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BOR - Papers in Press, published online ahead of print October 20, 2003.
Biol Reprod 2003, 10.1095/biolreprod.103.020024
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BIOLOGY OF REPRODUCTION 70, 439–447 (2004)
DOI: 10.1095/biolreprod.103.020024
© 2004 by the Society for the Study of Reproduction, Inc.

Gamete Biology

Increased Phosphorylation of a Distinct Subcellular Pool of Protein Phosphatase, PP1{gamma}2, During Epididymal Sperm Maturation1

Zaohua Huang, and Srinivasan Vijayaraghavan2

Biological Sciences Department, Kent State University, Kent, Ohio 44242


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The enzyme PP1{gamma}2 is a testis- and sperm-specific isoform of type 1 protein phosphatase (PP1), and it is the only isoform of PP1 in spermatozoa. The enzyme PP1{gamma}2 is essential for spermatogenesis and is also a key enzyme in the development and regulation of sperm motility. The carboxy terminus of the enzyme contains a consensus amino acid sequence for phosphorylation by cyclin-dependent kinases. Using antibodies specific to this phosphorylated amino acid sequence domain, we found that phosphorylated PP1{gamma}2 is present in bovine epididymal spermatozoa. The level of phosphorylated PP1{gamma}2 is significantly higher in motile caudal compared to immotile caput epididymal spermatozoa. A number of treatments, such as 2-chloro adenosine, cAMP analogues, cAMP phosphodiesterase inhibitors, and calcium, which stimulate sperm motility, did not alter the level of phosphorylated PP1{gamma}2. However, calyculin A, which is an inhibitor of protein phosphatase subtypes PP1 and PP2A, significantly increases the level of phosphorylated PP1{gamma}2 in both caput and caudal epididymal spermatozoa. Partial purification by column chromatography showed that phosphorylated PP1{gamma}2 is catalytically active. Phosphorylated PP1{gamma}2 is the only spontaneously catalytically active form of the enzyme in caudal sperm extracts. Western blot analysis shows that the enzyme cyclin-dependent kinase 2, one of the enzymes that phosphorylates the consensus domain at the carboxy terminus in PP1 isoforms, is present in spermatozoa. Western blot analysis of proteins extracted from purified head and tail fragments of spermatozoa showed that phosphorylated PP1{gamma}2 is present predominantly in the sperm head. Fluorescence immunocytochemistry also showed that phosphorylated PP1{gamma}2 is present predominantly in the posterior region of the sperm head. The distinct subcellular localization and changes in its level during sperm maturation suggest a possible role for sperm phosphorylated PP1{gamma}2 in signaling events during fertilization.

epididymis, fertilization, phosphatases, sperm maturation, sperm motility and transport


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Mammalian spermatozoa acquire their capacity for motility and fertilization during epididymal transit while concomitantly undergoing marked changes in chemical and physical properties [1, 2]. These changes are in shape, metabolic patterns, enzymatic activities, chemical and physical properties of the plasma membrane, ability to bind specific epididymal proteins, and in the female tract, ability to bind to the zona pellucida [1, 2]. Despite this knowledge and several decades of biochemical work, the biological significance of most of these changes in relation to development of motility and fertilization potential of spermatozoa remains unclear.

Sperm functions are regulated by intracellular "second messengers"—cAMP, calcium, and pH [1, 3–12]. These mediators presumably act through protein phosphorylation. Protein phosphorylation is a result of the regulated action of protein kinases and protein phosphatases. Sperm motility in immature spermatozoa can be initiated by treatments that stimulate protein kinase activity or inhibit protein phosphatase activity [13–15]. This suggests that the potential for motility already exists in immature epididymal spermatozoa. Low protein kinase and high protein phosphatase activities most likely limit motility in immature spermatozoa. A key enzyme regulating sperm motility development is PP1{gamma}2, which is one of the isoforms of type 1 serine/threonine phosphatase (PP1) [13–15]. High catalytic activity of sperm PP1{gamma}2 holds motility in check in immature caput epididymal spermatozoa. Protein phosphatase inhibitors initiate motility in caput epididymal spermatozoa and stimulate motility in caudal epididymal spermatozoa [13–15].

The ability of spermatozoa to bind and fertilize the egg develops in the epididymis in parallel with motility [1, 16, 17]. The same protein kinases and protein phosphatases responsible for regulating motility also likely play a role in signaling events during fertilization. Supporting this notion is the observation that many factors and treatments shown to be essential for maintaining and promoting the fertilizing ability of spermatozoa are also those that stimulate sperm motility [1, 2, 16]. These factors include bicarbonate, calcium, phosphodiesterase inhibitors, and protein phosphatase inhibitors [13–15]. For example, the protein phosphatase-inhibitors calyculin A and okadaic acid not only stimulate motility but also promote sperm hyperactivation [18, 19] and zona-induced sperm acrosome reaction [20]. Therefore, PP1{gamma}2, which is present both in the head and tail regions of spermatozoa [21], likely plays roles in signaling events during fertilization and in regulating sperm flagellar motion.

The two isoforms of PP1 (PP1{gamma}1 and PP1{gamma}2) are alternatively spliced isoforms generated from a single gene. These two PP1 variants are identical in all respects, except that PP1{gamma}2 has a unique, 21-amino-acid carboxy terminus extension. The PP1{gamma}1 is ubiquitous, but PP1{gamma}2 is expressed only in testis and spermatozoa [22–24]. Disruption of the gene for PP1{gamma}2 arrests spermatogenesis in mice [25]. The PP1{gamma}2 isoform, with its unique carboxy terminus extension, is present in all mammalian spermatozoa that we have tested so far—mouse, rat, hamster, bull, nonhuman primate, and human [13–15; unpublished data].

In somatic cells, the serine/threonine phosphatase PP1 participates in many functions, such as regulation of metabolism, cell cycle, cell signaling, and muscle contraction [26, 27]. The catalytic subunit of PP1, depending on the cell type, is bound to many targeting subunits and regulatory subunits [26–28]. Targeting subunits determine substrate specificity and localize PP1 to distinct subcellular compartments. The regulatory subunits either increase or suppress PP1 activity. Some of the PP1 regulatory subunits identified in somatic cells are inhibitor 1, inhibitor 2 (I2), inhibitor 3, and DARPP32 (dopamine- and cAMP-regulated phosphoprotein) [28, 29]. We have shown that sds22, which is a mammalian homologue of the yeast PP1-binding protein, is a PP1{gamma}2 regulatory protein in spermatozoa [21].

Enzymatic activity of PP1 can also be regulated by phosphorylation of the catalytic subunit. All isoforms of PP1, including PP1{gamma}2, contain a Thr-Pro-Pro-Arg (tppr) amino acid sequence segment at the carboxy terminus, which is a consensus sequence for phosphorylation by cyclin-dependent kinases (Cdks) [30]. The protein phosphatase PP1{alpha} and PP1{gamma}1 can be phosphorylated in vitro by cyclin-dependent kinases, Cdk1 and Cdk2 [31–33]. Increased PP1{alpha} phosphorylation, taking place during G2/M-phase and G1/S-phase transitions, is essential for normal cell cycle and mitosis [31–36]. In those studies, it was found that phosphorylation reduced catalytic activity of the enzyme [31–33]. This change in PP1 activity is thought to alter the balance between phosphorylation and dephosphorylation of key proteins involved in cell-cycle progression.

Research in our laboratory is devoted to understanding how PP1{gamma}2 activity is regulated in spermatozoa. Sperm PP1{gamma}2 fractionates into three distinct pools during purification by column chromatography. Characterization of these different forms of PP1{gamma}2 by Western blot analysis revealed that one of these was phosphorylated. This observation prompted the studies outlined in the present report. We have demonstrated, to our knowledge for the first time, that a portion of sperm PP1{gamma}2 localized to the sperm head is phosphorylated and that the level of phosphorylated PP1{gamma}2 increases during sperm maturation in the epididymis. The enzyme Cdk2, which is the protein kinase implicated in PP1 phosphorylation, is present in spermatozoa. The distinct localization in the head and changes in its levels during sperm passage through the epididymis suggest an important role for PP1{gamma}2 phosphorylation in sperm functions.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Preparation of Sperm Extracts

Testes with intact tunica from mature bulls were obtained from a local slaughterhouse. Spermatozoa were isolated from caudal and caput epididymis and washed as previously described [13] in buffer A (10 mM Tris-HCl [pH 7.2] containing 120 mM NaCl, 10 mM KCl, and 5 mM MgSO4). Sperm pellets were suspended in a homogenization buffer (buffer B; 10 mM Tris [pH 7.2] containing 1 mM EDTA, 1 mM EGTA, 10 mM benzamidine-HCl, 1 mM PMSF, 0.01 mM N-tosyl-L-phenylalanine chloromethylketone, and 5 mM ß-mercaptoethanol). The sperm suspension was sonicated with three 5-sec bursts of a Biosonic II sonicator (Bronwell Scientific, Rochester, NY) at maximum setting. The sperm sonicate was centrifuged at 16 000 x g for 10 min. The supernatants obtained were then supplemented with 10% glycerol and stored at -20°C until further use; hereafter, this preparation is referred to as sperm 16K extracts or sperm extracts. For the detection of phospho-PP1{gamma}2, we also prepared whole-sperm extracts. The sperm pellet was boiled with sample buffer without dithiothreitol for 5 min, then the solution was centrifuged at 16 000 x g for 10 min and the supernatant boiled again with 1% (v/v) ß-mercaptoethanol for 1 min. Hereafter, this preparation is referred to as whole-sperm extracts. Unless indicated otherwise, all the phospho-PP1{gamma}2 Western blot results were from the analysis of whole-sperm extracts.

Preparation of Sperm Head and Tail Fragments

Preparation of head and tail fragments following sonication and centrifugation is based on a previously published technique [37]. Briefly, testes with intact tunica from mature bulls were obtained from a local slaughterhouse. Spermatozoa isolated from caudal and caput regions of the epididymis were resuspended in buffered 0.9% NaCl twice and sonicated three times for 1 min each. The sperm sonicate centrifuged at 3000 x g at room temperature for 15 min resulted in the formation of three layers. The cloudy supernatant contained tail fragments. The buff-colored top layer was enriched in midpiece fragments, and the layer beneath this was a lighter-colored pellet composed of sperm heads. The top layer was scraped away with a spatula and the remaining pellet resuspended and centrifuged at 3000 x g to isolate the sperm heads. This procedure was repeated three times. Visual analysis by microscopy showed that the tail fragment preparation was essentially pure, with an occasional head fragment being visible. The head fragment preparation, although enriched in heads, also had a few midpiece fragments and, occasionally, whole spermatozoa.

Column Chromatography

Caudal sperm 16K extract (50 ml prepared from 5 x 1010 spermatozoa in buffer B) was passed through a diethylaminoethyl (DEAE)-cellulose (Amersham, Piscataway, NJ) column (0.5 x 13 cm) pre-equilibrated with buffer C (buffer B with 0.05 M KCl and additional protease inhibitors: pepstatin A, 1 µg/ml; aprotinin, 2 µg/ml; and leupeptin, 0.5 µg/ml). The column was washed with 20 ml of buffer C followed by elution with a linear gradient of 0.05–0.65 M KCl in buffer C. Flow-through and gradient fractions (0.185–0.35 M KCl) containing PP1{gamma}2 activity or PP1{gamma}2 immunoreactivity were pooled separately and concentrated using a Centricon-10 filter (Millipore Corp., Bedford, MA). The DEAE-cellulose flow-through containing PP1{gamma}2 was also applied to sulfopropyl (SP)-sepharose (5 ml, prepacked; Amersham) column pre-equilibrated with buffer C. The column was washed with 10 ml of buffer C followed by elution with a linear gradient of 0.05–0.65 M KCl in buffer C. The flow-through and gradient fraction after the SP-sepharose column were concentrated and analyzed for PP1{gamma}2 activity and immunoreactivity. The DEAE-cellulose gradient fraction containing PP1{gamma}2 was applied to SP-sepharose (5 ml, prepacked) column pre-equilibrated with buffer C. The column was washed with 10 ml of buffer C followed by elution with a linear gradient of 0.05–0.65 M KCl in buffer C. The flow-through and gradient fraction after SP-sepharose column were concentrated and analyzed for PP1{gamma}2 activity and immunoreactivity. All column procedures were conducted at 4°C. Total protein in caudal sperm extracts and in fractions obtained from column chromatography were measured with Coomassie brilliant blue dye reagent (Bio-Rad, Hercules, CA) as described previously [38].

Protein Phosphatase Activity Assay

Preparation of radiolabeled phosphorylase a and its use as a substrate for measurement of PP1 activity was based on a protocol described previously [13]. The substrate and aliquots from fractions obtained after column chromatography were incubated (in a total volume of 40 µl) at 30°C with 1 mM Mn2+ and with or without inhibitors for 10 min. At the end of this incubation, the reaction was terminated with 180 µl of 20% trichloroacetic 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. One unit of enzymatic activity is the amount of enzyme that catalyzed the release of 1 nmol/min of 32PO4. This assay measured both protein phosphatase PP1 and PP2A activities [13]. Phosphatase activity of PP1 was measured in the presence of the protein phosphatase inhibitor I2. Sperm extracts or column fractions were incubated with I2 (0.5 µg/ml) for 10 min at 30°C before addition of substrate. Enzyme activity sensitive to I2 was caused by PP1.

Western Blot Analysis

Sperm extracts and aliquots from fractions obtained from column chromatography (2–10 µg) were separated by SDS-PAGE through 12% acrylamide slab gels based on the protocol of Laemmli [39]. After electrophoresis, proteins were electrophoretically transferred to an Immobilon-P polyvinylidene fluoride membrane (Millipore, Billerica, 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] and 150 mM NaCl). The blots were then washed twice for 15 min each time with TTBS (TBS containing 0.1% Tween 20) followed by incubation with anti-PP1{gamma}2 (1:5000), anti-phospho-PP1 (1:2000), anti-Cdk1(1:1000), and anti-PSTAIR (1:500) antibodies. The PP1{gamma}2 antibody was commercially prepared (Zymed Laboratories, San Francisco, CA) using a synthetic carboxy terminus extension of PP1{gamma}2 (22 amino acids of the carboxyl terminus) as the antigen. The specificity of the antibody to detect PP1{gamma}2 has been documented previously [13, 21]. Phospho-PP1 antibody was prepared using residues 316–323 of PP1-C{alpha} as the antigen [32]. Antibodies were affinity purified with the synthetic peptides conjugated to a sulfo-link column [32]. We have used two Cdk1 antibodies. One (Santa Cruz Biotechnology, Inc., Santa Cruz, CA) is an affinity-purified rabbit polyclonal antibody raised against a 19-amino-acid peptide mapping at the carboxy terminus of Cdk1 of human origin. The other (EMD Biosciences, Inc., San Diego, CA) is an affinity-purified rabbit polyclonal antibody raised against a peptide mapping mouse Cdk1 (263–297 amino acid residues). The PSTAIR antibody (Sigma-Aldrich Corp., St. Louis, MO) is a mouse monoclonal antibody raised against BSA conjugated to a synthetic, 16-amino-acid oligopeptide containing the PSTAIR sequence. The antibody recognizes both Cdk1 and Cdk2. After washing, the blots were incubated with the appropriate secondary antibody conjugated to horseradish peroxidase at 1:2000 dilution for 1 h at room temperature. Blots were washed with TTBS twice for 15 min each time and four times for 5 min each time. Blots were then developed with an enhanced chemiluminescence kit (Amersham, Little Chalfont, UK).

Calyculin A, Isobutylmethylxanthine, and Calcium Treatment of Caudal and Caput Spermatozoa

Spermatozoa were isolated from caudal and caput epididymis and washed as previously described [13] in buffer A. Twice-washed spermatozoa were suspended in buffer A supplemented with 10 mM glucose. Sperm were treated with calyculin A (final concentration, 50 nM), isobutylmethylxanthine (IBMX; final concentration, 0.1 mM), or varying calcium concentrations (0.1–2 mM) in the presence or absence of 10 µM ionomycin. The total volume of the sperm suspension was 1 ml. The sperm suspension was incubated at 37°C for 15 min. After the incubation, spermatozoa were pelleted by centrifugation at 600 x g at 4°C. Whole-sperm extracts were prepared as previously described. The samples not used immediately were stored at -20°C.

Indirect Fluorescence Immunocytochemistry

Spermatozoa were isolated as described above, washed twice, and resuspended in PBS. Cells were fixed in 4% formaldehyde in PBS at 4°C for 30 min. The sperm solution was then treated with 0.2% Triton X-100. Fixed spermatozoa were attached to poly-L-lysine-coated coverslips. The coverslips were washed once with TTBS and three times with TTBS supplemented with 5% BSA and then incubated for 1 h 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 (Cy3; Jackson Laboratories, West Grove, PA) for 1 h at room temperature. The coverslips were washed five times with TTBS and then examined by fluorescence microscopy.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Phosphorylated PP1{gamma}2 Is Present in Caudal Epididymal Sperm Extracts

We first examined whether phosphorylated PP1{gamma}2 was present in bovine caudal epididymal spermatozoa. We used affinity-purified antibody raised against the phosphorylated amino acid sequence domain in the PP1{alpha} carboxy terminus GRPT(p)PPR, where T(p) indicates phosphorylated threonine residue. The ability of this antibody to detect phospho-PP1{alpha} and phospho-PP1{gamma}1 has been documented previously [32, 33]. Because testis- and sperm-specific PP1{gamma}2 has the sequence of TRPTPPR, which differs by only one amino acid from the corresponding sequence in PP1{alpha} but is the same as that of PP1{gamma}1, we anticipated that the antibody should recognize phosphorylated PP1{gamma}2. Western blot analysis of soluble caudal epididymal sperm extracts (sperm 16K extracts) with the phospho-PP1-specific antibody showed a single, 39-kDa band, which is presumably caused by phosphorylated PP1{gamma}2 (Fig. 1A, lane 2). Electrophoretic migration of this protein matched PP1{gamma}2 detected by antibody against the carboxy terminus region (Fig. 1A, lane 1). The antibody, raised against the unique carboxy terminus region in PP1{gamma}2, is expected to react against both the phosphorylated and nonphosphorylated forms of the enzyme. The phospho-PP1 antibody did not react against a bacterially expressed recombinant PP1{gamma}2 or PP1{gamma}2 fusion protein with a histidine tag (Fig. 1A, lanes 4 and 6). However, the PP1{gamma}2 carboxy terminus antibody recognized both these recombinant proteins (Fig. 1A, lanes 3 and 5).



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  		FIG. 1. Phosphorylated PP1{gamma}2 is present in caudal epididymal sperm extracts. A) Western blot analysis of sperm extracts and recombinant PP1{gamma}2 with phospho-PP1 antibody or PP1{gamma}2 antibody. Lanes 1 and 2: caudal sperm 16K extracts (20 µg each); lanes 3 and 4: his-PP1{gamma}2 (1 µg each); lanes 5 and 6: recombinant PP1{gamma}2 (1 µg each). Extracts and recombinant PP1{gamma}2 were developed with phospho-PP1 antibody (lanes 2, 4, and 6) or with PP1{gamma}2 antibody (lanes 1, 3, and 5). B) Western blot analysis of soluble sperm extracts (lanes 1 and 3; 1 x 107 cells each) and whole-sperm extracts (lanes 2 and 4; 1 x 107 cells each) probed with phospho-PP1 antibody (lanes 1, 2) or PP1{gamma}2 antibody (lanes 3, 4).

A significant proportion of sperm PP1{gamma}2 is present in the insoluble fraction of sperm sonicates [13, 21]. Thus, we next determined the amount of phospho-PP1{gamma}2 in soluble sperm compared to whole-sperm extracts. Data in Figure 1B show that soluble sperm extracts contain only a portion of the total amount of PP1{gamma}2 and phospho-PP1{gamma}2 in spermatozoa. The identity of the higher-molecular-weight band (~80 kDa) obtained in the blot of whole-sperm extracts (Fig. 1B, lane 2) is not known.

Phosphorylation of PP1{gamma}2 Increases During Epididymal Sperm Maturation

In somatic cells, PP1 is phosphorylated only during the G1/S-phase or G2/M-phase transitions of the cell cycle, and phosphorylation declines in other phases [31–33]. The presence of phosphorylated PP1{gamma}2 in terminally differentiated spermatozoa was therefore surprising. We have previously shown that sperm PP1{gamma}2 activity declines during sperm epididymal maturation [13]. Therefore, we examined whether the levels of phospho-PP1{gamma}2 changed during sperm epididymal maturation. It can be seen that the level of phosphorylated PP1{gamma}2 is much higher in caudal compared to caput epididymal spermatozoa (Fig. 2). This comparison is based on extracts prepared from equal number of caput and caudal epididymal spermatozoa, as evidenced by the equal amounts of total PP1{gamma}2 (Fig. 2).



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  		FIG. 2. Phosphorylation of PP1{gamma}2 increases during epididymal sperm maturation. Western blot of caudal and caput whole-sperm extracts from 1 x 107 cells in each lane probed with phospho-PP1 antibody (left) or PP1{gamma}2 antibody (right)

Protein Phosphatase Inhibitor Treatment Leads to Increased PP1{gamma}2 Phosphorylation in Caudal and Caput Epididymal Spermatozoa

Because motile caudal epididymal spermatozoa contain more phosphorylated PP1{gamma}2 compared to immotile caput spermatozoa, a logical question was whether phospho-PP1{gamma}2 levels are correlated with motility. If so, the levels of phospho-PP1{gamma}2 should increase in sperm treated with agents known to alter motility. Cyclic AMP and calcium are two key intracellular mediators of sperm motility. We first examined the effect of the phosphodiesterase-inhibitor IBMX, which initiates motility in caput and stimulates motility in caudal epididymal spermatozoa through elevation of intrasperm cAMP levels [7, 8]. No change was observed in phospho-PP1{gamma}2 levels in sperm treated with IBMX in both caput and caudal spermatozoa (Fig. 3A). Changes in intracellular calcium levels, induced by calcium ionophores, have a biphasic effect on motility—stimulation at low levels and inhibition at high levels of external calcium [5]. The levels of phospho-PP1{gamma}2 did not change as a function of increasing intracellular calcium level (Fig. 3B). Other compounds known to activate motility, such as 2-chloroadenosine [8, 11] and 8-bromo-cAMP [6], or compounds that inhibit motility, such as s-Ht31 [11], had no effect on the level of phospho-PP1{gamma}2 (data not shown).



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  		FIG. 3. Changes in sperm cAMP or calcium do not change the levels of phospho-PP1{gamma}2. (A and B). Protein phosphatase inhibitor treatment leads to an increase in phosphorylation of PP1{gamma}2 in caudal and caput epididymal spermatozoa (C and D). A) Western blot analysis of extracts from caudal (Cd) and caput (Cp) spermatozoa treated with 0.1 mM IBMX compared to control (no treatment) probed with phospho-PP1 (upper blot) and PP1{gamma}2 antibodies (lower blot). Each lane has 1 x 107 cells. B) Western blot analysis of calcium effect on sperm PP1{gamma}2 phosphorylation. Spermatozoa were treated with the indicated amounts of calcium and 10 µM ionomycin. Lane 1: control 1 (no treatment); lane 2: control 2 (10 µM ionomycin in DMSO [final concentration, 0.1%]); lane 3: control 3 (0.1% DMSO); lane 4: 0.2 mM calcium; lane 5: 0.5 mM calcium; lane 6: 1.0 mM calcium; lane 7: 2.0 mM calcium. After a 10-min incubation at 37°C, spermatozoa were collected by centrifugation at 600 x g, and whole-sperm extracts were prepared for Western blot analysis as described in Materials and Methods. C) Effect of calyculin A on PP1{gamma}2 phosphorylation in caudal epididymal spermatozoa. Extracts of control sperm (lane 1), sperm treated with DMSO (lane 2), and sperm treated with 50 nM calyculin A (lane 3) were separated on SDS-PAGE and subjected to Western blot analysis by probing with phospho-PP1 (top) or PP1{gamma}2 antibodies (bottom). Each lane has 1.5 x 107 cells (top) or 0.5 x 107 cells (bottom). Briefly, spermatozoa were incubated with or without calyculin A at 37°C for 15 min and then collected by centrifugation at 600 x g, and whole-sperm extracts were prepared for Western blot analysis as described in Materials and Methods. D) Effect of calyculin A on PP1{gamma}2 phosphorylation in caput epididymal spermatozoa. Extracts of control sperm (lane 1), sperm treated with DMSO (lane 2), and sperm treated with 50 nM calyculin A (lane 3) were separated on SDS-PAGE and subject to Western blot analysis by probing with phospho-PP1 (top) or PP1{gamma}2 antibodies (bottom). Each lane has 1.5 x 107 cells (top) or 0.5 x 107 cells (bottom). Spermatozoa were incubated with or without calyculin A at 37°C for 15 min and then collected by centrifugation at 600 x g, and whole-sperm extracts were prepared for Western blot analysis as described in Materials and Methods

The compounds calyculin A and okadaic acid inhibit the protein phosphatase subtypes PP1 and PP2A. Spermatozoa contain both PP1{gamma}2 and PP2A (unpublished data). Because protein phosphorylation is a balance between the activities of protein kinases and protein phosphatases, we examined whether inhibition of PP1{gamma}2 and PP2A activity would change phospho-PP1{gamma}2 levels. Figure 3 (C and D) shows that phospho-PP1{gamma}2 levels are increased in both caput and caudal epididymal spermatozoa treated with 50 nM calyculin A. This concentration of calyculin A induces motility in caput and stimulates motility in caudal epididymal spermatozoa [13, 14]

Phosphorylated PP1{gamma}2 Is the Only Spontaneously Active Form of PP1{gamma}2 in Caudal Epididymal Spermatozoa 16K Extracts

Studies are underway in our laboratory to identify protein regulators of PP1{gamma}2 in spermatozoa [21, 40, 41]. Data (Fig. 4A) from these studies showed that PP1{gamma}2 in caudal sperm 16K extracts could be separated into three distinct pools by cation- and anion-exchange columns (DEAE-cellulose and SP-sepharose columns, respectively). The three pools of PP1{gamma}2 are as follows: PP1{gamma}2 in the DEAE-cellulose flow-through further purified through SP-sepharose (Fig. 4A, lane 4) is pool 1, and PP1{gamma}2 released from the DEAE-cellulose column separated on SP-sepharose as flow-through (Fig. 4A, lane 6) and gradient fractions (Fig. 4A, lane 7) are pools 2 and 3, respectively.



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  		FIG. 4. Phosphorylated PP1{gamma}2 is the only spontaneously active form of PP1{gamma}2 in caudal epididymal spermatozoa 16K extracts. A and B) Western blot analysis of caudal sperm extracts and its column chromatographic fractions. Caudal sperm 16K extracts were passed through DEAE-cellulose and SP-sepharose columns, and purified fractions were collected as described in Material and Methods. The sperm 16K extracts and column chromatographic fractions were separated on SDS-PAGE and subject to Western blots analysis by probing with PP1{gamma}2 (A) or phospho-PP1 (B) antibodies. Lane 1: caudal sperm 16K extract (20 µg of protein); lane 2: DEAE-cellulose flow-through; lane 3: SP-sepharose flow-through from DEAE-cellulose flow-through; lane 4: SP-sepharose gradient fraction (0.125–0.275 M) from DEAE-cellulose flow-through; lane 5: DEAE-cellulose gradient fraction (0.185–0.35 M); lane 6: SP-sepharose flow-through from DEAE-cellulose gradient fraction (lane 5); lane 7: SP-sepharose gradient fraction (0.155–0.365 M) from DEAE-cellulose gradient fraction (lane 5). Lanes 2–7 contain protein amounts ranging from 2 to 10 µg. The bleached appearance in lane 3 in A and lanes 2, 3, and 7 in B is an artifact of scanning and/or visual illusion. C) Protein phosphatase activity of column chromatographic fractions. Protein phosphatase activity was measured using radiolabeled phosphorylase a as a substrate as described in Materials and Methods. Aliquots of sperm extracts and column fractions were used in the assay, which was performed in the presence or absence of PP1-inhibitor I2 (final concentration, 0.5 µg/ml). The values shown are the I2-sensitive activity attributed to PP1. The values are the means of a duplicate measurement of a single large-scale column run. Similar results were obtained in multiple trial column runs

We examined if any of these pools contained phospho-PP1{gamma}2. Figure 4 shows that PP1{gamma}2 absorbed in DEAE-cellulose (Fig. 4B, lane 5) contained phospho-PP1{gamma}2. The enzyme released from DEAE-cellulose by a salt gradient (0.185–0.35 M) was passed through SP-sepharose column. It can be seen that phospho-PP1{gamma}2 is excluded from the SP-sepharose column (Fig. 4B, lane 6). The other PP1{gamma}2-containing fractions, DEAE flow-through (Fig. 4A, lane 2) and its SP-sepharose gradient fraction (Fig. 4A, lane 4), do not contain phospho-PP1{gamma}2 (Fig. 4B, lanes 2 and 4). Activity data in Figure 4C show that phospho-PP1{gamma}2-containing fractions (Fig. 4, A and B, lanes 5 and 6), are the only catalytically active form of PP1{gamma}2 in caudal sperm extracts.

Cyclin-Dependent Kinases in Spermatozoa

Phosphorylation of PP1{gamma}2 most likely results from the actions of the cyclin-dependent kinases, Cdk1 or Cdk2. Both migrate as 34-kDa proteins in SDS-gel electrophoresis. These two cyclin-dependent kinases phosphorylate somatic cell PP1 isoforms of PP1{alpha} and PP1{gamma}1 both in vitro and in vivo. Cyclin-dependent kinase 1(Cdk1) has been found in developing spermatozoa in mouse testis [42]. However, it is not certain whether Cdk1 or Cdk2 is present in epididymal spermatozoa. We used two different commercial antibodies to examine whether Cdk1 is present in sperm extracts. Both these antibodies revealed a protein at 34 kDa, which is the expected molecular weight of Cdk1, in Jurkat cell lysates (used as a positive control) but not in bovine sperm extracts (Fig. 5, A and B). One of these antibodies showed a number of unidentified, cross-reacting proteins in sperm but not in Jurkat cell extracts (Fig. 5A). We next used antibodies against a conserved amino acid sequence domain PSTAIR of Cdk1 and Cdk2 [43–45]. The PSTAIR antibody, which was expected to react with both Cdk1 and Cdk2, showed a 34-kDa protein in sperm 16K, sperm pellet, and whole-sperm extracts. A proportion of the enzyme is insoluble, as seen in the analysis of extracts prepared from the insoluble fraction of sperm sonicates (Fig. 5C). A immunoreactive band at 34 kDa was also observed in Jurkat cell lysates (used as a positive control) (Fig. 5C).



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  		FIG. 5. Cyclin-dependent kinases in epididymal spermatozoa. A and B) Western blot analysis of caudal and caput sperm soluble extracts (16K) and insoluble extracts (pellet) show no immunoreactivity with two different Cdk1 antibodies (sample load is 50 µg of protein per lane) but strong immunoreactivity with positive control (Jurkat cell lysate, 10 µg of protein). C) Western blots of caudal soluble extracts (16K), insoluble extracts (pellet), and whole-sperm extracts show immunoreactivity with anti-PSTAIR antibody (sample load is 50 µg protein per lane). This anti-PSTAIR antibody also shows strong immunoreactivity with positive control (Jurkat cell lysate, 10 µg protein)

Subcellular Localization of Phospho-PP1{gamma}2

We next attempted to determine the subcellular localization of phospho-PP1{gamma}2 in spermatozoa. First, we isolated head and tail fragments from caudal spermatozoa. Western blot analysis shows that phospho-PP1{gamma}2 is present predominantly in sperm head compared to tail fragments (Fig. 6A). Figure 6A also shows that this comparison is based on extracts adjusted to equal amounts of immunoreactive PP1{gamma}2. The 80-kDa, cross-reacting protein seen in Figure 1B is also observed in the Western blot of the head but not of the tail fraction (not shown).



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  		FIG. 6. Subcellular localization of phospho-PP1{gamma}2. A) Western blot analysis of caudal head and tail preparations (50 µg of protein per lane) show their immunoreactivity with anti-phospho-PP1 (top) and anti-PP1{gamma}2 (bottom). B) Immunolocalization of PP1{gamma}2 and phospho-PP1{gamma}2 in bovine caudal epididymal spermatozoa. Phospho-PP1{gamma}2 labeling is shown in a single spermatozoon (middle left) and in a group spermatozoa (lower left). Control spermatozoa were incubated with second antibody alone. The fluorescence and bright-field images were obtained with a x100 oil immersion lens with a total magnification of x1000

Immunofluorescence was used to determine the localization of phospho-PP1{gamma}2 within spermatozoa. Immunostaining appeared localized to the posterior region of the sperm head (Fig. 6B, left lower and middle). No fluorescence was observed when preimmune rabbit serum or when second antibody alone was used. For comparison, immunostaining observed with PP1{gamma}2 antibody, which recognizes both phosphorylated and nonphosphorylated PP1{gamma}2, is also shown in Figure 6. It can be seen that PP1{gamma}2 is present along the entire length of the flagellum, with intense staining in the posterior region and the equatorial segment of the head.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
The ability of spermatozoa to move and fertilize the egg develops during their transit through the epididymis. Little protein synthesis occurs in epididymal spermatozoa. Therefore, it follows that regulation of protein function by posttranslational modification must be part of the biochemical changes underlying development of sperm function. Protein phosphorylation is a universal cellular mechanism that is used in the regulation of protein function. Protein phosphorylation is the result of the regulated actions of protein kinases and protein phosphatases. Protein kinases and protein phosphatases may themselves be regulated by phosphorylation. In somatic cells, PP1 plays critical roles in cell-cycle progression. The somatic cell isoform of PP1, PP1{alpha}, undergoes phosphorylation and dephosphorylation in a cell cycle-dependent manner. It is phosphorylated during the G1/S-phase and G2/M-phase transitions of the cell cycle [32–34]. In the present study, we have made the unexpected observation that PP1{gamma}2, the sperm-specific protein phosphatase, is phosphorylated in the terminally differentiated, posttesticular, epididymal spermatozoa. Antibody specific for the phosphorylated amino acid sequence domain GRPTPPR was used in the present study.

Initially, we considered the possibility that phosphorylated PP1{gamma}2 in posttesticular spermatozoa could be remnants from events that took place during sperm differentiation in the seminiferous tubules. However, this did not appear to be the case, because the level of phospho-PP1{gamma}2 increases during sperm passage through the epididymis (Fig. 2). It may be noted that caput and caudal epididymal spermatozoa contain roughly equal amounts of total PP1{gamma}2 (Fig. 2). Increased phosphorylation during sperm epididymal maturation suggests that not only the enzymes responsible for phosphorylation and dephosphorylation of PP1{gamma}2 are active in posttesticular spermatozoa but also that their activities are undergoing changes during sperm passage through the epididymis. Further evidence for an active turnover of the phosphate in PP1{gamma}2 is the observation that the level of phospho-PP1{gamma}2 increases in spermatozoa treated with the serine/threonine phosphatase-inhibitor calyculin A (Fig. 3, C and D). Because calyculin A is a potent inhibitor of the phosphatases PP1 and PP2A, it is likely that PP1{gamma}2 itself or PP2A could be the enzyme responsible of dephosphorylating phospho-PP1{gamma}2.

The phosphorylated amino acid sequence domain T(p)PPR in sperm PP1{gamma}2 and the somatic cell isoforms of PP1 is a consensus amino acid sequence for action by cyclin-dependent kinases [30]. The ability of Cdk1 and Cdk2 to phosphorylate PP1{alpha} and PP1{gamma}1 both in vitro and in vivo has been demonstrated [32–34]. Therefore, one or both of these protein kinases, if present in spermatozoa, likely could be responsible for PP1{gamma}2 phosphorylation. Our data suggests that Cdk1 may not be present in posttesticular spermatozoa. Two different, commercially obtained antibodies specific for Cdk1 did not detect a 34-kDa (the expected molecular size of Cdk1) protein in Western blot analysis of sperm extracts (Fig. 5). It should be emphasized that Cdk1 in developing sperm of the mouse testis has been documented using both Western blot analysis and enzyme assay [42]. Our failure to detect Cdk1 is unlikely to have resulted from the lack of cross-reactivity of these antibodies against bovine sperm Cdk1. These antibodies also failed to detect a 34-kDa protein in mouse epididymal sperm extracts (data not shown). The observation that PSTAIR antibodies, which are capable of reacting against Cdk1 and Cdk2, detect a 34-kDa protein in sperm extracts (Fig. 5) suggests that bovine epididymal spermatozoa contain Cdk2. It is possible that Cdk2 may be responsible for PP1{gamma}2 phosphorylation in spermatozoa. Based on intensity of staining in the Western blots, it appears that Cdk2 is present in much lower amounts in spermatozoa compared to somatic cell extracts (Fig. 5). Further studies are required to determine whether Cdk2, detected in Western blots of sperm extracts, is catalytically active within spermatozoa. At present, these studies are complicated by the inability of PSTAIR antibodies to immunoprecipitate Cdks and by the fact that a substantial portion of sperm Cdk2 is insoluble. If sperm Cdk2 is shown to be active, the next question to be answered is whether the increase of PP1{gamma}2 phosphorylation during sperm maturation is caused by an increase in Cdk2 activity, a decline in activity of the phosphatase responsible for dephosphorylating phospho-PP1{gamma}2, or both.

Notable differences exist concerning PP1 phosphorylation in somatic cells compared to that in spermatozoa. First, in somatic cells, PP1 is phosphorylated and dephosphorylated as the cell passes through the G1/S-phase and G2/M-phase transitions. However, in spermatozoa, phosphorylation of PP{gamma}2 increases with sperm epididymal development. Following this increase, it is not known if the enzyme is ever dephosphorylated. Second, phosphorylation of PP1 in somatic cells results in inhibition of PP1 activity, whereas in spermatozoa, phosphorylated PP1{gamma}2 appears to be the only catalytically active form of the enzyme. Third, phosphorylated PP1 is cytoplasmic in somatic cells, whereas phosphorylated PP1{gamma}2 is predominantly insoluble in spermatozoa. In both somatic cells and spermatozoa, the protein phosphatase-inhibitor calyculin A increases PP1 phosphorylation, suggesting that PP1 (PP1{gamma}2) or PP2A may be responsible for dephosphorylation.

Finally, the main question raised in the present study concerns the physiological importance of phosphorylated PP1{gamma}2 in spermatozoa. Although phosphorylation of PP1{gamma}2 increases in parallel with the initiation of sperm motility in the epididymis, the relationship between phosphorylation of PP1{gamma}2 and sperm motility is still unclear. It appears unlikely that the second-messengers cAMP or calcium, which are two of the intrasperm mediators of sperm motility, regulate PP1{gamma}2 phosphorylation. Thus, it is also unlikely that phospho-PP1{gamma}2 may have a direct role in motility. Further support for this conclusion comes from the observations of little phospho-PP1{gamma}2 detected in Western blot analysis of sperm extracts prepared from tail fragments and the lack of a strong signal in the flagellum in fluorescence immunocytochemistry. Localization of phospho-PP1{gamma}2 in the posterior region of the sperm head suggests that it may be required for signaling events during sperm contact with the egg. It is suspected that the equatorial segment and the posterior region of the sperm head may be involved in sperm binding and fusion to the egg plasma membrane [16, 17].

It may appear to be paradoxical that the levels of phospho-PP1{gamma}2, which is the only catalytically active form of caudal sperm PP1{gamma}2, increase in caudal spermatozoa, whereas the activity of the enzyme in soluble sperm extracts decreases in caudal compared to caput spermatozoa [13–15]. It should be emphasized that phospho-PP1{gamma}2 is but one of three forms of caudal sperm PP1{gamma}2. The two other forms of PP1{gamma}2 are inactive [20, 40, 41, 46]. One of these inactive forms is bound to a homologue of the yeast PP1-binding protein sds22 [20, 46]. A summary of the properties of these pools of PP1{gamma}2 is shown in Figure 7. In caput spermatozoa, a substantial portion of PP1{gamma}2, which is not phosphorylated, is not bound to sds22 and is in its catalytically active form [46]. The decrease in PP1{gamma}2 activity during sperm maturation is caused by changes in the association of nonphosphorylated PP1{gamma}2 with its protein regulators. Presumably, these changes more than compensate for the increase in the pool of catalytically active phospho-PP1{gamma}2.



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  		FIG. 7. Properties of three pools of PP1{gamma}2 in caudal and caput epididymal spermatozoa. Purification by column chromatography showed three distinct pools of PP1{gamma}2 in caudal and caput sperm extracts. Both contain PP1{gamma}2 bound to heat shock protein 90 (hsp90), which is inactive [40], and PP1{gamma}2 bound to protein 14-3-3, which shows PP1 activity [40]. The pool of PP1{gamma}2 bound to protein 14-3-3 is phosphorylated. Caudal sperm extracts contain a third pool of PP1{gamma}2, which is bound by sds22 and shows no PP1 activity [46], whereas caput sperm extracts contain a pool of PP1{gamma}2, which is in its free, catalytically active form [46]

We have recently found that phospho-PP1{gamma}2 may be bound to protein 14-3-3, a phosphoprotein-binding protein, which is also localized to the posterior region of the sperm head [40, 41; unpublished results]. Because phospho-PP1{gamma}2 is the only spontaneously active form of PP1{gamma}2 in caudal sperm extracts (Fig. 4), we speculate that phosphorylation of PP1{gamma}2 may be a biochemical mechanism to maintain a catalytically active pool of PP1{gamma}2 in this distinct subcellular location in spermatozoa. This increased phosphorylation and catalytically active PP1{gamma}2 may be necessary for signaling events during fertilization. We are actively pursuing studies to elucidate the role for this discrete pool of catalytically active phospho-PP1{gamma}2 in sperm function.


   ACKNOWLEDGMENTS
 
We thank Drs. Angus Nairn and Paul Greengard (Rockefeller University, New York, NY) for their generous gift of phospho-PP1 antibody and critical review of the manuscript. We also thank Dr. Balwant Khatra (University of California, Long Beach, CA) for his supply of phosphorylase b and phosphorylase kinase. We also thank Dr. Payaningal R. Somanath, Shannan Jack, Brian Sapola, John Ferrara, and Kimberly Meyers for their assistance in the research and discussions.


   FOOTNOTES
 
1 Supported by NIH grant RO1 HD38520. Back

2 Correspondence: Srinivasan Vijayaraghavan, Biological Sciences Department, Kent State University, Kent, Ohio 44242-0001. FAX: 330 672 3713; svijayar{at}kent.edu Back

Received: 5 June 2003.

First decision: 30 June 2003.

Accepted: 17 September 2003.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

  1. Bedford JM, Hoskins DD. The mammalian spermatozoon: morphology, biochemistry and physiology. In: Lamming GE (ed.), Marshall's Physiology of Reproduction. New York: Churchill Livingstone; 1990:379–568
  2. Cooper TG, Waites GM, Nieschlag E. The epididymis and male fertility. A symposium report. Int J Androl 1986 9:281-90[Medline]
  3. Hoskins DD, Brandt H, Acott TS. Initiation of sperm motility in the mammalian epididymis. Fed Proc 1978 37:112534-2542[Medline]
  4. Garbers DL, Kopf GS. The regulation of spermatozoa by calcium and cyclic nucleotides. Advances In Cyclic Nucleotide Research 1980 13:251-306[Medline]
  5. Hoskins DD, Acott TS, Critchlow L, Vijayaraghavan S. Studies on the roles of cyclic AMP and calcium in the development of bovine sperm motility. J Submicrosc Cytol 1983 15:21-27[Medline]
  6. Vijayaraghavan S, Critchlow LM, Hoskins DD. Evidence for a role for cellular alkalinization in the cyclic adenosine 3',5'-monophosphate-mediated initiation of motility in bovine caput spermatozoa. Biol Reprod 1985 32:489-500[Abstract]
  7. Vijayaraghavan S, Hoskins DD. Forskolin stimulates bovine epididymal sperm motility and cyclic AMP levels. J Cyclic Nucleotide Protein Phosphor Res 1985 10:499-510[Medline]
  8. Vijayaraghavan S, Hoskins DD. Regulation of bovine sperm motility and cyclic adenosine 3',5'-monophosphate by adenosine and its analogues. Biol Reprod 1986 34:468-477[Abstract]
  9. Goltz JS, Gardner TK, Kanous KS, Lindemann CB. The interaction of pH and cyclic adenosine 3',5'-monophosphate on activation of motility in Triton X-100 extracted bull sperm. Biol Reprod 1988 39:1129-1136[Abstract]
  10. Vijayaraghavan S, Hoskins DD. Changes in the mitochondrial calcium influx and efflux properties are responsible for the decline in sperm calcium during epididymal maturation. Mol Reprod Dev 1990 25:186-194[CrossRef][Medline]
  11. Vijayaraghavan S, Goueli SA, Davey MP, Carr DW. Protein kinase A-anchoring inhibitor peptides arrest mammalian sperm motility. J Biol Chem 1997 21: (272) 4747-4752
  12. Hamamah S, Gatti JL. Role of the ionic environment and internal pH on sperm activity. Hum Reprod 1998 13:20-30
  13. Vijayaraghavan S, Stephens DT, Trautman K, Smith GD, Khatra B, da Cruz e Silva EF, Greengard P. Sperm motility development in the epididymis is associated with decreased glycogen synthase kinase-3 and protein phosphatase 1 activity. Biol Reprod 1996 54:709-718[Abstract]
  14. Smith GD, Wolf DP, Trautman KC, da Cruz e Silva EF, Greengard P, Vijayaraghavan S. Primate sperm contain protein phosphatase 1, a biochemical mediator of motility. Biol Reprod 1996 54:719-727[Abstract]
  15. Smith GD, Wolf DP, Trautman KC, Vijayaraghavan S. Motility potential of macaque epididymal sperm: the role of protein phosphatase and glycogen synthase kinase-3 activities. J Androl 1999 20:47-53[Abstract/Free Full Text]
  16. Yanagimachi R. Mammalian fertilization. In: Knobil E, Neill JD (eds.), The Physiology of Reproduction. New York: Raven Press; 1994:189–378
  17. Eddy EM, O'Brian DA. The spermatozoon. In: Knobil E, Neill JD (eds.), The Physiology of Reproduction. Raven Press, New York; 1993:29–78
  18. Si Y, Okuno M. Role of tyrosine phosphorylation of flagellar proteins in hamster sperm hyperactivation. Biol Reprod 1999 61:240-246[Abstract/Free Full Text]
  19. Si Y. Hyperactivation of hamster sperm motility by temperature-dependent tyrosine phosphorylation of an 80-kDa protein. Biol Reprod 1999 61:247-252[Abstract/Free Full Text]
  20. Furuya S, Endo Y, Osumi K, Oba M, Nozawa S, Suzuki S. Calyculin A, protein phosphatase inhibitor, enhances capacitation of human sperm. Fertil Steril 1993 59:216-22[Medline]
  21. Huang Z, Khatra B, Bollen M, Carr DW, Vijayaraghavan S. Sperm PP1{gamma}2 is regulated by a homologue of the yeast protein phosphatase binding protein sds22. Biol Reprod 2002 67:1936-1942[Abstract/Free Full Text]
  22. Sasaki K, Shima H, Kitagawa Y, Irino S, Sugimura T, Nagao M. Identification of members of the protein phosphatase 1 gene family in the rat and enhanced expression of protein phosphatase 1{gamma} gene in rat hepatocellular carcinomas. Jpn J Cancer Res 1990 81:1272-1280[CrossRef][Medline]
  23. Kitagawa Y, Sasaki K, Shima H, Shibuya M, Sugimura T, Nagao M. Protein phosphatases possibly involved in rat spermatogenesis. Biochem Biophys Res Commun 1990 171:230-235[CrossRef][Medline]
  24. Da Cruz e Silva EF, Fox CA, Ouimet CC, Gustafsou E, Watson SJ, Greengard P. Differential expression of protein phosphatase 1 isoforms in mammalian brain. J Neurosci 1995 15:3375-3389[Abstract]
  25. Varmuza S, Jurisicova A, Okano K, Hudson J, Boekelheide K, Shipp EB. Spermiogenesis is impaired in mice bearing a targeted mutation in the protein phosphatase 1c{gamma} gene. Dev Biol 1999 205:98-110[CrossRef][Medline]
  26. Cohen P. The structure and regulation of protein phosphatases. Annu Rev Biochem 1989 58:453-508[CrossRef][Medline]
  27. Cohen PT. Protein phosphatase 1-targeted in many directions. J Cell Sci 2002 115:Pt 2241-256[Abstract/Free Full Text]
  28. Aggen JB, Nairn AC, Chamberlin R. Regulation of protein phosphatase-1. Chem Biol 2000 7:R13-R23[CrossRef][Medline]
  29. Oliver CJ, Shenolikar S. Physiologic importance of protein phosphatase inhibitors. Front Biosci 1998 3:D961-D972[Medline]
  30. Moreno S, Nurse P. Substrates for p34cdc2: in vivo veritas?. Cell 1990 61:549-551[CrossRef][Medline]
  31. Dohadwala M, da Cruz e Silva EF, Hall FL, Williams RT, Carbonaro-Hall DA, Nairn AC, Greengard P, Berndt N. Phosphorylation and inactivation of protein phosphatase 1 by cyclin-dependent kinases. Proc Natl Acad Sci U S A 1994 91:6408-6412[Abstract/Free Full Text]
  32. Kwon YG, Lee SY, Choi Y, Greengard P, Nairn AC. Cell cycle-dependent phosphorylation of mammalian protein phosphatase 1 by cdc2 kinase. Proc Natl Acad Sci U S A 1997 94:2168-2173[Abstract/Free Full Text]
  33. Liu CW, Wang RH, Dohadwala M, Schonthal AH, Villa-Moruzzi E, Berndt N. Inhibitory phosphorylation of PP1{alpha} catalytic subunit during the G1/S transition. J Biol Chem 1999 274:29470-29475[Abstract/Free Full Text]
  34. Brautigan DL. Flicking the switches: phosphorylation of serine/threonine protein phosphatases. Semin Cancer Biol 1995 6:211-217[CrossRef][Medline]
  35. Yamano H, Ishii K, Yanagida M. Phosphorylation of dis2 protein phosphatase at the C-terminal cdc2 consensus and its potential role in cell cycle regulation. EMBO J 1994 13:5310-5318[Medline]
  36. Berndt N. Protein dephosphorylation and the intracellular control of the cell number. Front Biosci 1999 4:D22-D42[Medline]
  37. Stambaugh R, Buckley J. Identification and subcellular localization of the enzymes effecting penetration of the zona pellucida by rabbit spermatozoa. J Reprod Fertil 1969 19:423-432[Abstract/Free Full Text]
  38. Bradford MM. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 1976 72:248-254[CrossRef][Medline]
  39. Laemmli UK. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 1970 227:680-685[CrossRef][Medline]
  40. Mishra S, Somanath PR, Vijayaraghavan S. Distinct pools of PP1{gamma}2 are regulated by sds22, protein 14-3-3 and hsp90 within spermatozoa. In: Annual meeting of Experimental Biology, American Society of Molecular Biology and Biochemistry; 2003; San Diego, CA. Abstract LB270
  41. Mishra S, Huang Z, Vijayaraghavan S. Binding to sds22 and inactivation of catalytic activity of the protein phosphatase PP1 gamma 2 occurs during sperm motility in the epididymis. In: 36th annual meeting, Society for the Study of Reproduction; 2003; Cincinnati, OH. Abstract 141.
  42. Zhu D, Dix DJ, Eddy EM. HSP70-2 is required for CDC2 kinase activity in meiosis I of mouse spermatocytes. Development 1997 124:3007-3014[Abstract]
  43. Minshull J, Golsteyn R, Hill CS, Hunt T. The A- and B-type cyclin associated cdc2 kinases in Xenopus turn on and off at different times in the cell cycle. EMBO J 1990 9:2865-2875[Medline]
  44. Hirai T, Yamashita M, Yoshikuni M, Tokumoto T, Kajiura H, Sakai N, Nagahama Y. Isolation and characterization of goldfish cdk2, a cognate variant of the cell cycle regulator cdc2. Dev Biol 1992 152:113-120[CrossRef][Medline]
  45. Yamashita M, Fukada S, Yoshikuni M, Bulet P, Hirai T, Yamaguchi A, Yasuda H, Ohba Y, Nagahama Y. M-phase-specific histone H1 kinase in fish oocytes. Purification, components and biochemical properties. Eur J Biochem 1992 205:537-543[Medline]
  46. Mishra S, Somanath PR, Huang Z, Vijayaraghavan S. Binding and inactivation of the germ cell-specific protein phosphatase PP1{gamma}2 by sds22 during epididymal sperm maturation. Biol Reprod 2003 69:1572-1579[Abstract/Free Full Text]



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