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BOR - Papers in Press, published online ahead of print September 22, 2004.
Biol Reprod 2004, 10.1095/biolreprod.104.034140
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BIOLOGY OF REPRODUCTION 72, 384–392 (2005)
DOI: 10.1095/biolreprod.104.034140
© 2005 by the Society for the Study of Reproduction, Inc.

Changes in Intracellular Distribution and Activity of Protein Phosphatase PP1{gamma}2 and Its Regulating Proteins in Spermatozoa Lacking AKAP41

Zaohua Huang2,3, Payaningal R. Somanath4, Rumela Chakrabarti3, Edward M. Eddy5, and Srinivasan Vijayaraghavan3

Department of Biological Sciences,3 Kent State University, Kent, Ohio 44242 Department of Molecular Cardiology,4 Center for Thrombosis and Vascular Biology, Lerner Research Institute, Cleveland Clinic Foundation, Cleveland, Ohio 44195 Gamete Biology Section,5 Laboratory of Reproductive and Developmental Toxicology, National Institute of Environmental Health Sciences/NIH, Research Triangle Park, North Carolina 27709


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The second messenger cAMP mediates its intracellular effects in spermatozoa through cAMP-dependent kinase (PKA, formally known as PRKACA). The intracellular organization of PKA in spermatozoa is controlled through its association with A-kinase-anchoring proteins (AKAPs). AKAP4 (A kinase [PRKA] anchor protein 4; also called fibrous sheath component 1 or AKAP 82) is sperm specific and the major fibrous sheath protein of the principal piece of the sperm flagellum. Presumably, AKAP4 recruits PKA to the fibrous sheath and facilitates local phosphorylation to regulate flagellar function. It is also proposed to act as a scaffolding protein for signaling proteins and proteins involved in metabolism. Akap4 gene knockout mice are infertile due to the lack of sperm motility. The fibrous sheath is disrupted in spermatozoa from mutant mice. In this article, we used Akap4 gene knockout mice to study the effect of fibrous sheath disruption on the presence, subcellular distribution, and/or activity changes of PKA catalytic and regulatory subunits, sperm flagellum proteins PP1{gamma}2 (protein phosphatase 1, catalytic subunit, gamma isoform, formally known as PPP1CC), GSK-3 (glycogen synthase kinase-3), SP17 (sperm autoantigenic protein 17, formally known as SPA17), and other signaling proteins. There were no changes in the presence and subcellular distribution for PP1{gamma}2, GSK-3, hsp90 (heat shock protein 1, alpha, formally known as HSPCA), sds22 (protein phosphatase 1, regulatory [inhibitor] subunit 7, formally known as PPP1R7), 14-3-3 protein (tyrosine 3-monooxygenase/tryptophan 5-monooxygenase activation protein), and PKB (thymoma viral proto-oncogene, also known as AKT) in mutant mice. However, the subcellular distributions for PKA catalytic subunit and regulatory subunits, PI 3-kinase (phosphatidylinositol 3-kinase), and SP17 were disrupted in mutant mice. Furthermore, there was a significant change in the activity and phosphorylation of PP1{gamma}2 in mutant compared with wild-type spermatozoa. These studies have identified potentially significant new roles for the fibrous sheath in regulating the activity and function of key signaling enzymes.

Akap4 gene knockout, epididymis, GSK-3, phosphatases, PP1{gamma}2, SP17, sperm, sperm motility and transport, testis


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The cyclic AMP (cAMP) pathway is a well-studied signal-transduction cascade. The second messenger cAMP mediates its intracellular effects through PKA (cAMP-dependent kinase, formally known as PRKACA). The holoenzyme of PKA is a tetramer of two catalytic subunits and two regulatory subunits. The catalytic subunits are encoded by three genes, C{alpha}, Cß, and C{gamma}, while the regulatory subunits are encoded by four genes, RI{alpha}, RIß, RII{alpha}, and RIIß. Each regulatory subunit contains an N-terminal dimerization domain, which is an autophosphorylation site and also the principal contact site for the catalytic subunit, and two cAMP-binding sites. Binding of two cAMP molecules to each regulatory subunit relieves the autoinhibitory contact and allows the dissociation and activation of the catalytic subunits, resulting in phosphorylation of protein substrates. There are two forms of the heterotetrameric PKA holoenzyme: type I (RI{alpha} and RIß dimers) and type II (RII{alpha} and RIIß dimers). Type I PKA is predominantly cytoplasmic and more sensitive to cAMP than type II [1–4]. Type II PKA usually associates with specific cellular structures and organelles [1–4]. The intracellular organization of PKA is controlled through the association with AKAPs (A-kinase-anchoring proteins) [1–4]. AKAPs are a structurally diverse but functionally similar family of proteins of over 50 members [1–4]. They usually have the following functional motifs: a conserved binding domain interacting with the PKA regulatory subunit dimer [1–4], a targeting domain directing the AKAP-PKA complex to specific subcellular locations [1–4], and docking sites for other signaling enzymes, such as kinases and phosphatases [1–4].

AKAP4 (A kinase [PRKA] anchor protein 4; also called fibrous sheath component 1 or AKAP 82) is the major fibrous sheath protein of the principal piece of the sperm flagellum [5, 6]. Morphologically there are three distinguishable sperm flagellum segments: the midpiece, the principal piece, and end piece, all with the axoneme as the core. In the midpiece, the accessory structures are the mitochondrial sheath and the outer dense fibers surrounding the core. In the principal piece, the fibrous sheath replaces the mitochondrial sheath, and outer dense fibers 3 and 8 are also substituted by the two longitudinal columns of the fibrous sheath. The end piece is the final segment of the flagellum with no accessory structures. Proper assembly of the fibrous sheath and the flagellum is critical for sperm motility and other biological functions. One suggested function of the fibrous sheath is to serve as a scaffold for proteins in signaling pathways involved in sperm maturation, motility, capacitation, hyperactivation, and glycolysis [5, 6].

It has been assumed that AKAP4 recruits PKA to the fibrous sheath and facilitates local phosphorylation to regulate flagellum functions [5]. AKAP4 can bind both PKA RI {alpha} and RII {alpha} regulatory subunits in vitro [7, 8]. Akap4 gene knockout mice have been generated successfully [5]. These mutant male mice were infertile, not due to the reduction in sperm numbers but to the immotility of spermatozoa [5]. In the knockout mice, the anlagen of the fibrous sheath formed but a definitive fibrous sheath did not develop. Also, the flagellum was shortened, and some of the proteins associating with the fibrous sheath were absent or significantly reduced in amount [5]. However, the other cytoskeletonal components of the flagellum appeared completely developed [5].

Akap4 gene knockout mice may be a valuable model to study the biological functions of the fibrous sheath and associated proteins. A number of sperm proteins are reported to be associated with the fibrous sheath, such as AKAP3 (A kinase [PRKA] anchor protein 3), GAPDS (glyceraldehyde 3-phosphate dehydrogenase-s), HK1-S (hexokinase 1, formally known as HK1), ropporin (rhophilin associated protein 1, formally known as ROPN1) and FSIP1, FSIP2 (fibrous sheath interacting protein 1, fibrous sheath interacting protein 2) [5, 6, 9]. Studies have suggested PP1{gamma}2 (protein phosphatase 1, catalytic subunit, gamma isoform, formally known as PPP1CC) and its regulator proteins play a key role in sperm motility and other biological functions [10–15], PP1{gamma}2 is localized along the whole flagellum [14], and SP17 (sperm autoantigenic protein 17, formally known as SPA17) is a flagellar and a fibrous sheath protein suspected to bind AKAPs [16–18]. The main emphasis of this article is to explore the effect of fibrous sheath disruption caused by Akap4 gene knockout on the subcellular distribution and/or activity changes of these signaling proteins thought to be important in sperm functions.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Preparation of Mouse Testes and Sperm-Soluble Extracts

The testes of Akap4 gene knockout and wild-type mice were isolated and weighed. They were homogenized in homogenization buffer (buffer A: 10 mM Tris [pH 7.2] containing 1 mM EDTA, 1 mM EGTA, 10 mM benzamidine-HCl, 1 mM phenylmethylsulfonyl fluoride, 0.01 mM N{alpha}-tosyl-phenylalanine-chloromethylketone, and 5 mM ß-mercapto-ethanol) using 1 ml buffer for 0.1 g tissue. The homogenized testes were centrifuged at 16 000 x g at 4°C for 10 min; the supernatant is referred to as testes extracts. The testes extracts were supplemented with 10% (v/v) glycerol and stored at –20°C until further use. Caudal epididymal spermatozoa were isolated from mutant and wild-type mice epididymis and washed twice with Whittingham media (buffer B): 99.3 mM NaCl, 2.7 mM KCl, 1.8 mM CaCl2·2H2O, 0.5 mM MgCl2·6H2O, 0.36 mM NaH2PO4, 25 mM NaHCO3, 25 mM sodium lactate, 0.50 mM sodium pyruvate, 5.55 mM glucose, 100 U/ml penicillin G-K salt, 50 µg/ml streptomycin sulfate. Spermatozoa were collected by centrifugation and the pelleted spermatozoa were suspended in buffer A. The sperm suspension was sonicated with three 5-sec bursts of a Biosonic {Pi} sonicator (Bronwell Scientific, Rochester, NY) at maximum setting. The sperm sonicate was centrifuged at 16 000 x g at 4°C for 10 min. The 16 000 x g supernatants were supplemented with 10% (v/v) glycerol and stored at –20°C until further use. This preparation is referred to as sperm-soluble extracts in this article. Samples of testes and sperm preparations for Western blot analysis were prepared by boiling with SDS sample buffer for 2 min. All procedures involving animals used in this study were approved by the National Institute of Environmental Health Sciences Animal Care and Use Committee and Kent State University Animal Care and Use Committee.

Protein Phosphatase Activity Assay

Preparation of radiolabeled phosphorylase a and its use as a substrate for measurement of PP1 (protein phosphatase 1) activity is based on a protocol described previously [10]. Aliquots from testes and spermatozoa-soluble extracts were incubated with the substrate (in a total volume of 40 µl) at 30°C with 1 mM Mn2+ and with or without PP1-specific inhibitor I2 for 10 min. At the end of this incubation, the reaction was terminated with 180 µl 20% trichloro acetic acid, after which the tubes were centrifuged for 5 min at 12 000 x g at 4°C. The supernatants were quantitated for 32PO4 released from phosphorylase a. This assay measures protein phosphatase activities of both PP1 and PP2A [10]. Protein phosphatase activity due to PP1 was measured in the presence of I2, the PP1-specific inhibitor.

Western Blot Analysis

Testes and sperm preparations were separated by 12% SDS-PAGE based on the protocol of Laemmli [19]. Proteins were then electrophoretically transferred to Immobilon-P, PVDF membrane (Millipore Corp., Bedford, MA). Nonspecific protein-binding sites on the membrane were blocked with 5% nonfat dry milk in Tris-buffered saline (TBS: 25 mM Tris-HCl, pH 7.4, 150 mM NaCl). The blots were then washed twice for 15 min each with TTBS (TBS containing 0.1% Tween 20) and then incubated with primary antibodies at 4°C overnight. After the wash, the blots were incubated with the appropriate horseradish peroxidase-conjugated secondary antibody, usually at a 1:2000 dilution, for 1 h at room temperature. Blots were washed with TTBS twice 15 min each and four times 5 min each. Blots were then developed with an ECL chemiluminescence kit (Amersham, Piscataway, NJ). The antibodies used in this article are as follows.

Mouse monoclonal antibodies against the PKA catalytic and regulatory subunits were purchased from BD Transduction Laboratories (San Jose, CA). The antigens used to produce these antibodies are amino acid residues 18–347 of human PKA {alpha} catalytic subunit, amino acid residues 225– 381 of mouse PKA RI regulatory subunit, the full length of human PKA RII {alpha} regulatory subunit.

AKAP4 antibody was prepared using a synthetic peptide corresponding to amino acids 191–204 from mouse AKAP4 [5].

PP1{gamma}2 antibody was purchased (Zymed Laboratories, San Francisco, CA) and prepared using a synthetic peptide corresponding to the 22 amino acids at the carboxy terminus of PP1{gamma}2 as the antigen. The ability of this antibody to recognize PP1{gamma}2 is well documented [10, 14, 15]

Phospho-PP1 antibody was prepared using residues 316–323 of the catalytic subunit of PP1{alpha} (protein phosphatase 1, catalytic subunit, alpha isoform, formally known as PPP1CA) as the antigen. Antibodies were affinity purified with the synthetic peptides conjugated to a sulfo-link column (Pierce, Rockford, IL). The ability of this antibody to detect phospho-PP1{gamma}2 is documented [15].

Hsp 90 (heat shock protein 1, alpha, formally known as HSPCA) monoclonal antibody was purchased (BD Transduction Laboratories) and was generated using amino acid residues 586–732 of human hsp90 as the antigen.

A polyclonal antibody to 14-3-3 protein (tyrosine 3-monooxygenase/ tryptophan 5-monooxygenase activation protein) was purchased (Zymed Laboratories) and was generated using a synthetic peptide corresponding to the 20 amino acids at the N-terminus of the human 14-3-3 beta/alpha protein. This sequence is identical in the delta/zeta isoforms.

The PI 3-kinase (phosphatidylinositol 3-kinase) and GSK-3 (glycogen synthase kinase-3) antibodies were purchased (Upstate Biotechnology, Lake Placid, NY). The PI 3-kinase antibody recognizes the p85 subunit of PI 3-kinase. The GSK-3 antibody was prepared against a peptide corresponding to residues 203–219 of Drosophila GSK-3/shaggy. This antibody recognizes both {alpha} and ß isoforms of GSK-3. Their abilities to recognize sds22 or PKB in spermatozoa are previously documented [10, 20].

The GSK-3{alpha} (glycogen synthase kinase-3, alpha isoform) antibody was purchased (Zymed Laboratories) and was prepared against a synthetic polypeptide corresponding to the carboxy terminus of GSK-3{alpha} (amino acids WQSTDATPTLTNSS) and was only used for the immunocytochemistry studies in this article.

Sds22 (protein phosphatase 1, regulatory [inhibitor] subunit 7, formally known as PPP1R7) and PKB (thymoma viral proto-oncogene, also known as AKT) antibodies were purchased (Zymed Laboratories) and were prepared using synthetic peptides corresponding to amino acid residues 329– 342 of sds22 and amino acid residues 466–479 of mouse PKB as the antigens. Their abilities to recognize sds22 or PKB in spermatozoa were previously documented [14, 20].

SP17 antibody was a generous gift from Dr. Michael O'Rand (Department of Cell and Developmental Biology, University of North Carolina at Chapel Hill, Chapel Hill, NC) [21].

In the Western blot analysis of this article, there was only one predominant band observed at the expected molecular weight, with the following exceptions: for phospho-PP1{gamma}2, there is a cross-reactive band at about 80 kDa for sperm pellets (not shown), similar to the one observed in a previous article [15]; for SP17, there is a cross-reactive band at about 36 kDa for sperm pellets (not shown).

Indirect Fluorescence Immunocytochemistry of Mouse Spermatozoa

Caudal spermatozoa were isolated as described above, washed twice, and resuspended in PBS. The cells were fixed in 4% formaldehyde in PBS at 4°C for 30 min, followed by the permeabilization with 0.2% Triton X-100. The fixed spermatozoa were attached to poly-L-lysine-coated coverslips. The coverslips were washed once with TTBS, three times with TTBS supplemented with 5% BSA, and incubated for 1 h at room temperature in a blocking solution containing 5% BSA and 5% normal goat serum in TTBS at room temperature. The coverslips were then incubated with primary antibody for 1 h at room temperature or overnight at 4°C, washed three times with TTBS, and incubated with corresponding secondary antibody conjugated to indocarbocyanine (Jackson Laboratories, West Grove, PA) for 1 h at room temperature. The coverslips were washed five times with TTBS and examined by FluoView 500 Confocal Fluorescence Microscope (Olympus, Melville, NY).

Immunohistochemistry of Mouse Testis

Testes of Akap4 gene knockout and wild-type mice were fixed in 4% paraformaldehyde in PBS at 4°C overnight. The testes were then transferred to 70% ethanol and dehydrated, permeabilized, and paraffinized using a Shandon Tissue Processor (Thermo Electron Corporation, Waltham, MA). Serial sections of the whole testes were prepared with a thickness of 10 µm. The sections were placed on poly-L-lysine-coated slides, deparaffinized, and rehydrated according to standard procedure. Nuclei were stained with DAPI (49, 69-diamidino-2-phenylindole) in mounting media. The slides were observed under a fluorescence microscope.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Akap4 Gene Knockout and Testes-Weight Comparison

Western blot assay was used to check the presence of AKAP4 in mice spermatozoa. As expected, AKAP4 was not detected in Akap4 gene knockout spermatozoa but was detected in wild-type spermatozoa. Furthermore, AKAP4 was only present in the sperm pellets of wild-type mice (Fig. 1A). This confirmed the previous article showing that AKAP4 was insoluble and it was present in pellets of sperm homogenates [22]. Testes weights were not different between knockout and wild-type mice (Fig. 1B).



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  		FIG. 1. Analysis of AKAP4 expression and testes-weight comparison in mutant and wild-type mice. A) Western blot analysis showed that AKAP4 was not present in the Akap4 gene knockout mice sperm preparations but was present in the wild-type sperm pellets. Sperm preparations corresponded to 1.5 x 105 cells were loaded in the lanes. B) Testes of mutant and wild-type mice were isolated, weighed, and compared. The values of testes weights are expressed as mean ± SD (sample size: n = 6 for both groups)

Changes in the Distribution of PKA in Akap4 Gene Knockout Mice

The RI {alpha} and RII {alpha} subunits of PKA are found in the flagellum of spermatozoa in different species [23–26]. PKA bound to the flagellum is generally insoluble and is presumably bound to AKAPs. Both RI {alpha} and RII {alpha} subunits of PKA are able to bind AKAP4 in vitro [7, 8]. Results in Figure 2 showed that significantly higher amounts of RII {alpha} and {alpha} catalytic subunit of PKA are present in soluble sperm extracts from mutant compared with wild-type mice. However, there is no difference in the presence and distribution of PKA RI subunit in spermatozoa from mutant and wild-type mice. It is noteworthy that a significant portion of PKA catalytic and regulatory subunits are in the insoluble fraction of sonicates of spermatozoa from both normal and mutant mice.



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  		FIG. 2. The presence of the catalytic subunit and regulatory subunits of PKA in testes and spermatozoa. Western blot analysis showed that the catalytic subunit and regulatory subunit RII {alpha} of PKA are present in both testes extracts and sperm pellets, but their amounts are significantly higher in soluble extracts of mutant compared with wild-type spermatozoa. Testis extracts (50 µg protein) and sperm preparations corresponding to 1.5 x 106 cells were loaded in the lanes

PP1{gamma}2 and Its Phosphorylation in Akap4 Gene Knockout Mice

PP1{gamma}2 is the predominant serine/threonine protein phosphatase in spermatozoa. It plays an important role in sperm motility and other biological functions [10–15]. In bovine spermatozoa, it is present in the principal piece and also in the head region [14]. Enzyme activity of PP1 can be regulated by phosphorylation of the catalytic subunit or by protein regulators. All isoforms of PP1, including PP1{gamma}2, contain a Thr-Pro-Pro-Arg amino acid sequence segment at the carboxy terminus, which is a consensus sequence for phosphorylation by cyclin-dependent kinases [27]. Here, we examined if the Akap4 gene knockout disrupted the PP1{gamma}2 subcellular distribution and/or its phosphorylation status. Western blot showed that there was no change in the amounts of PP1{gamma}2 in knockout and wild-type mice either in testes extracts or in soluble sperm extracts and pellets (Fig. 3A). Next, we measured PP1{gamma}2 catalytic activity. Enzyme activity in testes extracts was not different in knockout compared with wild-type mice (Fig. 3B). However, PP1 activity was about two-fold higher in soluble sperm extracts of mutant mice (Fig. 3C). Finally, we examined the status of PP1{gamma}2 phosphorylation. There was a notable decrease in PP1{gamma}2 phosphorylation in both soluble extracts and pellets of spermatozoa of knockout mice (Fig. 3A).



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  		FIG. 3. PP1{gamma}2 presence, PP1{gamma}2 phosphorylation, and PP1{gamma}2 activity in testes and spermatozoa from mutant and wild-type mice. A) Western blot analysis showed that the presence and amount of PP1{gamma}2 did not change between mutant and wild-type mice either in testes or sperm preparations. Although PP1{gamma}2 phosphorylation did not change in testes extracts, it decreased in extracts and pellets from mutant spermatozoa compared with wild-type spermatozoa. Testis extracts (50 µg protein) or sperm preparations corresponded to 1.5 x 106 cells were loaded. B) Measurement of protein phosphatase activity of PP1{gamma}2 in testis extracts were performed as described in Materials and Methods. Values from these measurements are expressed as mean ± SD (sample size: n = 4 for both groups) and each measurement was performed in duplicate. C) Measurement of PP1{gamma}2 protein phosphatase activity in sperm-soluble extracts were performed as described in Materials and Methods. The values are expressed as mean ± SD (sample size: n = 3 for both groups) and each measurement was performed in duplicate

Presence and Distribution of PP1{gamma}2 Regulators in Akap4 Gene Knockout Mice

Sds22, 14-3-3 protein, and hsp90 were recently shown to be protein regulators of PP1{gamma}2 in spermatozoa [14, 28, and unpublished data]. Sds22 is a mammalian homologue of yeast PP1 binding protein sds22 [14]. It belongs to a family of proteins that contain repeats of leucine-rich 22 amino acid segment. The 14-3-3 proteins belong to a family of abundant and widely expressed 28–33 kDa acidic polypeptides that spontaneously self-assemble as dimers [29– 31]. The 14-3-3 proteins bind to phosphoserine/threonine-containing motifs in a sequence-specific manner [29–31]. Hsp90 is distinguished from other chaperones in that its known substrates are mainly signal-transduction proteins. It acts as an ATP-dependent chaperone [32, 33]. We examined if these PP1{gamma}2 regulators were present in spermatozoa lacking AKAP4 and, if so, whether there were differences in their distribution in the pellet and soluble extracts. Sds22 and 14-3-3 protein were present in testes extracts and in both sperm-soluble extracts and pellets (Fig. 4); hsp90 was present in testes extracts and in sperm pellets (Fig. 4). There were no changes in the amounts or distribution in knockout compared with wild-type mice either in testes or spermatozoa for these three proteins (Fig. 4).



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  		FIG. 4. PP1{gamma}2 regulators in testes and spermatozoa from mutant and wild-type mice. Western blot analysis showed that the presence and amount of sds22, 14-3-3 protein, and hsp90 did not change between mutant and wild-type mice either in testes or sperm preparations. Testis extracts (50 µg protein) and sperm preparations corresponding to 1.5 x 106 cells were loaded in the lanes

PI 3-Kinase, PKB, and GSK-3 Presence in Akap4 Gene Knockout Mice

We have previously identified GSK-3 as one of the possible regulators of PP1{gamma}2 and demonstrated that it is important for sperm functions [10, 13, 20, 34]. GSK-3 is regulated by PI 3-kinase and PKB in a number of cellular functions and processes [35, 36]. In addition to GSK-3, PI 3-kinase/PKB signal-transduction pathways have also been identified involved in motility of spermatozoa [37]. We examined the presence and distribution of GSK-3 and its regulatory enzymes in spermatozoa lacking AKAP4. Western blot analysis showed that all three signaling enzymes were present in testes and spermatozoa of mutant and wild-type mice (Fig. 5). In wild-type mice, PI 3-kinase was present only in sperm pellets (Fig. 5), while in knockout mice, it is present both in sperm-soluble extracts and pellets (Fig. 5). PKB and GSK-3 were both present mainly in the pellets of spermatozoa (Fig. 5). There were no differences in the amounts or distribution of PKB and GSK-3 either in testes or sperm preparations.



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  		FIG. 5. PI 3-kinase, PKB, and GSK-3 in testes and spermatozoa from mutant and wild-type mice. Western blot analysis showed that PI 3-kinase presented in both testes extracts and sperm pellets but only in mutant sperm-soluble extracts; PKB presented in all the testes and sperm preparations. Both isoforms of GSK-3 were present in testes extracts and only in the sperm pellets of both groups. Testis extracts (50 µg protein) and sperm preparations corresponding to 1.5 x 106 cells were loaded in the lanes

Sperm Protein 17 Presence in Akap4 Gene Knockout Mice

Sp17 was first isolated in rabbit as the 17-kDa member of the rabbit serum antigen family of rabbit testes/sperm autoantigens [16, 17, 21]. Initial studies suggested the protein played a role during fertilization [21]. While SP17 is present in many different cell types, its expression is the highest in testes [17, 21]. SP17 is localized within the cytoplasm of the sperm head and through the whole tail region [16]. Significantly, SP17 contains an RII-like AKAP binding domain in the N-terminal half of its sequence [17, 18]. Interestingly, SP17 was present in soluble sperm extracts of knockout mice but not in soluble sperm extracts of wild-type mice (Fig. 6). SP17 was detected in the testes extracts and sperm pellets of both knockout and wild-type mice (Fig. 6).



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  		FIG. 6. SP17 presence in testes and spermatozoa from mutant and wild-type mice. Western blot analysis showed that SP17 was present in testes extracts and sperm pellets from both wild-type and mutant mice but was detected only in soluble extracts of mutant sperm. Testis extracts (50 µg protein) and sperm preparations corresponding to 1.5 x 106 cells were loaded in the lanes

Immunocytochemistry of Mouse Spermatozoa and Immunohistochemistry of Mouse Testes

As previously reported [5], there were distinct morphological differences in sperm flagellum between Akap4 gene knockout and wild-type mice (also see Fig. 7A). Spermatozoa from knockout mice have a shorter and thinner flagellum [5] and sometimes the tip of the flagellum is curled into multiple circles [5]. Immunofluorescence was used to determine the intrasperm localization of PP1{gamma}2, sds22, and GSK-3{alpha}. As seen in Figure 7A, PP1{gamma}2 was present both in the head and the entire length of the flagellum, including the midpiece, in both knockout and wild-type mice spermatozoa. Sds22 labeling showed a similar pattern as PP1{gamma}2. GSK-3{alpha} antibodies stained both knockout and wild-type mouse spermatozoa similarly. However, staining of the midpiece region of knockout-mouse spermatozoa was significantly more intense. Immunofluorescence also showed that hsp90 (data not shown) and 14-3-3 protein (data not shown) were flagellar proteins. Staining with the antibodies appeared to be specific because there was no fluorescence observed when the preimmune serum was used or when spermatozoa were incubated with secondary antibody alone.



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  		FIG. 7. Immunocytochemical analysis of spermatozoa and testis sections from mutant and wild-type mice. A) Fluorescence immunocytochemistry of spermatozoa from gene knockout (KO) and wild-type (WT) mice. Spermatozoa were labeled with antibodies to PP1{gamma}2, sds22, or GSK-3{alpha} as described in Materials and Methods. A x100 objective was used for the observations. A bar shown in one of the panels corresponds to 20 µm. B) Immunohistochemistry of testis sections from KO and WT mice performed as described in Materials and Methods. The photos of both phase contrast and DAPI staining are presented (1–6 are of mutant mice, 1–2 were taken with a x20 lens, 3–4 with a x40 lens, 5–6 with a x100 lens; 7–12 are of wild-type mice, 7–8 with a x20 lens, 9–10 with a x40 lens; 11–12 with a x100 lens. A 50-µm bar is shown in the phase contrast images of knockout mice

Fluorescent microscopy was used to examine morphology of Akap4 gene knockout and wild-type mice testes sections. The observations (Fig. 7B) showed no differences in the structure and organization of seminiferous tubules, seminiferous tubule diameter, sperm cells at different developmental stages, and spermatogenesis in mutant and wild-type mice.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Biogenesis of the mammalian sperm flagellum is a complex sequential process, starting early during spermiogenesis with the elaboration of a primary flagellum, a simple axoneme enveloped by a plasma membrane [38]. The primary flagellum is very thin, and ribosomes and other organelles are excluded from it. The proteins destined for the maturing flagellum were assumed to be synthesized in the spermatid cell body and then transported to their sites of assembly [38]. Early in spermiogenesis, anlagen of the fibrous sheath begins to be deposited near the distal terminal of the presumptive principal piece, and assembly proceeds proximally toward the cell body [38]. Outer dense fibers, on the other hand, begin to assemble at the head-tail junction and continue to be laid down in a proximal-to-distal direction [39]. The postmeiotically expressed Akap4 gene is on the mouse X chromosome [40]; this requires the AKAP4 mRNA and/or protein to be shared among conjoined X- and Y-bearing spermatids of a clonal syncytium via the intercellular bridges [41]. The precursor of AKAP4 must be synthesized in the cell body, transported down the axoneme to the distal portion of the principal piece, associated to form the transverse ribs and longitudinal columns, and stabilized into a detergent-resistant structure [22].

In spermatozoa of Akap4 gene knockout mice, the principal piece was reduced in diameter compared with wild-type spermatozoa [5]. In addition, the flagellum was shortened and the tip was sometimes curled or splayed apart into fine filaments, the outer dense fibers and axoneme were intact, and the smaller diameter and splaying of the flagellum was assumed to be caused by a reduction in size and integrity of the fibrous sheath. Formation of the fibrous sheath is incomplete without AKAP4 [5]. It would be instructive to determine the fate of signaling proteins in mutant spermatozoa lacking the fibrous sheath.

This article examined the presence, distribution, and/or activities of PKA catalytic subunit and regulatory subunits, the signaling proteins PP1{gamma}2, PI 3-kinase, GSK-3, and other regulatory proteins in spermatozoa lacking a fibrous sheath. The activities and phosphorylation of PP1{gamma}2 were studied in some detail. The presence and distribution of these proteins in testes extracts, in sperm-soluble extracts, and in insoluble sperm pellets were also determined. It is assumed that, if a protein were associated with the fibrous sheath, the disruption of the fibrous sheath due to the absence of AKAP4 most likely would change the presence and/or distribution of the protein in mutant spermatozoa.

Disruption of the fibrous sheath caused by Akap4 gene knockout significantly increases the amount of PKA RII {alpha} regulatory subunit and {alpha} catalytic subunit in sperm-soluble extracts of mutant mice (Fig. 2). This increase could be the portion of PKA normally bound to AKAP4 in wild-type spermatozoa. Previous studies have shown that the catalytic and regulatory subunits of PKA are mainly insoluble proteins, perhaps because they are bound to AKAPs [23–26]. The fact that a significant portion of PKA is insoluble in spermatozoa lacking AKAP4 may be a reflection of the portion of the enzyme bound to AKAP3. On the other hand, disruption of the fibrous sheath did not affect the presence and subcellular distribution of PP1{gamma}2 in testes and spermatozoa (Fig. 3A). In addition, phosphorylation and catalytic activity of PP1{gamma}2 did not change in the testis of mutant mice (Fig. 3B). However, phosphorylation of PP1{gamma}2 and the enzymatic activity of PP1{gamma}2 changed significantly for the mutant mice in sperm-soluble extracts (Fig. 3, A and C). It is known that phosphorylation inhibits PP1 activity [42–44], which is consistent with data in this article. The reason for this decrease in PP1{gamma}2 phosphorylation is yet unknown. One explanation is that low levels of ATP might be a contributory factor. In fact, ATP levels in mutant spermatozoa is about one half the levels in wild-type spermatozoa (unpublished data). Because the fibrous sheath anchors glycolytic enzymes, such as GAPDS and HK1-S [5, 6], disruption of the fibrous sheath might disrupt glycolysis and thus lower local ATP levels. With regard to the PP1{gamma}2 regulators sds22, 14-3-3 protein, and hsp90, there was no change in their presence and subcellular distribution either in testes or spermatozoa. These data suggest that PP1{gamma}2, sds22, 14-3-3 protein, and hsp90 are not bound to the fibrous sheath. One noteworthy fact is that, unlike bovine spermatozoa, which have hsp90 in both soluble sperm extracts and pellet preparations (unpublished data), spermatozoa from mice have hsp90 only in sperm pellet preparations. The underlying reason is still unknown. Indirect immunofluorescence determined that PP1{gamma}2, sds22, 14-3-3 protein, and hsp90 (data not shown) are all flagellar proteins in spermatozoa of mice (Fig. 7A).

The presence of PI 3-kinase in supernatant fraction of mutant sperm extracts suggests that PI 3-kinase may be an enzyme associated with the fibrous sheath. There is no difference in PKB and GSK-3 between mutant and wild-type mice, suggesting that these proteins are not associated with the fibrous sheath.

SP17 was first isolated in rabbit as the 17-kDa member of the rabbit serum antigen family of rabbit testis/sperm autoantigens [16, 17, 21]. SP17 was originally proposed to be a sperm-specific protein and thought to play a role in sperm-egg interactions by binding to the zona pellucida via two conserved heparin-binding motifs [16, 17, 21]. However, more recent data indicate that it may be expressed more widely [17]. SP17 is not expressed on the sperm surface but is localized within the cytoplasm of the sperm head and throughout the tail regions [16]. Transcripts for SP17 have been cloned and sequenced from a wide range of mammalian species, including rabbits, mice, rats, humans, macaques, baboons, and sheep and were found to be highly conserved [16, 21]. Analysis of the primary amino acid sequence of SP17 shows that the protein has several interesting features. In particular, the first 74 amino acid residues are almost totally conserved among all studied species and contain a region showing high sequence similarity to the N-terminus of the PKA regulatory subunit II [14, 18, 21]. The N-terminus of the PKA regulatory subunit is critical for PKA dimerization and interaction with AKAPs [1–4]. The presence of SP17 in the fraction of soluble extracts from spermatozoa suggests that SP17 may be associated with the fibrous sheath. But whether SP17 directly binds to AKAP4 remains to be determined.

A logical question is whether motility can be induced in mutant spermatozoa. A number of motility stimulants, such as cAMP analogs [5 and unpublished data], cAMP phosphodiesterase inhibitors, and protein phosphatase 1 inhibitor calyculin A [unpublished data] could not induce increase of motility in mutant spermatozoa. Thus, the lack of motility in spermatozoa lacking AKAP4 is not merely due to insufficient protein phosphorylation. Disruption of the fibrous sheath could be a serious structure defect. Further studies are needed to determine the reasons why spermatozoa lacking the fibrous sheath are immotile.

In summary, this article suggests that PI 3-kinase and SP17 may be bound to the fibrous sheath whereas PP1{gamma}2, GSK-3, hsp90, sds22, protein 14-3-3, and PKB are not. A change in the activity and phosphorylation of PP1{gamma}2 in spermatozoa lacking a fibrous sheath is intriguing. This suggests that the fibrous sheath is very important to maintain the phosphorylation level of PP1{gamma}2 in spermatozoa. Further research is required to ascertain the reason for this observation.


   ACKNOWLEDGMENTS
 
We thank Dr. Michael O'Rand for his generous gift of SP17 antibody; we thank Dr. Douglas Kline and Dr. Robert Clements (Department of Biological Sciences, Kent State University, Kent, OH) for their assistance in confocal fluorescence microscopy, and Dr. Gail Fraizer and Dr. Jennifer Marcinkiewicz (Department of Biological Sciences, Kent State University, Kent, OH) for their assistance in immunohistochemistry; we also thank Kimbery Myers, John Ferrara, and Mauris Nnamani for their assistance in the research and discussion.


   FOOTNOTES
 
1 Supported by NIH grant R01 HD38520. Back

2 Correspondence. FAX: 330 672 3713; zhuang1{at}kent.edu Back

Received: 5 July 2004.

First decision: 8 August 2004.

Accepted: 3 September 2004.


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 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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