WAVE1, an A-kinase anchoring protein, during mammalian spermatogenesis

Vanesa Y. Rawe1,4, João Ramalho-Santos1,2, Christopher Payne1, Hector E. Chemes3 and Gerald Schatten1

1 Pittsburgh Development Center, Magee–Women's Research Institute, Departments of Obstetrics, Gynecology and Reproductive Sciences, and Cell Biology and Physiology, University of Pittsburgh School of Medicine, Pittsburgh, PA 15213, USA, 2 Center for Neuroscience and Cell Biology, Department of Zoology, University of Coimbra, 3004-517 Coimbra, Portugal, 3 Laboratory of Testicular Physiology and Pathology, Center for Research in Endocrinology, National Research Council (CONICET), Endocrinology Division, Buenos Aires Children's Hospital, C1425EFD Buenos Aires, Argentina

4 To whom correspondence should be addressed at: Centro de Estudios en Ginecologia y Reproduccion, Viamonte 1438, (1055) Buenos Aires, Argentina. Email: vanerawe{at}hotmail.com


    Abstract
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
BACKGROUND: Proper compartmentalization of signalling cascades is paramount to many intracellular activities during spermatogenesis and sperm function. In the present study we focus on the A-kinase-anchoring protein (AKAP) WAVE1, a member of the Wiskott–Aldrich syndrome (WASP) family of adaptor proteins, to study its localization throughout mammalian spermatogenesis. METHODS: Using transmission electron microscopy, immunocytochemistry and western blotting, we examined the distribution of WAVE1 and putative partners during mammalian spermatogenesis. The localization and association of PKA RII, the regulatory subunit II of protein kinase A, tyrosine kinase Abl, and small GTPase RAC1 were also explored. RESULTS: WAVE1 localization in spermatocytes and round spermatids coincided with Golgi apparatus distribution, whereas in elongated spermatids and testicular sperm WAVE1 localized to the mitochondrial sheath. Following epididymal passage, WAVE1 was found exclusively on the mitochondrial sheath, suggesting that the protein may function in this region. WAVE1 and PKA RII co-localized along the mitochondrial sheath, PKA RII concentrates in the mid-piece, and RAC1 associated with the post-acrosomal region and the connecting piece. The distribution of WAVE1, PKA RII and RAC1 is conserved in mature mouse, bull, baboon and human sperm. CONCLUSIONS: The data support the possibility of a functional signalling unit established by WAVE1 and its associated proteins in the mid-piece of maturing sperm.

Key words: AKAP/Golgi/mitochondrial sheath/sperm/WAVE1


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
Key cellular processes, including growth, differentiation, metabolism and motility, are regulated by cyclic AMP (cAMP; Scott, 1991Go; Francis and Corbin, 1994Go). The primary target for cAMP is cAMP-dependent protein kinase (PKA) (Beebe and Corbin, 1986Go). PKA consists of a regulatory (R) subunit dimer and two catalytic (C) subunits (Diviani and Scott, 2001Go). In sperm, phosphorylation of proteins through the activation of PKA by cAMP elicits initiation and maintenance of flagellar movement required by mature sperm (Tash and Means, 1982Go; Brokaw, 1987Go). Although the underlying mechanisms are still unknown, sperm have been shown to contain distinct adenylyl cyclase PKA type I and II isozymes, and phosphodiesterases (Gordeladze and Hansson, 1981Go; Landmark et al., 1993Go, Buck et al., 1999Go; Salanova et al., 1999Go), implying that sperm have the machinery to generate and execute cAMP effects. Protein phosphatase 1 g2 (PP1g2) is also present in sperm and has been shown to influence sperm motility (Smith et al., 1996Go), suggesting that dephosphorylation events are important for sperm function.

Intracellular organization of PKA is controlled through its association with A-kinase-anchoring proteins (AKAP; Rubin, 1994Go; Colledge and Scott, 1999Go). Each member of the AKAP family of proteins contains two functional motifs: a conserved PKA-binding region and a targeting region that directs the PKA–AKAP complex to defined subcellular locations (Carr et al., 1991Go; Colledge and Scott, 1999Go; Edwards and Scott, 2000Go). One important aspect of AKAP is their ability to simultaneously interact with multiple enzymes, integrate signalling pathways, and regulate the phosphorylation of specific cellular substrates (Colledge and Scott, 1999Go). Anchoring of PKA through AKAP to distinct intracellular sites in sperm is believed to be essential for regulating sperm motility, as the disruption of the AKAP–PKA interaction results in motility arrest (Vijayaraghavan et al., 1997Go). Several AKAP have been identified in sperm, most notably in the flagellum (Carrera et al., 1994Go; Miki and Eddy, 1998Go; Turner et al., 1999Go; Vijayaraghavan et al., 1999Go), mid-piece (Lin et al., 1995Go), and acrosomal region (Vijayaraghavan et al., 1999Go). Thus, anchoring of PKA appears to be important for sperm function and, consequently, for fertility.

WAVE1, a member of the Wiskott–Aldrich syndrome (WASP) family of adaptor proteins, has recently been identified as an AKAP that targets to actin (Westphal et al., 2000Go). WASP family proteins provide a molecular bridge that functionally couples individual Rho GTPases to the Arp2/3 complex, a group of seven related proteins that nucleate actin during its polymerization (Higgs and Pollard, 1999Go; Machesky and Gould, 1999Go). Selective interaction of these small GTPases with specific PKA-binding proteins leads to distinct actin-remodelling events. Specifically, RAC1, a member of the Rho family of small GTPases, is known to induce WAVE in somatic cells (Ridley and Hall, 1992Go; Miki et al., 1998Go). The regulation of these events, however, is not completely understood. WAVE1 binds to both PKA and the Abl tyrosine kinase (Westphal et al., 2000Go), and is sequestered inside the nucleus in Swiss 3T3 and HEK 293 cells. Although the function and nuclear localization of WAVE1 are not entirely clear, one possibility is that WAVE1 participates in signalling events to help reorganize the cytoskeletal architecture through its interaction with PKA RII, Abl, p21 Arc (a component of the Arp2/3 complex) and the G-actin binding protein profilin (Mullins, 2000Go; Sasaki et al., 2000Go; Volkmann et al., 2001Go).

In the present study we focused on WAVE1, PKA RII and RAC1 localization and compartmentalization during mammalian spermatogenesis.


    Materials and methods
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
Chemicals
All chemicals were obtained from Sigma Chemical Co. (USA), unless otherwise stated.

Antibodies and live/dead sperm staining
Affinity-purified goat polyclonal antibody, raised against a peptide mapping near the carboxy terminus of WAVE1 (human origin), and affinity-purified rabbit polyclonal antibody against RAC1 p21 were obtained from Santa Cruz Biotechnology (USA). A monoclonal mouse antibody raised against sperm protein sp56, related to the guinea-pig acrosomal matrix protein AM67 (Foster et al., 1997Go), was obtained from QED Bioscience (USA). Goat polyclonal antibody against recombinant murine {alpha} and {beta} regulatory subunit II (RII) of PKA was obtained from Upstate Biotechnology (USA). When anti-WAVE1 and anti-PKA RII antibodies were used simultaneously, a mouse monoclonal anti-WAVE1 antibody was used (BD Transduction Laboratories). Affinity-purified sheep anti-tubulin antibody was obtained from Cytoskeleton, Inc. (USA). The Golgi apparatus was identified using the mouse monoclonal antibody anti-GM130 matrix protein clone 35 (BD Transduction Laboratories). To identify mitochondria, a monoclonal antibody (Molecular Probes) was used to identify subunit II of complex IV–cytochrome c oxidase (COX). To investigate the presence of Abl protein, the purified anti-mouse antibody 8E9 was used (BD Transduction Laboratories). For control purposes, blocking peptides for the WAVE1 antibody were also obtained (Santa Cruz Biotechnology, Inc.).

To test sperm viability and for quantification purposes, the live/dead sperm viability kitTM (Molecular Probes) was used on live cells before fixation for immunocytochemistry. The kit uses the combination of a membrane-permeant nucleic acid stain (SYBR 14TM dye, green) and the conventional dead-cell stain propidium iodide (red). Concentrations of reagents for optimal staining were used according to the manufacturer.

Isolation of mouse and human spermatogenic cells
Mouse spermatogenic cells were isolated from adult mouse testes using previously described methodology (Bellve, 1993Go). Briefly, the testes were dissected in a Petri dish with EKRB (enriched Krebs–Ringer bicarbonate) medium containing 120.1 mmol/l NaCl, 4.8 mmol/l KCl, 25.2 mmol/l NaHCO3, 1.2 mmol/l KH2PO4 (pH 7.2), 1.2 mmol/l MgSO4 7H2O, 1.3 mmol/l CaCl2, supplemented with 11.1 mmol/l glucose, 1 mmol/l glutamine, 10 ml/l MEM essential amino acid solution, 10 ml/l non-essential amino acid solution, 100 mg/ml streptomycin, and 100 IU/ml penicillin (K salt).

Dry collagenase was then added at a final concentration of 0.5 mg/ml, and the testes were incubated for 15–45 min at 32°C with gentle stirring. Once the seminiferous tubules were dispersed in the medium they were allowed to settle at the bottom of the dish, and the medium was aspirated and discarded. The tubules were then placed in fresh EKRB containing 1 mg/ml DNase I and 0.25 mg/ml trypsin and incubated for 15–45 min with stirring and gentle pipetting. Released spermatogenic cells were pelleted by centrifugation (10 min at 500 g) and washed twice in EKRB before being attached to poly-L-lysine-coated coverslips.

Human adult testes were obtained from the Cooperative Human Tissue Network (Philadelphia, PA, USA). Small pieces of tissue were transferred into TALP–HEPES medium and digested using collagenase, bovine serum albumin (BSA)–fraction-V, pyruvate and gentamycin for 30 min at 37°C (see below Cooper et al., 1989Go). Isolated cells were then fixed for immunocytochemistry as described above.

Isolation of epididymal sperm
Pieces of the baboon epididymal tissue were transferred into TALP–HEPES medium (modified Tyrode lactate medium with pyruvate and albumin) containing 114 mmol/l NaCl, 3.2 mmol/l KCl, 2 mmol/l CaCl2, 0.5 mmol/l MgCl2, 25 mmol/l NaHCO3, 0.4 mmol/l NaH2PO4, 10 mmol/l sodium lactate, 6.5 IU penicillin/ml, 25 mg/ml gentamycin, 6 mg/ml fatty acid-free bovine serum albumin, 0.2 mmol/l pyruvate, and buffered with 10 mmol/l Hepes at pH 7.4 (Bavister et al., 1983Go), and digested using 2 mg/ml collagenase II, 3 mg/ml BSA-fraction-V, 0.2 mmol/l pyruvate and 0.5 ml/ml gentamycin for 30 min at 37°C (Cooper et al., 1989Go). Isolated epididymal sperm were collected by centrifugation, washed in TL–HEPES and fixed for immunocytochemistry as described above.

Mouse, bull, baboon and human sperm
Mouse sperm were isolated from adult mouse epididymis and attached to poly-L-lysine-coated coverslips before fixation (see below).

Frozen bull semen (American Breeders Service) was thawed to room temperature, layered over a 2-part 45%, 90% Percoll gradient and centrifuged at 700 g for 15 min to isolate live sperm. Samples were sedimented onto poly-L-lysine-coated coverslips to be studied after immunocytochemistry.

For isolation of baboon sperm cells, EKRB medium was replaced with TALP–HEPES. Samples were obtained by penile electro-ejaculation (Bavister et al., 1983Go; Boatman and Bavister, 1984Go) at the Southwest National Primate Center. Following liquefaction at 37°C for 30 min, the sample was washed twice in TALP–HEPES by centrifugation at 400 g for 5 min.

Normal frozen human semen samples were obtained from Follas Laboratories (USA).

All testicular samples were obtained post-mortem from the CHTN under the purview of Magee–Women's Hospital Institutional Review Board. All animal procedures were approved by the Magee–Women's Institutional Animal Care and Use Committee.

Immunocytochemistry
For immunocytochemistry, coverslips with mouse and human spermatogenic cells, baboon epididymal cells and/or mouse, bovine, baboon and human ejaculated sperm were placed in phosphate-buffered saline (PBS) containing 2% formaldehyde and fixed for 1 h. Following fixation, the samples were permeabilized for 60 min in PBS containing 1% Triton X-100, and non-specific reactions were blocked by further incubation in PBS containing 2 mg/ml BSA and 100 mmol/l glycine. For labelling, the antibodies were solubilized in this blocking solution and incubated overnight at appropriate dilutions. After extensive washing in PBS containing 0.1% Triton X-100, the samples were labelled with either Alexa-488 or Alexa-594 (Molecular Probes) secondary antibodies for 1 h. DNA was stained with 4',6'-diamino-2-phenylindole (DAPI; Molecular Probes). Following these incubations, coverslips were mounted onto glass microslides with VectaShield mounting medium (Vector Laboratories, USA) and sealed with nail polish. Samples were examined with a Nikon Eclipse E-1000 epifluorescence microscope operated with Metamorph software. Negative controls were performed by omitting the first antibody and/or pre-absorbing it with blocking peptides. Image acquisition times were comparable to those of labelled samples. All experiments were repeated at least twice.

SDS–PAGE and western blotting
For identification of WAVE1 protein in mature bull, baboon and human normal sperm, extracts were prepared by overnight incubation as a suspension in extraction buffer (1 mol/l NaCl, 1 mmol/l EDTA, 10 mg/ml phenylmethylsulphonyl fluoride, 1% v/v Triton X-100, 20 mmol/l Tris–Cl, pH 7.0), with the clear supernatant collected following centrifugation.

For bovine protein isolation, a two-step extraction protocol was used. First, samples were incubated overnight with the same buffer described above. For the second step, an incubation was done in 62.5 mmol/l Tris–HCl pH 6.8, 2% sodium dodecyl sulphate (SDS), 0.01% Bromophenol Blue, 10% glycerol and 5% {beta}-mercaptoethanol. This buffer was used on the pellet that remained following the first extraction.

Samples for western blotting were run on 4–20% Tris–HCl gels (Ready Gels, Bio-Rad, USA) under reducing and denaturing conditions (20–30 mg protein/lane). Following electrophoresis, the gels were soaked in Towbin's transfer buffer (25 mmol/l Tris, 192 mmol/l glycine, 0.037% SDS, 20% methanol) and the proteins were then transferred onto polyvinylidene difluoride (PVDF) membranes using a SemiPhor semi-dry blotting apparatus (Hoefer Scientific Instruments) at a current of 0.8 A/cm2 for 2 h. The membranes were blocked with Tris-buffered saline + Tween (25 mmol/l Tris, 137 mmol/l NaCl, 2.7 mmol/l KCl, and 0.2% Tween) supplemented with 3% IgG-free BSA and 5% fetal calf serum for 1 h on a rotating platform. After blocking, the membranes were incubated overnight at 4°C with anti-WAVE 1 antibody. After extensive washing in PBS containing 1–3% Tween 20, the blots were incubated with anti-goat IgG conjugated to horseradish peroxidase. Protein bands were detected using the ECL Plus system (Amersham) and Kodak X-OMAT LS Film (Sigma). Blots were then stripped with 100 mmol/l {beta}-mercaptoethanol, 2% SDS, 62.5 mmol/l Tris–HCl pH 6.7 for 1 h at 50°C, followed by multiple washes. Incubations with secondary antibodies and detection with ECL reagents verified that primary antibodies had been removed. After blocking the membranes, they were incubated with anti-PKA RII (1:500) antibodies. Bovine aortic endothelial cells (BAEc) were used as controls in all cases. Negative controls were performed by the pre-absorption of anti-WAVE1 and anti-PKA RII antibody with the corresponding blocking peptide.

Transmission electron microscopy (TEM) of normal human testis
Testicular biopsies from fertile men (Brökelman, 1963; kindly provided by M.Dym, Georgetown University) were used in this study. Tissue fragments were fixed by immersion in 2.5% glutaraldehyde in 0.2 mol/l s-collidine buffer, post-fixed in 1.3% osmium tetroxide, dehydrated in a graded series of ethanol, and embedded in Epon 812. Thin sections with pale golden to silver interference colours were examined and photographed in a Zeiss 109 electron microscope (Zeiss, Germany) after double staining with uranyl acetate and lead citrate.


    Results
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
Distinct spatial WAVE1 localization during mouse and human spermatogenesis
Figure 1 shows various stages in the maturation of human spermatogenic cells. A well-developed Golgi apparatus is present in a paranuclear position in meiotic primary spermatocytes (Figure 1A). Early in spermiogenesis, proacrosomic granules develop within Golgi vesicles that approach the nucleus to form the acrosomic granule (Figure 1B and 1C). This subsequently spreads over the nucleus forming the acrosomal cap (Figure 1D). The Golgi complex, located consistently to one side of the acrosome, starts to migrate caudally at the beginning of elongation. In elongated spermatids, the acrosomes are fully formed while Golgi remnants and abundant mitochondria can be seen in the caudal spermatid cytoplasm (Figure 1E). Golgi vesicles migrate to this region, while the mitochondria arrange around the axoneme to form the sperm mid-piece. Using antibodies that recognize acrosomal matrix protein sp56 (Foster et al., 1997Go) and WAVE1 in testicular cells (Figure 2), we examined the localization of this AKAP in different stages of murine and human spermatogenesis. Early in spermiogenesis, Golgi-phase spermatids of mice and humans show well-formed acrosomic granules (red), while a bright signal of WAVE1 (green) can be found close by (Figure 2A, compare with Figure 1B and 1C). WAVE1 consistently distributes in close lateral apposition to the growing acrosome. When the acrosome is more developed (Figure 2B and 2B'), WAVE1 moves to the opposite side of the cell. The localization of WAVE1 during the initial stages of spermiogenesis resembles a Golgi-like pattern described in the mouse (Ramalho-Santos et al., 2001Go, see below). Figure 2C and C' show a more developed acrosome and the localization of WAVE1 in two regions: close to the acrosome with a configuration that resembles the Golgi, and in a more caudal area as punctate foci that suggest mitochondrial localization (compare with Figure 1B and D where individual mitochondria can be seen near the Golgi–acrosomal complex). To test whether WAVE1 co-localizes with mitochondria, a double staining was performed using antibodies against the mitochondrial protein cytochrome c oxidase (COX). In the inset of Figure 2C, yellow areas show some co-localization of WAVE1 and mitochondria (arrowheads). Figure 2D shows a tetrad of cap phase spermatids (compare with Figure 1D), with WAVE1 associated to the lateral aspect of the fully formed acrosomal caps. Later in development, WAVE1 distributes to the caudal pole of the cell, where spermatid mitochondria and remaining Golgi now reside (Figure 1E and 2E). In human and mouse elongated spermatids, as well as in human testicular sperm, WAVE1 shows a distinct localization in the mitochondrial sheath (Figure 2F, F' and G, G'), also seen in mature epididymal baboon sperm (Figure 4). At this stage of maturation, the acrosomal granule has spread to cover the anterior 2/3 of the nucleus.



View larger version (177K):
[in this window]
[in a new window]
 
Figure 1. Different maturational stages of human spermatogenic cells. (A) A primary meiotic spermatocyte with synaptonemal complexes (arrow) indicates a very well developed Golgi (G) in a paranuclear position (magnification x16 400). (B) In this very early round spermatid, pro-acrosomic granules (*) have developed within vesicles of the Golgi (magnification x13 200). (C) The acrosomic vesicle and granule (AG) have attached to the cranial pole of the nucleus in this round spermatid. The Golgi (G) remains in a lateral position, near the forming acrosome (magnification x15 750). (D) Cap phase spermatid. The acrosomic granule has spread over the anterior aspect of the nucleus while the Golgi (G) remains in close apposition to the acrosome (magnification x15 900). (E) Two elongated spermatids with advanced chromatin condensation and full development of the acrosome. Remnants of the Golgi (G) and numerous mitochondria (M) can be seen in the caudal cytoplasm just before mid-piece development. Scale bars = 1 µm.

 


View larger version (82K):
[in this window]
[in a new window]
 
Figure 2. WAVE1 localization during murine and human spermatogenesis. (A) In early spermatids, a bright signal of WAVE1 (green) can be observed adjacent to the acrosome granule (red). (B) Later during spermatogenesis, and once the acrosome is further developed (B'), WAVE1 initiates a migration towards the opposite side of the cell. (C) The acrosome has extended to the apical region of the cell, and WAVE1 is in close proximity (detached). The acrosome is in a more caudal area in C' (beginning of elongation). Note also the co-localization of WAVE1 and mitochondria (COX II) indicated with arrowheads in the inset. (D) A tetrad of cap phase spermatids with WAVE1 associated to the fully formed acrosomal caps. (E) Late in spermiogenesis, while the acrosome persists in a distinct apical area of the forming head, the majority of WAVE1 migrates completely to the opposite cell pole. (F and F') Mouse and human elongated spermatids show WAVE1 associated with the growing mitochondrial sheath. (G and G') Mouse and human testicular sperm show a clear localization of WAVE1 on the mitochondrial sheaths. Scale bar for AC' and E=3 µm (shown in E). Scale bar for FG'=3 µm (shown in G'). Scale bar in D=10 µm.

 


View larger version (44K):
[in this window]
[in a new window]
 
Figure 4. WAVE1 localization during epididymal passage. (A and A') Baboon epididymal sperm showing WAVE1 localizing on the mitochondrial sheath. When the mitochondrial sheath (observed by differential interference contrast microscopy) is absent or poorly formed (A'), WAVE1 (A) is not present (arrowhead). Bar = 5 µm.

 
Golgi marker GM130 and WAVE1 co-localize during mouse and human spermatogenesis
In order to determine whether WAVE1 indeed co-localizes with the Golgi apparatus during mouse spermatogenesis, we studied its localization in parallel with that of the Golgi marker GM130. Early during spermatogenesis, WAVE1 distributes around the nucleus as punctate foci, associated with GM130 and perhaps with mitochondria (meiotic spermatocyte in Figure 1A, arrowhead in Figure 3A and A'). Later in spermiogenesis, WAVE1 still associates with GM130 (Figure 3B, arrowhead), but is also enriched in the cytoplasmic lobe, presumably migrating towards the caudal portion of the cell (arrow Figure 3B). Figure 3C and C' show WAVE1 in two distinct regions: localizing with Golgi markers, and distributing to the opposite side of the cell. Elongating spermatids again show the co-localization of WAVE1 and GM130, as well as a concentration of WAVE1 at the opposing side. WAVE1 distributes along the forming mitochondrial sheath, and no longer associates with GM130 in this specific location (Figure 3D and D'). In testicular sperm, Golgi markers localize in the discarded cytoplasmic droplet, presumably as Golgi remnants, while WAVE1 associates entirely with the mitochondrial sheath (Figure 3E).



View larger version (94K):
[in this window]
[in a new window]
 
Figure 3. WAVE1 co-localization with Golgi markers during spermatogenesis. (A and A') Early in mouse and human spermatogenesis, a co-localization can be observed between WAVE1 and the Golgi marker GM130 (yellow area, arrowheads). WAVE1 can also be seen distributed around the cells in a punctuate and diffuse pattern (mouse). (B) Later in spermatogenesis, WAVE1 is still associated with GM130 (arrowhead) but it is also seen enriched in an opposite area where it starts to migrate towards the dorsal portion of the cell (arrow). (C and C') WAVE1 is seen in two distinct regions in the cell: localizing with Golgi markers and at the opposite pole of the cell. (D and D') Elongated spermatids can be seen with a very clear signal of WAVE1 on the forming mitochondrial sheath and no longer associated with GM130 (red). (E) In mouse testicular sperm, the Golgi marker is in the discarded cytoplasmic droplet, whereas WAVE1 associates entirely with the mitochondrial sheath. (F and G) In a similar stage during spermatogenesis, WAVE1 co-localizes with PKA RII and Abl tyrosine kinase. For A' and B, negative controls are shown in insets. Scale bar for AC' and D'=5 µm (shown in A). Scale bar in D=1 µm, in E=5 µm. Scale bar for F and G=5 µm (shown in G).

 
Interestingly, WAVE1 co-localizes with PKA RII (Figure 3F) and Abl tyrosine kinase in round spermatids (Figure 3G). It is important to notice that PKA RII staining resembles the distribution of WAVE1 throughout spermatogenesis (not shown) and in mature ejaculated sperm (see Figure 6). Abl tyrosine kinase, however, associates with WAVE1 throughout the round spermatid stage, but is not detected in mature sperm (not shown).



View larger version (61K):
[in this window]
[in a new window]
 
Figure 6. WAVE1, PKA RII and RAC1 distribution in mature sperm. Mature mouse, bull, baboon and human semen samples were studied by immunocytochemistry. (A) A bright signal of WAVE1 (green) can be seen on the mitochondrial sheaths in the majority of mature mouse sperm. (B) PKA RII (red) is visualized as a bright signal in the mitochondrial sheath and, to a lesser extent, also in the principal piece. (C) The small GTPase RAC1 (red) is observed in both equatorial and post-acrosomal regions of the sperm head. A faint staining of RAC1 is also observed on the mitochondrial sheath. In mature bull sperm, a consistent co-localization of WAVE1 and PKA RII is observed on the mitochondrial sheath (A' and B'). Similar to the mature mouse sperm, RAC1 is preferentially distributed in the equatorial and post-acrosomal regions (C'). Similar co-localization of WAVE1 and PKA RII is detected in mature baboon and human sperm (A'', B'', A''', B'''). RAC1 also localizes to the equatorial and post-acrosomal regions in baboon and human sperm. RAC1 co-localizes with WAVE1 in the mitochondrial sheath (insets in C'' and C'''). A, A' and A''' show tubulin in red. Insets in A, A', A'' and A''' show negative controls after the omission of the primary antibody. Insets in C, C', C'' and C''' show RAC1 (red) and WAVE1 (green). Bar = 10 µm.

 
WAVE1 distribution is retained during epididymal passage
We next tested whether baboon sperm have a similar WAVE1 pattern following epididymal passage. For this purpose, we obtained baboon epididymal and ejaculated sperm and compared its staining pattern with our previous findings. Our immunocytochemistry results show WAVE1 localizing on the mitochondrial sheath in a very distinctive way (Figure 4A). Individual mitochondria can almost be visualized following WAVE1 staining. This pattern is very different from the one observed after the use of Mitotracker greenTM, a vital mitochondrion-specific fluorescent probe that labels the entire mitochondrial sheath (data not shown). As expected, when the mitochondrial sheath is absent or poorly formed, WAVE1 signal is not present (Figure 4A and A', arrowheads). No differences are found between epididymal and ejaculated sperm (see Figure 6).

Spatial localization of WAVE1, PKA RII and RAC1 in mature sperm
We then investigated whether mature, ejaculated sperm contain WAVE1 and its partner PKA RII, given that the majority of Golgi and Golgi-associated proteins are lost from mature sperm. For this purpose, Western blots were performed and the presence of WAVE1 and PKA RII was confirmed in bull, baboon, and human normal semen samples (Figure 5). Strong bands corresponding to WAVE1 (~80 kDa) and PKA RII (51 kDa) are detected in ejaculated sperm. Also, a band with higher molecular mass is detected in baboon sperm. Bands with lower molecular mass likely correspond to degradation products.



View larger version (45K):
[in this window]
[in a new window]
 
Figure 5. SDS–PAGE and western blotting of WAVE1 and PKA RII in mature sperm. (A and B) Strong bands corresponding to WAVE1 (~80 kDa) and PKA RII (~51 kDa) are detected in ejaculated bull, baboon and human sperm. Also, a band with higher molecular mass is observed when baboon sperm proteins were probed for WAVE1. Lower molecular mass bands, likely corresponding to degradation products, are detected during WAVE1 blotting when using baboon sperm and control somatic cells (bovine aortic endothelial cells, BAE). A total of 20–30 µg of protein was loaded per lane. Negative controls are shown after the preincubation of the antibodies with the corresponding blocking peptide.

 
In order to determine the spatial localization of WAVE1, PKA RII and RAC1 in mature sperm, semen samples from mice, bulls, baboons and humans were examined by immunocytochemistry. Table I shows the amount of live sperm studied, as well as the percentages of cells detected by the live/dead sperm kitTM, listing the corresponding label of each protein. Although some staining of WAVE1 has been visualized in dead sperm, only sperm with green DNA staining (alive) were considered positive during cell counting. One thousand sperm were counted from three different animal (mouse, bull, and baboon) and human samples.


View this table:
[in this window]
[in a new window]
 
Table I. Percentages of live mature sperm with signal for WAVE1, PKA RII and RAC1

 
Figure 6 shows the staining patterns of WAVE1, PKARII and RAC1 in mature mouse, bull, baboon and human sperm. As expected, WAVE1 localizes to the mitochondrial sheath in the majority of mature mouse sperm (Figure 6A). PKA RII also distributes to the mitochondrial sheath and the principal piece (Figure 6B). Because RAC1 activates WAVE1 during actin reorganization in somatic cells (Miki et al., 1998Go), we examined its localization with respect to WAVE1 here in sperm. A preferentially localized RAC1 distributes to the equatorial and post-acrosomal regions of the sperm head, whereas WAVE1 localizes to the mitochondrial sheath (Figure 6C, inset). RAC1 is also observed along the mitochondrial sheath, but the distribution is faint.

In mature bull sperm, WAVE1 and RAC1 localization is similar to those found in mice (Figure 6A and 6CFigure 6A and 6C). PKA RII (Figure 6B' and inset in C') is seen in the mitochondrial sheath, but is not evident in the principal piece.

A conserved distribution of WAVE1 and its partners is observed in mature baboon and human sperm. WAVE1 and PKA RII localize in the mitochondrial sheath, with PKA RII also distributing to the principal piece (Figure 6A'', 6B'', 6A''', 6B'''). RAC1 localizes in the equatorial and post-acrosomal regions, as well as in the mitochondrial sheaths (6C'' and 6C'''). The insets in Figure 6C'' and 6C''' show that RAC1 co-localizes with WAVE1 on the mitochondrial sheaths in the sperm of baboons and humans. Abl tyrosine kinase is not detected in the ejaculated sperm of any of the species studied (not shown).


    Discussion
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
One of the most extensively characterized signalling pathways utilizes the second messenger cAMP, which binds to PKA and results in the phosphorylation of proteins at serine and threonine residues. AKAP help sequester PKA isoforms and shift the signalling from the cytoplasm to the cytoskeleton, organelles, or other structures. Compartmentalization of these proteins, therefore, might be especially important in cells with a paucity of cytoplasm, such as sperm.

In the present study, we demonstrate the presence and retention of the AKAP WAVE1 in immature spermatogenic cells, in maturing sperm isolated during epididymal passage, and in mature ejaculated sperm of different mammalian species, including humans. In spermatogenic cells, WAVE1 closely associates with the Golgi apparatus and sperm mitochondria, and co-localizes with PKA RII, Abl and RAC1.

We find that during the early stages of mouse and human spermiogenesis, WAVE1 primarily localizes in a Golgi-like pattern, similarly to what has been described for other AKAP in different cell types (Schillace et al., 2002Go; Shanks et al., 2002Go; Liu et al., 2003Go). Immunofluorescence studies with the Golgi marker GM130 show a co-localization with WAVE1 during Golgi migration. Trafficking from the Golgi apparatus is involved in acrosome formation, and requires a very active actin cytoskeleton (Clermont and Tang, 1985Go; Ramalho-Santos et al., 2001Go; Kierszenbaum et al., 2003Go). It is therefore not surprising that WAVE1 concentrates near the Golgi, where it can participate in the reorganization of the actin cytoskeleton and associate with other actin-related proteins, such as profilin. In spermatocytes and spermatids, another population of WAVE1 is identified as bright punctuate foci either juxtaposed with the nuclei (Figure 2B', 2C', 3A and 3B), or distributed towards the cytoplasmic lobe of the cells. According to our immunofluorescence staining, this population of WAVE1 is present in mitochondria (Figure 2C inset), and in the forming mitochondrial sheaths. Whereas WAVE1 binds PKA in the mitochondria of the mid-piece, other AKAP localize to the principal piece of the flagellum such as AKAP82 in humans (Turner et al., 1999Go) and AKAP110 in bull and man (Vijayaraghavan et al., 1999Go). The dual association of WAVE1 with Golgi could reflect the synthesis and sorting of WAVE1. Alternatively, its association with Golgi and mitochondria could indicate that WAVE1 might utilize one or more of its targeting domains to localize scaffolded signalling proteins to specific subcellular compartments. The ability of AKAP to target different cellular compartments resides in unique targeting domains specific for distinct subcellular structures. For example, D-AKAP1 contains dual targeting domains that are utilized for its association with either mitochondrial or endoplasmic reticulum targeting domains, based upon amino-terminal splicing (Huang et al., 1999Go).

AKAP are known to bind proteins that facilitate the formation of macromolecular complexes on specific cellular structures. Previous studies have reported that WAVE1 binds PKA RII, Abl tyrosine kinase, the Arp 2/3 complex and the G-actin binding protein profilin in somatic cells. This current study now shows that during mammalian spermatogenesis, WAVE1 localization resembles PKA RII distribution, and that Abl tyrosine kinase associates with WAVE1 through the round spermatid stage in close proximity with the Golgi apparatus and mitochondrial sheath.

Previous studies in mouse male germ cells have identified other AKAP associated with the mitochondrial sheath. S-AKAP84 RII binding protein (Lin et al., 1995Go) is expressed primarily in the male germ cell lineage. The protein accumulates as spermatids undergo nuclear condensation and tail elongation. S-AKAP84 distributes to the outer mitochondrial membrane of the mitochondrial sheath, and binds to AMY-1 (a c-Myc binding protein) and PKA RII (Furusawa et al., 2001Go). Other reports have identified AKAP 220 as a protein that localizes to the outer mitochondrial membrane of the sperm mid-piece, most likely through an association with the cytoskeleton (Lieberman et al., 1988Go; Reinton et al., 2000Go).

By tethering PKA to distinct subcellular organelle compartments, such as the Golgi or mitochondria, WAVE1 may contribute to phosphorylation of protein substrates involved in the regulation of spermiogenesis (including vesicle trafficking through the Golgi), and/or various sperm activities, such as flagellar motility and mitochondrial respiration. Recent reports by Fujinoki et al. (2003)Go have identified a 36 kDa protein that undergoes serine phosphorylation in a cAMP-dependent manner, and which likely regulates sperm activation to the sperm mid-piece. Thus, WAVE1 association with Golgi markers in spermatogenic cells, and WAVE1 localization in the mid-piece on mature sperm, may serve to specify the appropriate subcellular compartments of spermatogenic cells for different sperm functions. The data presented here suggest the existence of WAVE1/PKA RII signalling units in the Golgi and mitochondria of spermatids, and in the mid-piece of mature sperm.

RAC1, a member of the Rho family of small GTPases, is known to induce WAVE1 activity (Ridley and Hall, 1992Go; Miki et al., 1998Go). The regulation of these events, however, is not completely known. Our immunofluorescence studies show that RAC1 localizes in the post-acrosomal region and mid-piece of mature sperm. It seems likely that there is a population of RAC1 that does not bind directly to WAVE1 (post-acrosomal region, Figure 5), raising the question of how WAVE1 is regulated by RAC1. Miki et al. (2000)Go demonstrated that IRSp53, a substrate for insulin receptor 12 with unknown function, is an important link between RAC and WAVE. In Swiss3T3 cell lysates, activated RAC1 binds to the amino terminus of IRSp53, and the carboxy-terminal SH3 domain of IRSp53 binds to WAVE1 to form a tri-molecular complex. The authors found that IRSp53 is essential for RAC1 to induce membrane ruffling, perhaps by recruiting WAVE1, which stimulates actin polymerization mediated by the Arp2/3 complex.

The discovery of actin-related proteins at distinct cellular locations in all stages of mammalian spermatogenesis suggests that the participation of WAVE1, PKA RII and Abl tyrosine kinase may be necessary for proper sperm architecture design. As previously suggested, anchoring of PKA may also be important for sperm function. The exact functional role of these proteins remains open to future investigation.


    Acknowledgements
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
We would like to thank David McFarland and Anthony Martin for their technical support. We appreciate the generous contributions of Dr K.Dee Carey for providing us the baboon material from the Southwestern Primate Center, San Antonio, Texas; Dr John McCarey, now at the University of Texas, San Antonio; and Dr Lee Caperton, as well as the support of Deborah Randall. This work was supported by the Americas Fellowship RSANET (NICHD, NIH) to V.Y.R. and NIH grants to G.S. A grant from FCT, Portugal (POCTI/ESP/38049/2001) supported J.R.-S.


    References
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
Bavister BD, Boatman DE, Leibfried L, Loose M and Vernon MW (1983) Fertilization and cleavage of rhesus monkey oocytes in vitro. Biol Reprod 28, 983–999.[ISI][Medline]

Beebe SJ and Corbin JD (1986) Cyclic nucleotide-dependent protein kinases. Enzymes 17, 43–111.

Bellve AR (1993) Purification, culture, and fractionation of spermatogenic cells. Methods Enzymol 225, 84–113.[ISI][Medline]

Boatman DE and Bavister BD (1984) Stimulation of rhesus monkey sperm capacitation by cyclic nucleotide mediators. J Reprod Fertil 71, 357–366.[ISI][Medline]

Brokaw CJ (1987) A lithium-sensitive regulator of sperm flagellar oscillation is activated by cAMP-dependent phosphorylation. J Cell Biol 105, 1789–1798.[Abstract]

Buck J, Sinclair ML, Schapal L, Cann MJ and Levin LR (1999) Cytosolic adenylyl cyclase defines a unique signaling molecule in mammals. Proc Natl Acad Sci USA 96, 79–84.[Abstract/Free Full Text]

Carr DW, Stofko-Hahn RE, Fraser ID, Bishop SM, Acott TS, Brennan RG and Scott JD (1991) Interaction of the regulatory subunit (RII) of cAMP-dependent protein kinase with RII-anchoring proteins occurs through an amphipathic helix binding motif. J Biol Chem 266, 14188–14192.[Abstract/Free Full Text]

Carrera A, Gerton GL and Moss SB (1994) The major fibrous sheath polypeptide of mouse sperm: structural and functional similarities to the A-kinase anchoring proteins. Dev Biol 165, 272–284.[CrossRef][ISI][Medline]

Clermont Y and Tang XM (1985) Glycoprotein synthesis in the Golgi apparatus of spermatids during spermiogenesis of the rat. Anat Rec 213, 33–43.[ISI][Medline]

Colledge M and Scott JD (1999) AKAPs: from structure to function. Trends Cell Biol 9, 216–221.[CrossRef][ISI][Medline]

Cooper TG, Yeung C-H and Meyer R (1989) Immature rat epididymal epithelial cells grown in static primary monolayer culture on permeable supports. I. Vectorial secretion. Cell Tiss Res 256, 567–572.[ISI][Medline]

Diviani D and Scott JD (2001) AKAP signaling complexes at the cytoskeleton. J Cell Sci 114, 1431–1437.[Abstract/Free Full Text]

Edwards AS and Scott JD (2000) A-kinase anchoring proteins: protein kinase A and beyond. Curr Opin Cell Biol 12, 217–221.[CrossRef][ISI][Medline]

Foster JA, Friday BB, Maulit MT, Blobel C, Winfrey VP, Olson GE, Kim KS and Gerton GL (1997) AM67, a secretory component of the guinea pig sperm acrosomal matrix, is related to mouse sperm protein sp56 and the complement component 4-binding proteins. J Biol Chem 272, 12714–12722.[Abstract/Free Full Text]

Francis SH and Corbin JD (1994) Structure and function of cyclic nucleotide-dependent protein kinases. Annu Rev Physiol 56, 237–272.[CrossRef][ISI][Medline]

Fujinoki M, Kawamura T, Toda T, Ohtake H, Ishimoda-Takagi T, Shimizu N, Yamaoka S and Okuno M (2003) Identification of 36-kDa flagellar phosphoproteins associated with hamster sperm motility. J Biochem (Tokyo) 133, 361–369.[Abstract/Free Full Text]

Gordeladze JO and Hansson V (1981) Purification and kinetic properties of the soluble Mn21-dependent adenylyl cyclase of the rat testis. Mol Cell Endocrinol 23, 125–136.[CrossRef][ISI][Medline]

Higgs HN and Pollard TD (1999) Regulation of actin polymerization by Arp2/3 complex and WASp/Scar proteins. J Biol Chem 46, 32531–32534.[CrossRef]

Huang LJ, Wang L, Ma Y, Durick K, Perkins G, Deerinck TJ, Ellisman MH and Taylor SS (1999) NH2-Terminal targeting motifs direct dual specificity A-kinase-anchoring protein 1 (D-AKAP1) to either mitochondria or endoplasmic reticulum. J Cell Biol 145, 951–959.[Abstract/Free Full Text]

Landmark BF, Oyen O, Skalhegg BS, Fauske B, Jahnsen T and Hansson V (1993) Cellular location and age-dependent changes of the regulatory subunits of cAMP-dependent protein kinase in rat testis. J Reprod Fertil 99, 323–334.[ISI][Medline]

Lieberman SJ, Wasco W, MacLeod J, Satir P and Orr GA (1988) Immunogold localization of the regulatory subunit of a type II cAMP-dependent protein kinase tightly associated with mammalian sperm flagella. J Cell Biol 5, 1809–1816.[CrossRef]

Lin RY, Moss SB and Rubin CS (1995) Characterization of S-AKAP84, a novel developmentally regulated A kinase anchor protein of male germ cells. J Biol Chem 270, 27804.[Abstract/Free Full Text]

Furusawa M, Ohnishi T, Taira T, Iguchi-Ariga SMM and Hiroyoshi A (2001) AMY-1, a c-Myc-binding Protein, Is Localized in the Mitochondria of Sperm by Association with S-AKAP84, an Anchor Protein of cAMP-dependent Protein Kinase. J Biol Chem 276, 36647–36651.[Abstract/Free Full Text]

Kierszenbaum AL, Rivkin E and Tres LL (2003) Acroplaxome, an F-actin-keratin-containing plate, anchors the acrosome to the nucleus during shaping of the spermatid head. Mol Biol Cell 11, 4628–4640.[CrossRef]

Machesky LM and Gould KL (1999) The Arp2/3 complex: a multifunctional actin organizer. Curr Opin Cell Biol 1, 117–121.[CrossRef]

Miki K and Eddy EM (1998) Identification of tethering domains for protein kinase A type I alpha regulatory subunits on sperm fibrous sheath protein FSC1. J Biol Chem 273, 34384–34390.[Abstract/Free Full Text]

Miki H, Suetsugu S and Takenawa T (1998) WAVE, a novel WASP-family protein involved in actin reorganization induced by Rac. EMBO J 17, 6932–6941.[Abstract/Free Full Text]

Miki H, Yamaguchi H, Suetsugu S and Takenawa T (2000) IRSp53 is an essential intermediate between Rac and WAVE in the regulation of membrane ruffling. [Letter.] Nature 7, 732–735.[CrossRef]

Mullins RD (2000) How WASP-family proteins and the Arp2/3 complex convert intracellular signals into cytoskeletal structures. Curr Opin Cell Biol 12, 91–96.[CrossRef][ISI][Medline]

Ramalho-Santos J, Moreno RD, Wessel GM, Chan EK and Schatten G (2001) Membrane trafficking machinery components associated with the mammalian acrosome during spermiogenesis. Exp Cell Res 267, 45–60.[CrossRef][ISI][Medline]

Reinton N, Collas P, Haugen TB, Skalhegg BS, Hansson V, Jahnsen T and Tasken K (2000) Localization of a novel human A-kinase-anchoring protein, hAKAP220, during spermatogenesis. Dev Biol 223, 194–204.[CrossRef][ISI][Medline]

Ridley AJ and Hall A (1992) The small GTP-binding protein rac regulates growth factor-induced membrane rufling. Cell 70, 401–410.[ISI][Medline]

Rubin CS (1994) A-kinase anchor proteins and the intracellular targeting of signals carried by cAMP. Biochim Biophys Acta 1224, 467–479.

Salanova M, Chun SY, Iona S, Puri C, Stefanini M and Conti M (1999) Type 4 cyclic adenosine monophosphatespecific phosphodiesterases are expressed in discrete subcellular compartments during rat spermiogenesis. Endocrinology 140, 2297–2306.[Abstract/Free Full Text]

Sasaki N, Miki H and Takenawa T (2000) Arp2/3 complex-independent actin regulatory function of WAVE. Biochem Biophys Res Commun 272, 386–390.[CrossRef][ISI][Medline]

Scott JD (1991) Cyclic nucleotide-dependent protein kinases. Pharmacol Ther 50, 123–145.[CrossRef][ISI][Medline]

Liu J, Li Hua and Papadopoulos V (2003) PAP7, a PBR/PKA-RIalpha-associated protein: a new element in the relay of the hormonal induction of steroidogenesis. J Steroid Biochem Mol Biol 85, 275–283.[CrossRef][ISI][Medline]

Schillace RV, Andrews SF, Liberty GA, Davey MP and Carr DW (2002) Identification and characterization of myeloid translocation gene 16b as a novel a kinase anchoring protein in T lymphocytes. J Immunol 168, 1590–1599.[Abstract/Free Full Text]

Shanks RA, Steadman BT, Schmidt PH and Goldenring JR (2002) Identification of a distinct Golgi apparatus targeting motif in AKAP350. J Biol Chem 3, 40967–40972.[CrossRef]

Smith GD, Wolf DP, Trautman KC, da C, Greengard P and Vijayaraghavan S (1996) Primate sperm contain protein phosphatase 1, a biochemical mediator of motility. Biol Reprod 54, 719–727.[Abstract]

Tash JS and Means AR (1982) Regulation of protein phosphorylation and motility of sperm by cyclic adenosine monophosphate and calcium. Biol Reprod 26, 745–763.[Abstract]

Turner RMO, Eriksson RLM, Gerton GL and Moss SB (1999) Relationship between sperm motility and the processing and tyrosine phosphorylation of two human sperm fibrous sheath proteins, pro-hAKAP82 and hAKAP82. Mol Hum Reprod 5, 816–824.[Abstract/Free Full Text]

Vijayaraghavan S, Goueli SA, Davey MP and Carr DW (1997) Protein kinase A-anchoring inhibitor peptides arrest mammalian sperm motility. J Biol Chem 272, 4747–4752.[Abstract/Free Full Text]

Vijayaraghavan S, Liberty GA, Mohan J, Winfrey VP, Olson GE and Carr DW (1999) Isolation and molecular characterization of AKAP110, a novel, sperm-specific protein kinase A-anchoring protein. Mol Endocrinol 13, 705–717.[Abstract/Free Full Text]

Volkmann N, Amann KJ, Stoilova-McPhie S, Egile C, Winter DC, Hazelwood L, Heuser JE, Li R, Pollard TD and Hanein D (2001) Structure of Arp2/3 complex in its activated state and in actin filament branch junctions. Science 293, 2456–2459.[Abstract/Free Full Text]

Westphal RS, Soderling SH, Alto NM, Langeberg LK and Scott JD (2000) Scar/WAVE-1, a Wiskott-Aldrich syndrome protein, assembles an actin-associated multi-kinase scaffold. EMBO J 19, 4589–4600.[Abstract/Free Full Text]

Submitted on May 27, 2004; resubmitted on July 16, 2004; accepted on August 18, 2004.