Targeting of a Protein Kinase into an Intact Flagellum. AN AURORA/Ipl1p-LIKE PROTEIN KINASE TRANSLOCATES FROM THE CELL BODY INTO THE FLAGELLA DURING GAMETE ACTIVATION IN CHLAMYDOMONAS J. Biol. Chem., July 28, 2000; 275(31): 24106 - 24114. [Abstract] [Full Text] [PDF]

				D. W. Carr, A. Fujita, C. L. Stentz, G. A. Liberty, G. E. Olson, and S. Narumiya Identification of Sperm-specific Proteins That Interact with A-kinase Anchoring Proteins in a Manner Similar to the Type II Regulatory Subunit of PKA J. Biol. Chem., May 11, 2001; 276(20): 17332 - 17338. [Abstract] [Full Text] [PDF]

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