Association of sperm protein 17 with A-kinase anchoring protein 3 in flagella

AKAP3 (a.a. 1–200) cDNA was synthesized from mouse testis total RNA by reverse transcription PCR and verified by sequencing. The cDNA was ligated into pGST-4T-1 expression vector (Amersham Pharmacia Biotech) and GST-fusion proteins of AKAP3, AKAP4 and GST alone expressed as described [14]. Fusion proteins were separated from crude bacterial extracts (1–3 μl) by incubation with 25 μl glutathione-agarose resin (Sigma Chemical Co., St Louis, MO) for 30 minutes, 4°C. Non-specifically bound bacterial proteins were removed by extensive washing with PBS.

Lysates were prepared from washed human spermatozoa by incubation in CHAPS/PBS buffer (127 mM NaCl, 6.7 mM Na₂PO₄, 3.25 mM NaH₂PO₄, pH 7.0 and 5 mM CHAPS (3-[(cholamidopropyl) dimethylammonio]-1-propane sulfonate)) containing Protease Inhibitor Cocktail (Sigma Chemical Co., St. Louis MO) for 30–60 minutes, 4°C. Following this, spermatozoa were sonicated, centrifuged 700 g for 5 minutes and the supernatant removed. Aliquots of spermatozoa lysate (400 μg) were then added to agarose immobilized GST-AKAP3, GST-AKAP4 or GST alone, the total volume adjusted to 0.5 ml with PBS and proteins allowed to bind for 2 hours at 4°C. After washing extensively with PBS to remove unbound proteins, SDS sample buffer containing 5% β-mercaptoethanol was added and proteins eluted by boiling for 5 minutes.

SDS-PAGE and Western blots were carried out according to methods previously described [1‐3]. Blots were blocked with TBST (50 mM Tris pH 7.4, 150 mM NaCl with 0.05% Tween 20) containing 2% non-fat milk for 30–60 minutes, room temperature (RT) and probed with either rabbit anti-recombinant human Sp17 (anti-Sp17) IgG (1:2000) or normal rabbit IgG (1:2000) in TBST containing 1% non-fat milk for 1–2 hours. Antibody binding was detected with alkaline phosphatase conjugated goat anti-rabbit IgG Fc (1:1000) in the same buffer for 1 hour at room temperature. Blots were developed with NBT/BCIP (nitro blue tetrazolium/5-bromo-4chloro-3-indolyl phosphate) as substrate. Chemiluminescent staining was performed using Supersignal West Pico Chemiluminescent Substrate (Pierce, Rockford, IL) according to the manufacturer's instructions.

Human spermatozoa (gift of Dr. S. Beyler, University of North Carolina Hospital, Chapel Hill, NC) were washed in BWW medium by centrifugation at 500 g for 5 minutes, permeabilized with 1% Triton X-100, 15 minutes, washed twice and resuspended in PBS. Aliquots of spermatozoa were air dried onto slides and then fixed in methanol for 10 minutes (-20°C). For indirect immunofluorescence, spermatozoa were probed with rat antiserum to recombinant human AKAP3 (rFSP95, gift of Dr J. Herr, University of Virginia, VA; [22]) at 1:50 dilution and anti-Sp17 IgG at 1:100. Antibody binding was detected with Alexa Fluor-488 labeled goat anti-rat IgG (Molecular Probes, Eugene OR) and Alexa Fluor-568 labeled goat anti-rabbit IgG (Molecular Probes, Eugene, OR) at 1:5000 dilution. DNA was visualized with DAPI diluted 1:250,000 from a 5 mg/ml stock.

Fluorescent images were obtained from a Zeiss Axiophot microscope equipped with Plan-apochromat 63× 1.4 NA (numerical aperture), Plan-neofluar 100× 1.3 NA and 40× 0.75 NA objectives, chroma filter sets (Chroma Technology Corp., Brattleboro, VT), and Zeiss filter set 25 (emission at 460, 530 and 610 nm) that allows detection of co-localized Alexa Fluor 488 and Alexa Fluor 568 probes (Molecular Probes, Eugene OR). Images were recorded with a Zeiss AxioCam using AxioVision software (Zeiss MicroImaging, Thornwood, NY) and exported to Adobe Photoshop^® 5.0 to assemble the figures.

Mouse spermatozoa extracts were prepared from epididymal mouse spermatozoa allowed to swim out of the epididymis at 37°C, pelleted at 800 g and washed twice in PBS. Spermatozoa were lysed in sample buffer and loaded at 5 × 10⁶ /well. Western blots of fibrous sheath preparations [26], prepared from CD-1 mouse epididymal spermatozoa, were kindly provided by Dr. D. O'Brien, University of North Carolina at Chapel Hill.

Although Sp17 has been immunoprecipitated from the soluble fraction of spermatozoan lysates if they are prepared with SDS [2, 10], the majority of Sp17 is present in the insoluble fraction where it is inaccessible except under strong detergent or denaturing conditions. Figure 1 demonstrates the distribution of Sp17 between the soluble and pellet fractions in a CHAPS extract of human spermatozoa. The amount of Sp17 in a soluble fraction of spermatozoa will differ depending upon the extraction conditions, detergents and buffers employed. As reported previously [4] for human spermatozoa, two groups of immunoreactive proteins are present, 22–24 kDa and 19–20 kDa (fig. 1 arrows). To investigate Sp17 in the fibrous sheath of flagella and its association with the most abundant AKAPs in spermatozoa, namely AKAP3 (AKAP110) and AKAP4 (AKAP82), which make up most of the insoluble fibrous sheath [27], we utilized recombinant proteins and in vitro pull down assays. Recombinant AKAP3, encompassing the RII binding domain (a.a. 121–141 [28]), and recombinant AKAP4, containing the dual specific RI/RII binding domain (a.a. 220–229 [14]), were synthesized as GST fusion proteins. The GST fusion proteins were immobilized on glutathione-agarose resin and added to CHAPS extracts of human spermatozoa (400 μg) that had been sonicated to release the Sp17. Western blots of bound and eluted proteins from the immobilized GST fusion proteins, probed with anti-Sp17, revealed that AKAP3 bound and precipitated a significant level of Sp17 while AKAP4 did not (Fig. 2A). As far as we could determine, most of the CHAPS-sonicated solubilized Sp17 bound to AKAP3.

This result demonstrates that Sp17 has specificity for particular AKAPs and does not indiscriminately bind all AKAPs. Moreover when recombinant GST alone was used in the binding assay, no Sp17 was precipitated indicating that GST does not interact with Sp17 (Fig. 2A). Amido black staining of mock pull down assays in which no spermatozoan lysate was added revealed similar levels of recombinant GST protein in each assay (Fig. 2A). Therefore differential levels of Sp17 binding to AKAP3 and AKAP4 were not the result of differences in the amount of AKAP protein added to the assays.

Similar binding experiments are impossible to perform with native AKAP3 and AKAP4 because these proteins are highly insoluble. Therefore we utilized spermatozoa from AKAP4 knockout (KO) mice to analyze the interaction of Sp17 with AKAPs in vivo. These mice were generated through targeted disruption of the Akap4 gene resulting in complete loss of AKAP4 [15]. In spermatozoa from these mice, the FS forms incompletely, the principal piece is shorter and they do not exhibit progressive motility [15]. Interestingly the loss of AKAP4 also resulted in a significant reduction or absence of other proteins typically associated with the fibrous sheath: AKAP3, RII alpha and GAPDS [15]. Since AKAP4 binds AKAP3 [16] and we have shown that Sp17 binds AKAP3 in vitro, a reduction in the binding of Sp17 might therefore be expected in AKAP4 KO spermatozoa. To assess this we performed Western blots of spermatozoa lysates from 5 × 10⁶ wild type and AKAP4 KO spermatozoa. Probing with antibodies to Sp17 demonstrated Sp17 in both populations of spermatozoa. Significantly though there was a reduction in the amount of Sp17 present in AKAP4 KO spermatozoa when compared to wild type (Fig. 2B). To quantitate this change in protein level, densitometric scanning of the blot was performed using GelExpert software (NucleoTech Corporation). A 27% reduction in signal intensity was observed in AKAP4 knockout compared to wild type spermatozoa (figure 2C).

Since AKAPs 3 and 4 are located in the fibrous sheath (FS) [27] we examined whether Sp17 was also present. Isolation of the FS from spermatozoa has been achieved by its lack of solubility in successive extractions with Triton X-100, potassium thiocyanate and 4 M urea [26]. Western blots of FS preparations probed with anti-Sp17 demonstrated that Sp17 was present and similar levels of Sp17 were found in equivalent numbers of lysed spermatozoa in SDS sample buffer (Fig. 2D).

To determine whether or not AKAP3 and Sp17 co-localize in flagella, double labeling experiments with rat anti-AKAP3 and rabbit anti-Sp17 were performed. As published previously AKAP3 is found in the principal piece of human spermatozoa (FSP95; [22]) and Sp17 is found in the head and tail of monkey [29] and mouse [10] spermatozoa. As shown in figure 3A Sp17 (red) is found in the principal piece, mid piece and in scattered patches throughout the head region. AKAP3 (green, fig. 3B) is found in the principal piece of human spermatozoa. Figure 3C demonstrates the co-localization of AKAP3 and Sp17 along the length of the principal piece where the AKAP3 signal (green) and the Sp17 signal (red) merge into small aggregates of yellow staining. When spermatozoa were probed with pre-immune antibodies for either AKAP3 or Sp17, no fluorescence was detectable and spermatozoa labeled with Alexa Fluor 488 or Alexa Fluor 568 labeled goat anti-rabbit IgG or anti-rat IgG secondary antibodies only, did not fluoresce (data not shown).