ORIGINAL ARTICLE 394J o u r n a l o fJ o u r n a l o f

CellularPhysiologyCellularPhysiology

Sperm Ecto-Protein Kinase

and Its Protein Substrate:Novel Regulators of MembraneFusion During Acrosome Reaction

ARUNIMA MAITI,1 DEBJANI NATH,2 SANDHYA R. DUNGDUNG,1

AND GOPAL C. MAJUMDER1,3*1Indian Institute of Chemical Biology, Jadavpur, Kolkata, West Bengal, India2Department of Zoology, University of Kalyani, Kalyani, Nadia, West Bengal, India3Centre for Rural and Cryogenic Technologies, Jadavpur University, Jadavpur, Kolkata, West Bengal, India

Previously we have purified and characterized a unique plasma membrane-specific cyclic AMP-independent ecto-protein kinase(ecto-CIK) as well as its ecto-phosphoprotein substrate (MPS) using caprine sperm model. This study reports for the first time the role ofthe sperm external surface protein phosphorylation system on sperm acrosome reaction, which is essential for fertilization. Calciumionophore A23187 has been used to trigger the sperm acrosome reaction in vitro. Treatment of sperm cells with CIK antibody (dil: 1:500)causes a significant decrease (approx. 50%) in percentage of acrosome reacted sperm. Onset of the acrosome reaction causes aremarkable increase in the rate of acrosin release from the cells in the medium. However, CIK antibody inhibits significantly (approx. 50%)the acrosin release. The level of membrane-bound MPS as estimated by ELISA and the degree of its phosphorylation catalyzed by theendogenous ecto-CIK, increase significantly with the progress of the acrosome reaction. Both the parameters increase by approximately100% during the 20 min of the reaction. MPS antibody (1:100 dilution) markedly decreases (approx. 75%) percentage of acrosome-reactedsperm. MPS antibody as well shows high efficacy to inhibit acrosin release from spermatozoa. The results demonstrate that thecell–surface protein kinase and its protein substrate are essential for membrane fusion component of acrosome reaction. The data areconsistent with the view that MPS regulates acrosomal membrane fusion with the overlying plasma membrane by the mechanism of itsphosphorylation and dephosphorylation.

J. Cell. Physiol. 220: 394–400, 2009. � 2009 Wiley-Liss, Inc.

Contract grant sponsor: Department of Atomic Energy, Trombay,Mumbai.Contract grant sponsor: Department of Science and Technology,New Delhi.Contract grant sponsor: Indian council of Medical Research, NewDelhi.Contract grant sponsor: Council of Scientific and IndustrialResearch, New Delhi, India.

*Correspondence to: Gopal C. Majumder, Indian Institute ofChemical Biology, Jadavpur, Kolkata 700 032, West Bengal, India;Centre for Rural and Cryogenic Technologies, Jadavpur University,Jadavpur, Kolkata 700 032, West Bengal, India.E-mail: majumdergc42@yahoo.co.in

Received 13 October 2008; Accepted 27 February 2009

Published online in Wiley InterScience(www.interscience.wiley.com.), 13 April 2009.DOI: 10.1002/jcp.21778

Since the appearance of the first two reports on the localizationof a protein kinase (ecto-kinase) on the external surface ofmammalian cells (Mastro and Rosengurt, 1976; Schlaegerand Kohler, 1976), many articles have been publisheddemonstrating various types of ecto-kinase in a variety of celltypes (for review Nath et al., 2008). Preliminary studies ofseveral investigators using the cell-bound uncharacterizedecto-kinase models, have implicated that these ecto-enzymesmay participate in the regulation of cellular physiology such ascytokine functions (Al-Nadaki et al., 1999), neuraldifferentiation (Pawlowska et al., 1993), myogenesis (Chen andLo, 1991), etc. However, precise biochemical identity of theecto-kinases and their specific membrane-bound ecto-proteinsubstrates is largely unknown, as no study has yet beenreported on the purification of these enzymes/substrates toapparent homogeneity. Previous studies from our laboratoryprovided several lines of evidences for the occurrence of acAMP-independent protein kinase (ecto-CIK) on the externalsurface of goat epididymal spermatozoa that causesphosphorylation of the endogenous membrane-boundphosphoproteins that are oriented externally (Halder andMajumder, 1986; Halder et al., 1986; Mitra et al., 1994). In ourinitial studies, one of the major approach to establish the ‘‘ecto’’nature of the kinase as well as the phosphoprotein was theapplication of well documented cell surface probes such asp-chloromercuriphenylsulphonic acid (PCMPS) anddiazonoium salt of sulphanilic acid (DSS) that strongly inhibitsperm surface protein phosphorylatin event of the intact cellswithout penetrating the cell membrane (Halder and Majumder,1986). Our recent studies have described for the first time thepurification to apparent homogeneity of an ecto-protein kinase(ecto-CIK) (Nath et al., 2008) as well as its phosphoproteinsubstrate (ecto-MPS) (Maiti et al., 2004) located on the sperm

� 2 0 0 9 W I L E Y - L I S S , I N C .

external surface using caprine (Capra indicus) sperm as themodel. The isolated kinase is a dimmer possessing two subunits:63 and 55 kDa. The CIK is a strongly basic protein. CIK is aunique membrane protein-specific kinase, which specializes forphosphorylating the serine and threonine residues of the outercell–surface phosphoproteins. The specific activity of CIK isremarkably higher in spermatozoa as compared to other tissuesand body fluids tested (Nath D. and Majumder G.C.,unpublished data), thereby showing that CIK has high degree ofsperm specificity. The major protein substrate (MPS) of thesperm ecto-kinase is a 100-kDa phosphoprotein (Maiti et al.,2004). The ecto-protein kinase is primarily localized in theacrosomal cap area of the external surface of the mature sperm

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head as demonstrated by indirect immunofluorescence studies(Nath et al., 2008). ‘‘Ecto’’ nature of CIK as well as MPS has beenestablished conclusively using indirect immunofluorescencestudies based on antibodies of these pure antigens and studieson activities of these molecules utilizing their monovalentantibodies (Maiti et al., 2004, 2008; Nath et al., 2008). Althoughearlier investigators have provided several lines of evidences forthe occurrence of ecto-protein kinases in a variety ofmammalian cells (Mastro and Rosengurt, 1976; Schlaeger andKohler, 1976; Majumder, 1981; Halder and Majumder, 1986;Dey and Majumder, 1990; Walter et al., 2000; Guthmann et al.,2002; for review Nath et al., 2008), the above-mentionedfindings from our laboratory provide confirmatory evidence forthe localization of an ecto-protein kinase and its proteinsubstrate on a cell surface. The data demonstrate that ecto-CIKthrough its substrate protein: MPS plays a vital role in theregulation of sperm forward progression and velocity (Maitiet al., 2004, 2008; Nath et al., 2008). MPS serves as a significantpromoter of sperm forward progression.

The present study investigates the role of the purifiedecto-CIK and its phosphoprotein substrate: MPS in spermacrosome reaction: another important sperm function which isinitiated upon contact of sperm cells with the ovum. Duringacrosome reaction the outer acrosomal membrane fuseswith the sperm plasma membrane thereby causing releasefrom the acrosomal sac, hydrolytic enzymes such as acrosin,hyaluronidase, etc. that are essential for successful fertilization(Zaneveld and De Jonge, 1991; Yanagimachi, 1995; Breitbartand Naor, 1999). This study demonstrates that the novelecto-protein kinase and its protein substrate: MPS play vital rolein the acrosomal membrane fusion event.

Materials and MethodsReagents

The following reagents were obtained from Sigma ChemicalCompany (St. Louis, MO): ATP (horse muscle), polyethylene glycol(average molecular weight 20 kDa), ethylene glycol bis-(b-aminoethyl ether) N-N0-tetra acetic acid (EGTA), phenyl methylsulphonyl fluoride (PMSF), b-mercaptoethanol, Triton X-100,DEAE cellulose, gelatin, HRP-conjugated anti-rabbit IgG,FITC-conjugated anti-rabbit IgG, Tween-20, H2O2, complete andincomplete Fraunds adjuvant, sodium pyruvate, calcium ionophoreA23187, glutaraldehyde, sodium cacodylate, Bismarck Brown,Rose Bengal, Ficoll, p-phenylenediamine, benzoyl-l-arginineethyl ether (BAEE) and bovine serum albumin (BSA). [g-32P]-Orthophosphate (carrier free) was supplied by Bhabha AtomicResearch Centre (Trombay, Mumbai). [g-32P]ATP was prepared inour laboratory according to Halder and Majumder (1986).Polybuffer 74 was obtained from Pharmacia Fine Chemicals(Uppsala, Sweden).

Isolation of mature spermatozoa

Goat epididymal spermatozoa were isolated within 2 h of slaughter(Rana and Majumder, 1987; Halder et al., 1990). The caudaepididymis was minced and suspended in a modified Ringer’ssolution (RPS medium: 119 mM NaCl, 5 mM KCl, 1.2 mM MgSO4,10 mM glucose, 16.3 mM potassium phosphate, 50 U penicillin/ml,pH 6.9) with gentle stirring. The mature spermatozoa were thenfiltered through 4–5 layers of cheesecloth and sedimented bycentrifugation at 500g for 5 min and then washed two times in RPSmedium. The isolated spermatozoa were highly pure as judged byphase contrast microscopy. There was no detectablecontamination with other cells or cell debris.

Phosphorylation of sperm ecto-phosphoproteins

Freshly cut sperm preparations were incubated with [g-32P] ATPto measure the rate of phosphorylation of external cell surface

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phosphoproteins by endogenous ecto-protein kinase. Thestandard assay medium contained intact spermatozoa (10–20� 106), 5 nmol of [g-32P] ATP (containing 2–5� 106 cpm),2 mmol of MgCl2 and 0.2 mmol of EGTA in a total volume of 0.2 mlRPS medium. Incubation was carried out at 378C for 1 min andreaction was stopped by addition of 0.2 ml of 10 mM ATP/150 mMpotassium phosphate and 5 ml of 10% trichloroacetic acid wasadded. After 60 min the resulting cell suspension was filteredthrough a Whatman No. 1 filter paper disc and then washed with40 ml 5% trichloroacetic acid. The discs were then counted for32P radioactivity in a liquid scintillation spectrometer (Majumder,1981; Halder and Majumder, 1986; Nath and Majumder, 1999).

Isolation of goat sperm plasma membrane

Highly purified plasma membranes were isolated from the maturecauda sperm by an aqueous two-phase polymer method (Rana andMajumder, 1989). Membrane purity was high as determined byestimating marker enzymes (alkaline phosphatase, 50-nucleotidase,acrosin, cytochrome oxidase, and glucose-6-phosphatase) and byelectron microscopy. The membrane preparation was dispersed in25 mM potassium phosphate buffer, pH 7.0, containing 1 mM PMSF,2 mM b-mercapto ethanol, 1 mM EDTA and 30% (v/v) glycerol(Buffer A) and finally stored at �208C. The protein content of theplasma membrane was estimated using BSA as standard(Bensadown and Weinstein, 1976).

Purification of membrane-bound ecto-CIK and MPS

The ecto-CIK was purified to homogeneity from plasma membraneof mature goat cauda epididymal spermatozoa according to Nathet al. (2008). The physiological protein substrate of caprine spermecto-CIK was purified from isolated plasma membrane (Maiti et al.,2004) with minor modifications. The plasma membrane proteinswere first phosphorylated by the endogenous ecto-CIK usingthe assay medium that contained 250 nmol [g-32P]-ATP [4–5�108 counts/min (cpm)], 100 mmol MgCl2, 10 mmol EGTA, and 7.5–10 mg of plasma membrane in a total volume of 10 ml 50 mM Tris–HCl, pH 8.5. The reaction mixture was incubated for 1 min at 378C.The reaction was arrested with 100 ml of 125 mM potassiumphosphate buffer (pH 7) containing 4 mM ATP. The cell membranewas sedimented by centrifugation at 15,000g for 15 min. Thelabeled membrane proteins were then solubilized from themembrane with 1% Triton X-100 in 5 mM potassium phosphatebuffer (pH 7) containing 1 mM PMSF, 1 mM EDTA, 2 mM b-mercaptoethanol and 20% (v/v) glycerol (buffer A). The solubilized32P-labeled plasma membrane proteins were then subjected tosequential Sephacryl S-300 molecular sieve chromatography,DEAE-cellulose ion-exchange chromatography andchromatofocusing, to obtain purified 32P-MPS. The isolated32P-labelled MPS (approx. 2.3� 104 cpm/nmol) was preserved inbuffer A (Maiti et al., 2004) at �208C.

Production of antibody

Anti-serum against the purified ecto-CIK was raised in rabbit byfour successive injections at 1st, 7th, 15th, and 21st day. Firstinjection was given subcutaneously using 500 mg of protein incomplete Freund’s adjuvant. In second and third injections 200 mgprotein was used in incomplete Freund’s adjuvant. Fourth injectioncontained 400 mg of CIK in incomplete Freund’s adjuvant. Bloodwas collected from the ear vein on 27th day of inoculation andserum was prepared and stored at �708C. Non-immune bloodserum was collected from the same animal before startinginoculation programmed (Ouchterlony, 1958). Theimmunoglobulin of the immune serum was precipitated twicewith 50% ammonium sulfate. The final precipitate was dissolved inPBS (pH 8.0) and dialyzed overnight against the same buffer.

The same procedure was carried out for the production of MPSantibody in rabbit.

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ELISA

For determining the antibody titer value, 50 ml of protein solution(purified MPS/ecto-CIK containing 100 ng protein/Triton X-100solubilized plasma membrane) in PBS (10 mM sodium phosphatepH 7.5 containing 0.9% NaCl) was added in the wells of microtiterplates and incubated overnight at 48C. After washing with PBS, thewells were blocked with PBS containing 3% BSA and incubated at378C for 1 h. Then the 1st antibody (MPS antibody/CIK antibody)in PBS containing 1% BSA was added at different dilutions.Incubation and washing were done as before followed by theaddition of HRP-conjugated goat anti rabbit IgG (2nd antibody at adilution of 1:1,000 in PBS containing 1% BSA). The plate was thenincubated at 378C for 1 h. Finally color development was doneby using 3 mM orthophenyldiamine (OPD) in 24 mM citricacid–50 mM sodium hydrogen phosphate containing 0.04% H2O2

(pH 5.1–5.4) in PBS (Wisdom, 1976). Development of color wasstopped after 30 min with 4(N) H2SO4 and absorbance wasmeasured at 492 nm by ELISA reader.

Indirect Immunofluorescence of MPS

Goat spermatozoa derived from cauda epididymis were collectedin PBS, pH 7.4. The sperm suspensions were centrifuged at 500g for5 min at 48C. The resulting sperm pellet containing approximately5� 106 sperm, was incubated in PBS containing 1% BSA for 30 minat 48C. After washing, the sperm pellets were incubated with MPSantibody (1:100) in PBS containing 1% BSA at 48C for 1 h. Thecontrol experiment was run in same way where the sperm cellswere incubated with preimmune sera. After washing with PBS,FITC-conjugated anti-rabbit IgG was added at a dilution of 1:40 andincubated again at 48C for 1 h. The cells were further washed in PBSand mounted in PBS, pH 8 containing 90% glycerol, sodium azideand 1 mg/ml p-phenylendiamine to reduce photo bleaching duringobservation. The fluorescence was visualized through the Leitzfluorescence microscope.

Acrosome reaction of goat spermatozoa

The highly motile goat spermatozoa (5� 106 cells) were incubatedfor 1 h. with ecto-CIK antibody and MPS antibody (1:10, 1:100,1:500). The controls were treated with same amount ofpreimmune sera. Effect of MPS antibody on acrosome reaction wasdetermined by treating the cells with different dilutions of antibodyfor 1 h at room temperature prior to the reaction. Then thespermatozoa were washed with PBS for 2–3 times bycentrifugation and finally dispersed in capacitation medium, pH 7.4which is a modified Biggers–Whitten–Whittinham medium(BWW) (Biggers et al., 1971). The cells were then incubated at378C for 3 h. Finally calcium ionophore A23187 (10mM) was addedto these incubated cells to trigger Caþþ influx which is aprerequisite for the induction of acrosome reaction in vitro and theincubation was then continued for 15 min at 378C to permitcompletion of the acrosomal reaction when the acrosomemembrane fuses with the plasma membrane overlaying theacrosome. The resulting porous membrane will permit release ofthe glycoprotein and hydrolytic enzymes from the acrosomal sac.The acrosome reaction was stopped with the addition of 3%gluteraldehyde in sodium cacodylate (0.1 M). Acrosomal status wasthen assessed using the following two methods.

Assessment of acrosome reaction. The conventional‘‘acrosome reaction’’ is based on the detection of the acrosomalglycoproteins following interaction with Rose Bengal (De Jonge et al.,1988). The cell suspension following termination of acrosome reactionwith 3% glutaraldehyde was incubated at room temperature for 2 h.Samples were centrifuged at 800g for 3 min and the supernatant wasaspirated. The pellet was resuspended in PBS and washed twice withthe same. Smear was prepared with a drop of suspension and air-dried.The slides were stained with 0.8% Bismarck brown in deionized water(pH 1.8 with 2 N HCl) at 378C for 25 min and rinsed with distilleddeionized water. Finally the slides were stained for 25 min in 0.8% RoseBengal in 0.1 M cacodylate buffer, pH 6.0 for detection of the

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glycoprotein content of the intact acrosomal sac. The slides were thenwashed with deionized water, dehydrated in an alcohol series, clearedin xylene and mounted with paramount and cover slip. A total of 200–500 spermatozoa were evaluated and recorded as either ‘‘acrosomereacted (un-intact) sperm’’ (no colored spot on tip of sperm head) or‘‘acrosome un-reacted (intact)’’ (with a colored spot on the tip ofsperm head). The experiments were repeated five times.

Assessment of acrosin liberated. This method is based on therelease of acrosin a protease from acrosomal sac to the medium. Thesperm suspensions following the above-mentioned acrosome reactionwere centrifuged at 500g for 5 min to sediment the sperm cells.Treatment of the cells with preimmune sera and PBS-BSA served as thecontrol. Supernatant fluids were then used as acrosin source and pH ofthe medium was adjusted to 3–3.5 to dissociate enzyme inhibitorcomplex. The acrosin activity was measured (Polakoski and Zaneveld,1976) by adding 0.2 ml of above-mentioned supernatant fluid to0.5 ml substrate (6 mM BAEE-HCl) solution buffered with 2.3 ml 0.1 MTris–Cl (pH 9). Solutions were incubated at 258C and rapidly mixed in a3 ml cuvette of 1 cm light path. The change of absorbance at 253 nm wasmeasured for a period of 30 min against a blank sample containing onlybuffer and substrate. One unit of acrosin corresponds to the hydrolysisof 1 mmol substrate per minute, that is, an increase in absorbance of0.385/min. The daily variability of the assay was normalized by use of acryopreserved, partially purified human acrosin extract (Naz et al.,1992).

ResultsEffect of ecto-CIK antibody on acrosome reactionand acrosin release

The effect of CIK antibody was assessed on the spermacrosome reaction (Fig. 1, Table 1). Prior to the addition ofcalcium ionophore approximately 12% of the sperm cellsunderwent acrosome reaction whereas following treatmentwith the ionophore, nearly 40% of the untreated spermatozoashowed acrosome reaction. Treatment of sperm cells with CIKantibody (dil: 1:500) caused a significant decrease (approx. 50%)in percentage of acrosome reacted sperm compared to thePBS-BSA treated or control sera-treated sperm. The controlrabbit serum did not show any significant effect on thepercentage of acrosome reacted sperm as compared to thePBS-BSA control.

Another well-defined biochemical index for assessingacrosome reaction is the release of acrosin from the acrosomalsac of spermatozoa. Figure 2 shows the time course of theacrosin release from the sperm acrosome during the acrosomereaction. In absence of Caþþ ionophore, rate of release ofacrosin in the medium was very low. Onset of the acrosomereaction, that is, after the addition of Caþþ ionophore in thepreincubated cells, caused a remarkable increase in the rate ofreleased acrosin in the medium from the normal sperm, themajor amount of this release being nearly complete during thefirst 15 min of incubation. However, CIK antibody treatments(1:100, 1:500, and 1:1,000 dilutions) caused a significantdecrease in the release of this enzyme: 1:100 dilution beingmost effective in this respect. The control rabbit IgG fromnormal rabbit serum did not show any significant effect on theacrosome release of sperm as compared to the PBS-BSAcontrol (Table 1). Approximately 50% acrosin release wasinhibited, by antibody at a dilution of 1:500.

Studies on MPS

Localization of MPS on sperm surface. Distributionof MPS on the sperm surface was analyzed by the indirectimmunofluorescence technique. Binding of the MPSantibody on sperm surface was visualized by the binding ofFITC-conjugated IgG with MPS antibody. MPS antibody wasfound to bind intensely with acrosomal area of sperm head incauda sperm cells (Fig. 3b). The other parts of the spermatozoashowed little fluorescence. Negative control using the same

Fig. 1. Effect of ecto-CIK antibody on acrosome reaction of goat cauda spermatozoa as monitored by the Rose Bengal staining method.Acrosome reaction was carried out under the standard assay conditions and the cells after staining with Rose Bengal were observed undermicroscope at 1,000T magnification. A: Sperm cells treated with preimmune sera. B: Cells treated with ecto-CIK antibody. (~) Representsacrosome reacted (acrosome not intact) sperm or ( ) represent acrosome un-reacted (acrosome intact). The ‘‘acrosome unreacted’’ cell has awelldefinedtinycoloredspotonthetipof thespermheadwhereasthe ‘‘acrosomereacted’’ cellhasnosuchcoloredspot.The insetsshowingspermcells at higher magnification give clearer vision of the acrosome reacted and unreacted cells.

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amount preimmune rabbit serum instead of MPS antibody, ledto no detectable florescence on the head of cauda sperm cells(Fig. 3a). This observation provides support to the view thatMPS is localized primarily on the acrosomal region of themature goat sperm head.

Time course: MPS level and its phosphorylation. Theconcentration of MPS was estimated in sperm cells beforeand during acrosome reaction at different time intervalsfollowing initiation with calcium ionophore. It was found thatconcentration of MPS increases significantly with time up to20 min of incubation. During this period the membrane-boundMPS as estimated by ELISA, increase by nearly 100% (Fig. 4).

TABLE 1. Effect of CIK antibody on caprine sperm acros

TreatmentsAcrosome reacted

(%)a, mean W

CIK antibody (1:500 dil) 22 W 2.43Preimmune sera (1:500 dil) 45 W 1.10PBS-BSA control 39 W 4.62

Assays were performed using sperm collected from at least six differenAcrosin activity was expressed as mU of acrosin/107 sperm cells.aControl values in absence of calcium ionophore were: 12� 1.2% for acsupernatant.

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Degree of sperm surface protein phosphorylation catalyzedby the endogenous ecto-CIK, was estimated in sperm cellsbefore and during acrosome reaction at different time intervals.It was found that the degree of phosphorylation ofphosphoproteins bound to the plasma membrane increasednearly twofold during 20 min of the acrosome reactiontriggered by calcium ionophore (Fig. 5). Maiti et al. (2004) havereported that MPS is the primary phosphoprotein (approx.90%) that undergoes phosphorylation on the sperm outersurface by the endogenous ecto-CIK. The observed alterationof sperm surface protein phosphorylation is thus primarilyrelated to MPS. This view is further supported by the

ome reaction and acrosin release

spermSD

Acrosin released in supernatanta,mean W SD

42.7 W 34.1185.47 W 9.2279.22 W 4.28

t tissues.

rosome reacted sperm and 23� 5.6 mU for acrosin released in the

Fig. 2. Effect of CIK antibody on the release of acrosin duringacrosome reaction. Highly motile spermatozoa were preincubated inthe BWW medium for 180 min prior to the addition of calciumionophore for the induction of acrosome reaction. Acrosin releasedfrom the sperm samples was assayed as described in Materials andMethods Section. Acrosin was measured as change of OD at 253 nm(Q) change of OD before addition of ionophore A23187 (10 mM); ( )change of OD after addition of ionophore in preimmune sera treatedsperm; (*) 1:1,000 dil of antibody; ( ) in 1:500 dil of antibody; (&)with 1:100; (*) change of OD in absence of ionophore. The datashown are mean W SEM.

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observation that during acrosomal reaction there is elevatedlevel of MPS on sperm surface.

Effect of MPS antibody on acrosome reaction and acrosinrelease. Photographs of spermatozoa following treatmentswith control sera and MPS antibody were similar to thosealready shown in Figure 1. As indicated in Table 2,approximately 40% of the control sperm cells undergoacrosome reaction. Treatment of the cells with MPS antibody(1:100 dilution) caused a marked decrease in percentage ofacrosome reacted sperm compared to the PBS-BSA treatedcontrol or control rabbit IgG treated sperm. MPS antibody at1:500 and 1:100 dilutions, inhibited acrosome reaction to theextent of nearly 50% and 75%, respectively when observedunder a microscope at 1,000� magnification. The controlrabbit serum did not show any significant effect on the

Fig. 3. Immunofluorescence staining of goat epididymal spermatozoa. Cefollowed by FITC-labeled goat-anti-rabbit IgG according to the procedurethus were examined by fluorescence microscope at 1,000T magnification

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percentage of acrosome reacted sperm as compared to thePBS-BSA control.

Treatment of spermatozoa with MPS antibody as well causedsignificant decrease in the concentration of acrosin released inthe supernatant fluid as compared to PBS-BSA or preimmuneserum treated sperm controls (Table 2). It was found thatacrosin activities were approximately 50% and 75% less atantibody dilutions: 1:500 and 1:100, respectively. The controlrabbit serum did not show any significant effect on above twocases as compared to the PBS-BSA control.

Discussion

Testicular spermatozoa following their transit throughepididymis acquire forward motility and are stored in the lastpart (cauda) of this organ. Finally during ejaculation these cellsare largely diluted with reproductive fluids derived from variousaccessory sex organs before being ejaculated into vaginalcompartment of the female reproductive system. During transitfrom vagina to fallopian tube the male gametes undergo aprocess called ‘‘capacitation’’ which is a prerequisite for theacrosome reaction. This reaction is initiated following thebinding of spermatozoa to the zona layer of ovum in vivo(Ho and Suarez, 2001). Acrosome reaction is a secretory eventinvolving the specific fusion of the outer acrosomal membranewith the sperm plasma membrane overlaying the principal pieceof the acrosome and it is essential for the fertilization process.As a result of this membrane fusion, the plasma membraneon the top of acrosome becomes porous thereby permittingrelease of proteins including hydrolytic enzymes (e.g., acrosin,hyaluronidase, etc.) from the acrosomal sac of the acrosome(Zaneveld and De Jonge, 1991). Sperm–egg interactiontriggers Caþþ influx in sperm, which in turn activates a series ofbiochemical events leading to the phenomenon of membranefusion (Florman et al., 1998). It can be induced in vitro incapacitated spermatozoa by incubation with solubilized zonapellucida, progesterone, epidermal growth factor, atrialnatriuretic peptide or by Ca2þ/2Hþ/ionophore A23187(Breitbart et al., 1997). The biochemical mechanism ofacrosomal membrane fusion event is not well understood.Several intra-sperm protein kinases have been implicated tomediate the event by the mechanism of proteinphosphorylation and dephsophorylation (Breitbart and Naor,1999). Some of these kinase are: tyrosine kinase (Leyton andSaling 1989; Burks et al., 1995; Kalab et al., 1998; Seshagiri et al.,2007), protein kinase C (De Jonge et al., 1991; Rotem et al.,1992; Naor and Breitbart 1997; Seshagiri et al., 2007), proteinkinase A (Spungin and Breitbart, 1996; Vijayaraghavan et al.,

lls were treated with (a) preimmune sera (b) polyclonal MPS antibodydescribed in ‘‘Materials and Methods’’ Section. Spermatozoa obtained.

Fig. 4. Relationship of MPS concentration with acrosome reactionof spermatozoa. MPS concentration (by ELISA of solubilizedmembrane) were determined in isolated plasma membrane ofacrosome reacted and unreacted cell population, according to theprocedure describe of five such experiments.

TABLE 2. Effect of MPS antibody on caprine sperm acrosome reaction and

acrosin release

Treatments

Acrosome reactedsperm (%)a,mean W SD

Acrosin releasedin supernatanta,

mean W SD

MPS antibody (1:500 dilution) 20 W 2.1 42.73 W 4.11MPS antibody (1:100 dilution) 10 W 1.2 20.45 W 2.9Control (preimmune sera) 42 W 1.1 86 W 1.15PBS-BSA control 40 W 2.6 79.22 W 1.8

Assays were performed using sperm collected from at least 6 different tissues. Acrosinactivity was expressed as mU of acrosin/107 sperm cells.aControl values in absence of calcium ionophore were similar to those shown in Table 1.

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1997; Moseley et al., 2005; Morales et al., 2007) and Rho-kinase(de la Sancha et al., 2007; Fiedler et al., 2008). Little is knownabout the biochemical identity of the specific substrates of thesekinases. Consequently the precise role of cytosolic proteinkinases/phosphoproteins in the acrosome reaction is largelyunknown. As elaborated in the ‘‘Introduction’’ Section, a novelprotein kinase (CIK) and its endogenous membrane-boundprotein substrate (MPS) have been shown to occur on spermhead overlying the acrosome (Maiti et al., 2004, 2008; Nathet al., 2008). This study reports for the first time, the role ofthe well-defined sperm surface ecto-protein kinase and itsphosphoprotein substrate in the regulation of acrosomereaction triggered by Caþþ ionophore in vitro using the caprinesperm model.

For assessing the roles of ecto-CIK as well its substrate: MPSon acrosomal reaction, we have used two methods: a ‘‘direct’’method which is based on the release of acrosin: the proteolytic

Fig. 5. Relationship of membrane protein phosphorylationwith acrosome reaction of spermatozoa. Membrane proteinphosphorylation was determined in isolated plasma membrane ofcells during the acrosome reaction at different time intervals,according to the procedure described in ‘‘Materials and Method’’Section. The results showed the mean W SEM of five separateexperiments.

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enzyme from the acrosomal sac and an ‘‘indirect’’ methodcommonly known as ‘‘Rose Bengal’’ method which is based onthe staining of the residual amount of acrosomal content leftinside the sac following membrane fusion. Treatment of spermcells with CIK antibody caused a significant decrease inpercentage of acrosome reacted sperm (Fig. 1, Table 1). Onsetof the acrosome reaction caused a profound increase in the rateof acrosin release from the sperm cells and this enzyme releaseis inhibited significantly by CIK antibody (Fig. 2, Table 1). MPSantibody showed high efficacy to inhibit the acrosome reactionand the acrosin release from spermatozoa (Table 2). Nearlyidentical finding by the parallel set of experiments (direct/indirect methods) demonstrate conclusively that the spermexternal surface protein kinase as well as its substrate proteinparticipate in the sperm acrosome reaction.

It is of interest to note that the time course of the acrosomereaction induced by the addition of calcium ionophore, is wellcorrelated with significant increase of the level of membrane-bound MPS (Fig. 4). Biochemical basis of this altered level of MPSduring acrosomal reaction is not clear. In one of our earlierpublication (Maiti et al., 2008), we have reported that MPSpresent in the cytosol (that penetrated the biomembranethrough the cell electroporation technique) finally getslocalized to the external sperm surface. It is thus possible thatduring the acrosomal reaction more of cytosolic MPS getstranslocated to the outer cell surface. Alternatively during theacrosomal reaction phase there may be a major restructuring ofthe cell membrane leading to greater availability of exposedMPS on the external cell surface. This enrichment of MPSfollowing acrosome reaction strengthens the above view.Asmentioned above, Caþþ influx in the spermatozoa followingsperm interaction with the zona layer of the ova is the initialtrigger for the acrosome reaction (Barros et al., 1996). As Caþþ

ionophore has been used in this investigation to initiate theacrosome reaction in vitro, the findings of this study have morerelevance in the context of the ‘‘membrane fusion’’ componentof acrosome reaction, that is, on the fusion of the outeracrosomal membrane with the sperm plasma membraneoverlaying the acrosome (Yanagimachi, 1994, 1995). It is ofinterest to note that both the CIK (Nath et al., 2008) and itsprotein substrate: MPS (Fig. 3) are located on the outer surfaceof sperm head overlaying the acrosome. It thus appears thatCIK/MPS complex residing on the sperm plasma membranedirectly participates in the acrosomal membrane fusionprocess. The finding that the acrosome reaction is accompaniedby elevated phosphorylation of the ecto-MPS catalyzed by theendogenous ecto-CIK (Fig. 4) is consistent with the view thatthis phosphoprotein may serve as a pivotal regulator ofmembrane fusion event by the mechanism of its phosphorylationand dephosphorylation. This view is supported by theobservation that a phosphoprotein phosphatase as well occurson the sperm external surface that catalyses dephosphorylationof the sperm ecto-phosphoproteins phosphorylated by theecto-CIK (Barua et al., 1985, 1999). Further studies are nownecessary to delineate the biochemical mechanism of the outer

400 M A I T I E T A L .

cell surface enzyme-substrate directed fusion of the twobiomembranes: one sitting on top of the other and itscorrelation with the Caþþ-directed intracellular signalingevents. As reported earlier from our laboratory (Maiti et al.,2004; Nath et al., 2008), ecto-CIK and its substrate protein playan important role in the regulation sperm flagellar forwardmotility. This novel cell surface protein phosphorylationcomplex appears to play bifunctional role for the regulation ofboth sperm motility and acrosomal reaction. At present little isknown regarding the biochemical mechanism of their dualfunctions.

Acknowledgments

Research Fellowship offered to Ms. Arunima Maiti byDepartment of Atomic Energy, Trombay, Mumbai, is thanked.We take this opportunity to thank Department of Science andTechnology, Indian council of Medical Research, New Delhi,and Council of Scientific and Industrial Research, India forfinancially supporting this work. We also wish to express ourgratitude to Prof Samir Bhattachryya and Prof. Siddhartha RoyDirectors of Indian Institute of Chemical Biology, Kolkata, India,for taking interest and encouraging us for successful completionof this work.

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