Identification of capacitation associated tyrosine phosphoproteins in buffalo (Bubalus bubalis) and cattle spermatozoa

Animal Reproduction Science 123 (2011) 40–47

Contents lists available at ScienceDirect

Animal Reproduction Science journal homepage: www.elsevier.com/locate/anireprosci

Identification of capacitation associated tyrosine phosphoproteins in buffalo (Bubalus bubalis) and cattle spermatozoa G. Jagan Mohanarao, S.K. Atreja ∗ Division of Animal Biochemistry, National Dairy Research Institute, Karnal, Haryana 132001, India

a r t i c l e

i n f o

Article history: Received 18 May 2010 Received in revised form 28 October 2010 Accepted 25 November 2010 Available online 2 December 2010 Keywords: Sperm Capacitation Tyrosine phosphorylation Buffalo Cattle

a b s t r a c t At ejaculation mammalian sperm lack fertilizing ability as they are released in a functionally immature form. The capacity to fertilize eggs is only acquired after they have been educated in the female reproductive tract and this phenomenon is termed as capacitation. Sperm capacitation includes a cascade of biochemical modifications, including cholesterol efflux, Ca2+ influx and cAMP/PKA-dependent/independent protein tyrosine phosphorylation which is specifically considered as the biochemical marker for capacitation. The identification of tyrosine phosphoproteins shall be useful in delineating their physiological role in different events associated with sperm capacitation. The present study was conducted to identify the tyrosine phosphoproteins in the capacitated buffalo and cattle spermatozoa using 2D immunoblotting and mass spectrometry. Among several proteins identified in the buffalo capacitated sperm, serine/threonine-protein phosphatase PP1-gamma catalytic subunit, MGC157332 protein, alpha-enolase, 3-oxoacid CoA transferase 2 and actin-like protein 7A were identified as new tyrosine phosphorylation substrates in mammalian spermatozoa. Cattle sperm also contain proteins such as serine/threonine-protein phosphatase PP1-alpha catalytic subunit and membrane metalloendopeptidase-like 1 which have not been reported as tyrosine phosphorylated in any other species. Though the presence of serine/threonine-protein phosphatase PP1-alpha catalytic subunit was demonstrated for the first time in mammalian sperm, further studies are required for its existence and possible role in different sperm functions. © 2010 Elsevier B.V. All rights reserved.

1. Introduction Ejaculated spermatozoa are able to move actively but cannot immediately fertilize an egg. As spermatozoa traverse through the female genital tract, they undergo multiple physiological and biochemical modifications collectively referred to as capacitation (Yanagimachi, 1994) which was first reported nearly six decades ago by Austin

∗ Corresponding author at: Reproductive Biochemistry Lab, Division of Animal Biochemistry, National Dairy Research Institute, Karnal, Haryana 132001, India. Tel.: +91 184 2259131; fax: +91 184 2250042; mobile: +91 9416822587. E-mail addresses: [email protected] (G. Jagan Mohanarao), [email protected] (S.K. Atreja). 0378-4320/$ – see front matter © 2010 Elsevier B.V. All rights reserved. doi:10.1016/j.anireprosci.2010.11.013

(1951) and Chang (1951) independently. Capacitation is associated with two major physiological changes namely hyperactive motility (a whiplash-like flagellar movement with large-amplitude, asymmetric bends) that facilitates the sperm to penetrate through cumulus mass (Ho and Suarez, 2000; Yanagimachi, 1970) and acrosome reaction, another prerequisite event that enables the acrosomereacted spermatozoon to penetrate the zona pellucida and fertilize the egg (Shi and Roldan, 1995; Ward and Storey, 1984). The biochemical changes that are associated with the capacitation process are an efflux of cholesterol leading to an increase in membrane fluidity, increase in bicarbonate (HCO3 − ), intracellular pH (pHi ), Ca2+ and cAMP levels, and changes in protein phosphorylation and protein kinase activity (Baldi et al., 2000). Capacitation can

G. Jagan Mohanarao, S.K. Atreja / Animal Reproduction Science 123 (2011) 40–47

also be reproduced in vitro spontaneously in a defined medium. In fact, most of the knowledge about various aspects of mammalian sperm capacitation has originated from in vitro studies. Capacitation of bovine and buffalo spermatozoa can occur in vitro in medium supplemented with a glycosaminoglycan, heparin (Parrish et al., 1988; Roy and Atreja, 2008a). The postulated mechanism is that heparin functions as a ligand for yet an uncharacterized receptor localized in the sperm plasma membrane and possibly stimulates the intracellular elevation of calcium, pH, cAMP and reactive oxygen species which are necessary to initiate the signaling pathways coupled with capacitation (Galantino-Homer et al., 1997; Parrish et al., 1988, 1994; Roy and Atreja, 2008a; Visconti et al., 1998). Heparin might also involve in the removal of seminal plasma proteins, known as decapacitation factors, adsorbed on the plasma membrane (Miller et al., 1990; Therien et al., 1995). Phosphorylation of proteins is a post-translational modification event that acts as one of the cell’s regulatory mechanisms to control various cellular processes (Hunter, 2000; Pawson, 2004). As spermatozoa are terminally matured cells and devoid of any major transcriptional and translational activity one can justify the importance of post-translational modifications such as protein phosphorylation/dephosphorylation in regulating important sperm functions like capacitation, hyperactive motility and acrosome reaction, which are required for the spermatozoon to reach, bind, penetrate and fuse with the oocyte. Although both serine/threonine phosphorylation and tyrosine phosphorylation of proteins have been reported in spermatozoa, the tyrosine phosphorylation of a number of protein substrates has been associated with capacitation in most of the mammalian spermatozoa and considered as a hallmark event of capacitation (Ficarro et al., 2003; Galantino-Homer et al., 1997; Jha and Shivaji, 2002; Kalab et al., 1998; Leclerc et al., 1996; Roy and Atreja, 2007, 2008b; Visconti et al., 1995a,b). In recent years, 2D immunoblotting analysis of sperm proteins followed by identification using mass spectrometry has been successfully used to identify capacitation associated tyrosine phosphoproteins in human (Ficarro et al., 2003), mouse (Arcelay et al., 2008) and hamster (Kota et al., 2009; Kumar et al., 2006) spermatozoa. A number of tyrosine phosphoproteins have been detected in capacitated buffalo spermatozoa among which few proteins were shown to be tyrosine phosphorylated in a cAMP/PKA dependent or independent pathway (Roy and Atreja, 2007, 2008b). Although it has been well established that capacitation of a sperm cell is required before fertilization virtually in every mammalian species, the molecular mechanisms and signal transduction pathways involved in this process are not clearly understood. In this regard identification of tyrosine phosphoproteins especially in the large ruminant species like cattle and buffalo shall be useful in delineating their physiological role in different events associated with sperm capacitation. In the present study, attempts were made to identify the tyrosine phosphoproteins in capacitated buffalo and Karan Fries cattle (crossbred) spermatozoa using 2D immunoblotting and mass spectrometry.

41

2. Materials and methods 2.1. Chemicals Urea, thiourea, bovine serum albumin (BSA, fraction V), acrylamide, sodium orthovanadate, protease inhibitor cocktail, goat anti-mouse immunoglobulin G peroxidase conjugate, X-ray films (X-OMAT-AR, Kodak) and X-ray film developer and fixer (Kodak) were purchased from Sigma Chemical Company (St. Louis, MO, USA). Dithiothreitol (DTT), bis-acrylamide, ammonium persulfate, CHAPS (3-[(3-cholamidopropyl) dimethylammonio]1-propanesulfonate), ampholytes, immobilized pH gradient strips (IPG strips, pH 5–8), protein molecular weight marker and Coomassie stain were procured from Bio-Rad laboratories (Herculus, CA, USA). Polyvinylidine difluoride (PVDF) membrane and enhanced chemiluminiscent reagent (ImmobilonTM western chemiluminiscent HRP substrate) were purchased from Millipore (Billerica, MA, USA). Monoclonal antiphosphotyrosine antibody (clone 4G10) was purchased from Millipore (Temecula, CA, USA). All other chemicals used in this study were of analytical grade. 2.2. Sperm culture medium The medium used for washing and culturing of sperm was a modified Tyrode’s bicarbonate-buffered medium designated as sp-TALP [composed of 100 mM NaCl, 10 mM HEPES, 3.1 mM KCl, 0.4 mM EDTA, 0.4 mM MgCl2 ·6H2 O, 0.3 mM NaH2 PO4 ·2H2 O, 21.6 mM Na lactate, 2 mM CaCl2 ·2H2 O, 1 mM Na pyruvate, 25 mM NaHCO3, BSA (1 mg/mL for washing, 6 mg/mL for culturing), pH 7.4, osmolarity: 265–270 mOsmol/kg]. The medium was prepared as described by Galantino-Homer et al. (1997). The medium was devoid of sodium bicarbonate and BSA wherever spermatozoa had to be maintained under noncapacitating conditions. 2.3. Semen collection and processing The Murrah buffalo bulls and Karan Fries cattle (crossbred) were housed at Artificial Breeding Research Complex, National Dairy Research Institute, Karnal, India, under uniform nutritional conditions and semen was collected twice a week with an Artificial Vagina (IMV, France) maintained at 40 ◦ C. Immediately after collection the volume of the semen was measured in a conical tube graduated at 0.1 mL intervals and mass motility was assessed by light microscopy at 10× magnification. Within 10–20 min of collection, the semen was transported to the laboratory in a thermos flask maintained at 37 ◦ C, split into 0.5 mL fractions, and diluted with sp-TALP medium in 1:6 ratio in 15 mL polypropylene conical centrifuge tubes. The samples were then subjected to three washings by centrifugation at 275 × g for 5 min to remove seminal plasma. The final sperm pellet was resuspended with 2 mL of sp-TALP and the sperm suspensions of all the tubes were combined. The concentration of sperm was determined by haemocytometer and adjusted to 100 × 106 cells/mL by further dilution with sp-TALP. Semen quality parameters like pro-

42

G. Jagan Mohanarao, S.K. Atreja / Animal Reproduction Science 123 (2011) 40–47

gressive motility and viability were assessed (Therien and Manjunath, 2003; Tomar, 1997). Final sperm suspensions with >80% forward progressive motility and viability were only used in this study. 2.4. Sperm culture Buffalo and cattle spermatozoa were in vitro capacitated as described earlier (Parrish et al., 1988; Roy and Atreja, 2007). Briefly, heparin (10 ␮g/mL), a known capacitating agent, added to 250 ␮L aliquots of sp-TALP taken in 1.5 mL polypropylene microcentrifuge tubes and sperm suspension at 100 × 106 cells/mL in 250 ␮L fractions were added to the tubes to have a final volume of 500 ␮L and sperm concentration of 50 × 106 cells/mL. The tubes representing buffalo and cattle sperm suspensions with the cap open were incubated immediately at 38.5 ◦ C for 6 h and 4 h, respectively, in a cell culture incubator with 5% CO2 in air and at high humidity (>90%). At hourly intervals sperm motility was assessed by taking 10 ␮L drop of sperm suspension on a pre-warmed glass slide duly covered with a cover slip and observed under an inverted bright field microscope at 200×. The extent of capacitation was also determined by a double staining procedure to differentiate between acrosome-reacted and non-reacted cells (Sidhu et al., 1992). The sperm samples after various periods of incubations were incubated further with LPC (100 ␮g/mL), a known agent to induce acrosome reaction (AR) in capacitated cells only, according to the method of Parrish et al. (1988). The percentage of spermatozoa undergoing AR was determined as a measure of capacitation. Spermatozoa collected at 0 h were treated as noncapacitated and those that were collected after 6 h of incubation were treated as capacitated. 2.5. Protein extraction The noncapacitated and capacitated sperm suspensions of 50 × 106 cells each were taken in 1.5 mL centrifuge tubes and washed twice to remove sp-TALP medium with 1 mL of Tris–sucrose buffer, pH 7.0 with 1 mM sodium orthovanadate (Na3 VO4 , TS-V) by centrifuging the tubes at 2250 × g for 4 min each. The sperm pellet was resuspended in 125 ␮L of lysis buffer (7 M urea, 2 M thiourea, 4% CHAPS, 50 mM DTT, 0.2% ampholytes pH 3–10, 0.002% bromophenol blue, 1 mM Na3 VO4 , protease inhibitor cocktail), vortexed for 1 min and kept on ice for 90 min. The sperm cell lysate was then centrifuged at 12,000 × g for 20 min and the solubilized protein supernatant was collected in a new microcentrifuge tube and stored at −20 ◦ C until use. 2.6. 2D PAGE and immunoblotting The total protein extracts of noncapacitated and capacitated spermatozoa were loaded onto IPG strips (pH 5–8) by passive rehydration for 12 h. Isoelectric focusing (IEF) was carried out by using Protean IEF Cell apparatus (BioRad, Richmond, CA) and the focusing was conducted for a total of 11,800 V h using voltage stepping: 30 min at 300 V, 45 min at 1000 V, 3 h at 4000 V. After IEF run was completed IPG strips were equilibrated in equilibration buffer

I (6 M urea, 0.375 M Tris–HCl, pH 8.8, 2% SDS, 20% glycerol, and 2% DTT) followed by a second equilibration in equilibration buffer II which contains 2.5% iodoacetamide in place of DTT for 15 min each at room temperature under gentle agitation. IPG strips were then transferred onto a polymerized 10% SDS PAGE gel and embedded into the 1% agarose gel containing a trace amount of bromophenol blue. SDS PAGE was run at a constant voltage of 40 V. For each experimental condition (noncapacitated and capacitated) two gels were run simultaneously to ensure equivalent electrophoretic conditions. After electrophoresis, one gel was subjected to staining with Coomassie stain and the other gel was transferred to Immobilon-P PVDF membrane as described earlier (Otter et al., 1987). Protein transfer efficiency was assessed either by staining the membrane with 0.5% ponceu S dye in 1% glacial acetic acid (Salinovich and Montelaro, 1986) or by analyzing the transfer of prestained molecular weight marker onto the PVDF membrane. The membrane was then blocked with 5% (w/v) non-fat dried milk prepared in Tris-buffered saline with Tween-20 and sodium orthovanadate (TBS-TV: 20 mM Tris, 150 mM NaCl, pH 7.6; 0.1% (v/v) Tween-20, 1 mM Na3 VO4 ) for overnight at 4 ◦ C. The membrane was incubated with monoclonal antiphosphotyrosine antibody [Millipore: Clone 4G10, diluted (1:10,000) in TBS-TV] for 2 h at room temperature. After washing with TBS-T, membrane was incubated with goat anti-mouse IgG-peroxidase conjugate [Sigma: A2554, diluted (1:70,000) in TBS-TV] for 1 h and washed again with TBS-T. Subsequently, the bound peroxidase activity was visualized by the enhanced chemiluminescent reagent (ImmobilonTM western chemiluminescent HRP substrate) according to manufacturer’s instructions using Kodak XOMAT-AR X-ray films. In a control experiment, the specificity of the antiphosphotyrosine antibody was examined by pre-incubating antibody with 10 mM O-phospho-l-tyrosine for 1 h prior to use for immunoblotting. 2.7. Image analysis of 2D gels and mass spectrometry The Coomassie stained 2D gels and X-ray films were scanned at 300 dpi using Imagescanner III (GE healthcare, Uppsala, Sweden). The digitized image of a 2D gel and corresponding X-ray film were overlaid in Adobe Photoshop 7.0 and by decreasing or increasing the opacity, the images were aligned accurately. After the protein spots on X-ray film image was marked with arrows, the X-ray film image was hidden and arrows identified corresponding protein spots on 2D gel image. These protein spots were then excised from 2D gel and submitted for MALDI MS/MS analysis at Proteomics International Pty Ltd., Nedlands, Western Australia, Australia. 3. Results 3.1. Capacitation associated tyrosine phosphoproteins in buffalo spermatozoa When buffalo and cattle sperm were incubated over a period of 6 h and 4 h, respectively, in the presence of heparin and observed under microscope, out of the total motile

G. Jagan Mohanarao, S.K. Atreja / Animal Reproduction Science 123 (2011) 40–47

43

Fig. 1. Analysis of total proteins of noncapacitated and capacitated buffalo spermatozoa by 2D immunoblotting. Sperm proteins were extracted using lysis buffer, rehydrated into immobilized pH gradient strips, isoelectro focused, resolved by SDS PAGE subsequently stained with Coomassie stain (A). The other set of gels representing noncapacitated and capacitated were developed by antiphosphotyrosine antibody (B and C, respectively). The images were superimposed and tyrosine phosphoproteins from the 2D gel were cored and microsequenced by MALDI MS/MS. The identified proteins were labeled with black arrows in (A) and (C).

sperm, more than 80% of sperm showed progressive motility initially. After 2–3 h of incubation, however, the buffalo sperm motility pattern changed from the progressive type to characteristic whiplash type and after 3–4 h of incubation, a head-to-head agglutination of spermatozoa was observed which suggested that capacitation was achieved. These changes were observed early in the case of cattle sperm. The percentage of capacitated sperm obtained in our studies ranges between 50% and 55%. The protein profiles of both noncapacitated and capacitated buffalo spermatozoa found to be identical after they have been resolved into a number of proteins following 2D PAGE. A representative 2D gel is shown in Fig. 1A. Tyrosine phosphoproteins were detected in the capacitated spermatozoa, when the tyrosine phosphorylation status of the resolved proteins was analyzed by immunoblotting using monoclonal antiphosphotyrosine antibody (clone 4G10), which were labeled with arrows on X-ray film image (Fig. 1C). In the noncapacitated spermatozoa, however, none of the proteins was detected as tyrosine phosphorylated (Fig. 1B). The control immunoblots did not give any non-specific signal and confirmed the specificity of monoclonal antiphosphotyrosine antibody used in the study. After superimposing the X-ray film image with the 2D gel the corresponding protein spots were excised

from the gel and submitted for MALDI MS/MS to identify the tyrosine phosphoproteins. The protein spots that were excised from the 2D gel were also labeled as shown in Fig. 1A. The MALDI MS/MS identification of proteins demonstrated that, out of 16 tyrosine phosphoproteins detected on the immunoblot of the capacitated spermatozoa, 5 proteins were found to be present in multiple isoforms (Fig. 1C; Table 1). Among the proteins identified proacrosin binding protein (similar to sp32), ubiquinolcytochrome c reductase core protein I, outer dense fiber protein 2, glutathione S-transferase mu 3, tektin-3 and voltage-dependent anion-selective channel protein 2 have already been reported as tyrosine phosphorylation substrates whereas proteins such as serine/threonine-protein phosphatase PP1-gamma catalytic subunit, MGC157332 protein, alpha-enolase, 3-oxoacid CoA transferase 2 and actin-like protein 7A were identified as new tyrosine phosphorylated proteins in mammalian spermatozoa (Table 1). Proteins like proacrosin binding protein (similar to sp32), serine/threonine-protein phosphatase PP1-gamma catalytic subunit, MGC157332 protein, outer dense fiber protein 2 and 3-oxoacid CoA transferase 2 were found to be present as phosphorylated isoforms having different isoelectric points with same molecular mass.

44

G. Jagan Mohanarao, S.K. Atreja / Animal Reproduction Science 123 (2011) 40–47

Table 1 Capacitation associated tyrosine phosphoproteins identified by MALDI MS/MS in buffalo and cattle spermatozoa. Spot Ida

Proteins identified in buffalo sperm

BH1 BH2 BH3 BH4 BH5 BH6 BH7 BH8 BH9 BH10 BH11

Similar to sp32 (Proacrosin binding protein) Serine/threonine-protein phosphatase PP1-gamma catalytic subunitb MGC157332 proteinb Ubiquinol-cytochrome c reductase core protein I Outer dense fiber protein 2 Glutathione S-transferase mu 3 Alpha-enolasec 3-oxoacid CoA transferase 2b Tektin-3 Actin-like protein 7Ab Voltage-dependent anion-selective channel protein 2

Spot Ida

Proteins identified in cattle sperm

CH1

Serine/threonine-protein phosphatase PP1-alpha catalytic subunitb Membrane metallo-endopeptidase-like 1b A-kinase anchor protein 4 Major fibrous sheath protein Outer dense fiber protein 2 Outer dense fiber of sperm tails 2 Succinate dehydrogenase (ubiquinone) flavoprotein subunit, mitochondrial Dihydrolipoyl dehydrogenase (DLD)

CH2 CH3 CH4 CH5 CH6 CH7 CH8 a b

Spot Id as labeled in Figs. 1C and 2C. New tyrosine phosphoproteins in mammalian spermatozoa.

3.2. Capacitation associated tyrosine phosphoproteins in cattle spermatozoa The capacitation associated tyrosine phosphoproteins in Karan Fries cattle spermatozoa were identified by incubating 4 h in presence of heparin. The total proteins of cattle noncapacitated and capacitated spermatozoa following 2D PAGE resolved into a number of proteins and the protein profiles found to be identical. A representative 2D gel of cattle sperm proteins is shown in Fig. 2A. In the noncapacitated spermatozoa, we could not detect any protein tyrosine phosphorylation (Fig. 2B); however, as anticipated number of tyrosine phosphorylated proteins were detected in the capacitated spermatozoa which were labeled with arrows on X-ray film image as shown in Fig. 2C. It is noteworthy to mention here that the tyrosine phosphoproteins detected in cattle spermatozoa were mostly in the high molecular weight range (35–95 kDa) as compared to the buffalo spermatozoa (25–80 kDa). Each of the protein spots on the 2D PAGE gel that matched with the Xray film was carefully excised from the gel and submitted for MALDI MS/MS for protein identification. The protein spots that were excised from the 2D gel were labeled as shown in Fig. 2A. The proteins such as A-kinase anchor protein 4 (AKAP4), major fibrous sheath protein, outer dense fiber protein 2, outer dense fiber of sperm tails 2, succinate dehydrogenase (ubiquinone) flavoprotein subunit and dihydrolipoyl dehydrogenase (DLD) were found to be bovine orthologues of human, mouse and hamster spermatozoa known to undergo tyrosine phosphorylation whereas serine/threonine-protein phosphatase PP1-alpha catalytic subunit and membrane metallo-endopeptidaselike 1 were identified as new tyrosine phosphoproteins in

mammalian spermatozoa (Table 1). Proteins with multiple isoforms in the case of AKAP4, major fibrous sheath protein, outer dense fiber protein 2 and dihydrolipoyl dehydrogenase were identified in capacitated cattle spermatozoa. 4. Discussion Capacitation coupled with an increase in tyrosine phosphorylation of a number of protein substrates and has been considered as the consistent biochemical marker for capacitation (Arcelay et al., 2008; Ficarro et al., 2003; Galantino-Homer et al., 1997; Jha and Shivaji, 2002; Kalab et al., 1998; Leclerc et al., 1996; Roy and Atreja, 2007; Visconti et al., 1995a,b). 2D immunoblotting followed by mass spectrometry have been successfully employed to identify capacitation associated tyrosine phosphoproteins in human (Ficarro et al., 2003), mouse (Arcelay et al., 2008) and hamster (Kota et al., 2009; Kumar et al., 2006) spermatozoa. The capacitation associated increase in tyrosine phosphorylation has also been observed in buffalo and cattle spermatozoa when they were incubated in presence of heparin or l-arginine (Roy and Atreja, 2007, 2008b, 2009). Hence identification of tyrosine phosphoproteins shall be useful in delineating their physiological role in different events associated with buffalo and cattle sperm capacitation. In the present study, protein tyrosine phosphorylation was not observed in the noncapacitated sperm in agreement with the earlier reports in mouse (Arcelay et al., 2008) and hamster (Kota et al., 2009) spermatozoa. Earlier studies in buffalo and cattle spermatozoa reported several tyrosine phosphoproteins in presence of heparin (Roy and Atreja, 2007, 2008b, 2009). In those studies, the detection of tyrosine phosphoproteins even in the control spermatozoa (sp-TALP alone) can be attributed by the presence of BSA and bicarbonate in the sp-TALP medium which are known to induce capacitation in a number of species (Da Ros et al., 2004; Harrison, 2004; Luconi et al., 2005; Osheroff et al., 1999). The axoneme of the mammalian sperm tail consist of filamentous proteins called tetkins and is surrounded by a unique, specialized, and very prominent cytoskeletal structure, consisting of nine fibers known as outer dense fibers (ODF). In a recent study, Mariappa et al. (2010) demonstrated that a high percentage of tyrphostin-A47 (a tyrosine kinase inhibitor) treated hamster spermatozoa exhibited circular motility, which was associated with a distinct hypo-tyrosine phosphorylation of flagellar proteins such as outer dense fiber protein-2 and tektin-2 thus showing the critical importance of flagellar protein tyrosine phosphorylation during capacitation and hyperactivation. It has also been reported that male mice null for TEKT3 produce sperm with reduced motility and forward progression, and increased flagellar structural bending defects (Roy et al., 2009). Similarly the tyrosine phosphorylated ODF2 and tetkin-3 might also be required during capacitation of buffalo spermatozoa to achieve hyperactivated motility. The presence of glutathione S-transferases was clearly demonstrated on goat spermatozoa where they function as detoxifying enzymes, and the inability of goat sperm to fertilize oocytes when treated with anti-GST antibodies (Aravinda et al., 1995; Gopalakrishnan et al., 1998;

G. Jagan Mohanarao, S.K. Atreja / Animal Reproduction Science 123 (2011) 40–47

45

Fig. 2. Analysis of total proteins of noncapacitated and capacitated cattle spermatozoa by 2D immunoblotting. Sperm proteins were extracted using lysis buffer, rehydrated into immobilized pH gradient strips, isoelectro focused, resolved by SDS PAGE subsequently stained with Coomassie stain (A). The other set of gels representing noncapacitated and capacitated were developed by antiphosphotyrosine antibody (B and C, respectively). The images were superimposed and tyrosine phosphoproteins from the 2D gel were cored and microsequenced by MALDI MS/MS. The identified proteins were labeled with black arrows in (A) and (C). The protein spots that could not be identified by MALDI MS/MS were labeled with white arrows in (C).

Gopalakrishnan and Shaha, 1998; Hemachand et al., 2002). The tyrosine phosphorylation of GST isoform mu 5 has been reported during mouse (Arcelay et al., 2008) and hamster (Kota et al., 2009) sperm capacitation whereas our results are consistent with human where GST mu 3 was mapped as capacitation associated tyrosine phosphorylation target (Ficarro et al., 2003). It is predicted that the phosphotyrosine form of GST mu 3 might play an important role in regulating oxidative balance during capacitation. In mammals, all the four isoforms of type 1 serine/threonine protein phosphatase (PP1␣, PP1␤/␥, PP1␥1 and PP1␥2) expressed in the testis whereas PP1␥2 is the only isoform expressed on spermatozoa (Bollen and Stalmans, 1992; Han et al., 2007). High protein phosphatase activity is correlated with low sperm motility, whereas low catalytic activity is associated with vigorous motility in bovine and monkey spermatozoa (Smith et al., 1996, 1999; Vijayaraghavan et al., 1996). Recently it has been reported that Ser/Thr phosphatase inhibitors overcome the block by SFK (Src family of protein tyrosine kinases) inhibitors to all mouse sperm capacitation parameters, including in vitro fertilization (Krapf et al., 2010). Similarly in the present study the tyrosine phosphorylation of serine/threonineprotein phosphatase gamma by protein tyrosine kinase

might inactivate its dephosphorylating activity and lead to the motility initiation and hyperactivation of buffalo sperm during capacitation. Hinsch et al. (2001, 2004) demonstrated that VDACs (voltage-dependent anion channel), VDAC2 and VDAC3 are present in bovine sperm and VDAC2 was shown to localize to the anterior head. Recent studies in human and mouse sperm revealed that VDAC2 undergo tyrosine phosphorylation during capacitation as it was similarly observed in the buffalo spermatozoa (Arcelay et al., 2008; Ficarro et al., 2003). The tyrosine phosphorylation of VDAC2 probably affects the activity of channel. It was suggested earlier that proacrosin binding proteins immobilize and stabilize proacrosin until the acrosome reaction (Baba et al., 1994; Hardy et al., 1991; Yi et al., 1992). During capacitation, maturation of some proacrosin to acrosin could be facilitated by tyrosine phosphorylation of a 32-kDa protein, sp32 (Dube et al., 2005). In the same lines, the tyrosine phosphorylation of a proacrosin binding protein (similar to sp32) with molecular mass of 60 kDa was observed during capacitation of buffalo spermatozoa. At present it is difficult to predict the likely roles of MGC157332 protein, 3-oxoacid CoA transferase 2 and actin-like protein 7A during buffalo sperm capacitation.

46

G. Jagan Mohanarao, S.K. Atreja / Animal Reproduction Science 123 (2011) 40–47

The proteins that have been identified in cattle sperm are mostly either cytoskeletal structural proteins or enzymes (Table 1). The fibrous sheath is a unique cytoskeletal structure which is largely comprised of A-kinase anchor proteins (AKAPs) (Bajpai et al., 2006; Mandal et al., 1999; Vijayaraghavan et al., 1999). AKAP4 anchors cAMPdependent protein kinase A (PKA) to the fibrous sheath of the spermatozoon where the kinase is likely to be required for the regulation of spermatozoal motility (Brown et al., 2003; Edwards and Scott, 2000). Akap4 deletion results in shorter sperm flagellum and an incomplete fibrous sheath formation causing infertility due to loss of motility (Miki et al., 2002). The tyrosine phosphorylation of AKAP4 and major fibrous sheath protein was documented earlier in mouse (Brito et al., 1989; Johnson et al., 1997; Moss et al., 1999), human (Carrera et al., 1996; Ficarro et al., 2003; Mandal et al., 1999), and hamster (Jha and Shivaji, 2002; Kota et al., 2009) spermatozoa. So it is no surprise to identify these proteins in cattle spermatozoa during capacitation. Most interestingly, in the present study, we have identified the presence of serine/threonine-protein phosphatase PP1-alpha catalytic subunit and it was found to be tyrosine phosphorylated during capacitation. However, further studies are highly required to confirm its presence and possible role in different sperm functions. Recently, it has been demonstrated that pyruvate dehydrogenase complex (PDHc) components, pyruvate dehydrogenase A2 (PDHA2) and dihydrolipoyl dehydrogenase (DLD) exhibit capacitation dependent tyrosine phosphorylation in hamster spermatozoa and inhibition of their activities causes significant inhibition of hyperactivation or acrosome reaction or both (Kumar et al., 2006; Mitra et al., 2005; Mitra and Shivaji, 2004). In the present study, the tyrosine phosphorylation of DLD might also be required for capacitation associated hyperactivated motility in cattle spermatozoa. The component of the electron transport chain succinate dehydrogenase (ubiquinone) flavoprotein subunit has also been reported as tyrosine phosphorylated during hamster sperm capacitation (Kota et al., 2009). In conclusion, as a first step to understand the physiological roles of different tyrosine phosphoproteins during buffalo and cattle sperm capacitation, we have identified 11 and 8 tyrosine phosphoproteins respectively. Our results have demonstrated that though cattle and buffalo are closely related ruminant species, the set of proteins that undergo tyrosine phosphorylation during capacitation are highly different. Additional investigations are necessary for the existence and functional significance of serine/threonine-protein phosphatase PP1-alpha catalytic subunit in cattle sperm as it was identified for the first time in mammalian sperm. The functional characterization of the identified tyrosine phosphoproteins and their role in capacitation associated physiological changes are currently in progress. Acknowledgements The authors thank the Director, National Dairy Research Institute, Karnal 132001, India, for providing financial assistance and necessary facilities during the course of the study. This study was partly supported by world bank

funded NAIP project (C4/C30014). The authors also thank Dr. T.K. Mohanty, Senior Scientist, Dairy Cattle Breeding Division, for providing assistance in collecting semen samples.

References Aravinda, S., Gopalakrishnan, B., Dey, C.S., Totey, S.M., Pawshe, C.H., Salunke, D., Kaur, K., Shaha, C., 1995. A testicular protein important for fertility has glutathione S-transferase activity and is localized extracellularly in the seminiferous tubules. J. Biol. Chem. 270, 15675–15685. Arcelay, E., Salicioni, A.M., Wertheimer, E., Visconti, P.E., 2008. Identification of proteins undergoing tyrosine phosphorylation during mouse sperm capacitation. Int. J. Dev. Biol. 52, 463–472. Austin, C.R., 1951. Observations on the penetration of sperm into the mammalian egg. Aust. J. Sci. Res. B 4, 581–596. Baba, T., Niida, Y., Michikawa, Y., Kashiwabara, S., Kodaira, K., Takenaka, M., Kohno, N., Gerton, G.L., Arai, Y., 1994. An acrosomal protein, sp32, in mammalian sperm is a binding protein specific for two proacrosins and an acrosin intermediate. J. Biol. Chem. 269, 10133–10140. Bajpai, M., Fiedler, S.E., Huang, Z., Vijayaraghavan, S., Olson, G.E., Livera, G., Conti, M., Carr, D.W., 2006. AKAP3 selectively binds PDE4A isoforms in bovine spermatozoa. Biol. Reprod. 74, 109–118. Baldi, E., Luconi, M., Bonaccorsi, L., Muratori, M., Forti, G., 2000. Intracellular events and signaling pathways involved in sperm acquisition of fertilizing capacity and acrosome reaction. Front. Biosci. 5, E110–123. Bollen, M., Stalmans, W., 1992. The structure, role and regulation of type 1 protein phosphatase. Crit. Rev. Biochem. Mol. Biol. 27, 227–281. Brito, M., Figuero, J., Maldonado, E.U., Vera, J.C., Burzio, L.O., 1989. The major component of the rat sperm fibrous sheath is a phosphoprotein. Gamete Res. 22, 205–217. Brown, P.R., Miki, K., Harper, D.B., Eddy, E.M., 2003. A-kinase anchoring protein 4 binding proteins in the fibrous sheath of the sperm flagellum. Biol. Reprod. 68, 2241–2248. Carrera, A., Moos, J., Ning, X.P., Gerton, G.L., Tesarik, J., Kopf, G.S., Moss, S.B., 1996. Regulation of protein tyrosine phosphorylation in human sperm by a calcium/calmodulin-dependent mechanism: identification of A kinase anchor proteins as major substrates for tyrosine phosphorylation. Dev. Biol. 180, 284–296. Chang, M.C., 1951. Fertilizing capacity of spermatozoa deposited into the fallopian tubes. Nature 168, 697–698. Da Ros, V.G., Munuce, M.J., Cohen, D.J., Marin-Briggiler, C.I., Busso, D., Visconti, P.E., Cuasnicu, P.S., 2004. Bicarbonate is required for migration of sperm epididymal protein DE (CRISP-1) to the equatorial segment and expression of rat sperm fusion ability. Biol. Reprod. 70, 1325–1332. Dube, C., Leclerc, P., Baba, T., Reyes-Moreno, C., Bailey, J.L., 2005. The proacrosin binding protein, sp32, is tyrosine phosphorylated during capacitation of pig sperm. J. Androl. 26, 519–528. Edwards, A.S., Scott, J.D., 2000. A-kinase anchoring proteins: protein kinase A and beyond. Curr. Opin. Cell Biol. 12, 217–221. Ficarro, S., Chertihin, O., Westbrook, V.A., White, F., Jayes, F., Kalab, P., Marto, J.A., Shabanowitz, J., Herr, J.C., Hunt, D.F., Visconti, P.E., 2003. Phosphoproteome analysis of capacitated human sperm. Evidence of tyrosine phosphorylation of a kinase-anchoring protein 3 and valosin-containing protein/p97 during capacitation. J. Biol. Chem. 278, 11579–11589. Galantino-Homer, H.L., Visconti, P.E., Kopf, G.S., 1997. Regulation of protein tyrosine phosphorylation during bovine sperm capacitation by a cyclic adenosine 3 ,5 -monophosphate-dependent pathway. Biol. Reprod. 56, 707–719. Gopalakrishnan, B., Aravinda, S., Pawshe, C.H., Totey, S.M., Nagpal, S., Salunke, D.M., Shaha, C., 1998. Studies on glutathione S-transferases important for sperm function: evidence of catalytic activity independent functions. Biochem. J. 329, 231–241. Gopalakrishnan, B., Shaha, C., 1998. Inhibition of sperm glutathione Stransferase leads to functional impairment due to membrane damage. FEBS Lett. 422, 296–300. Han, Y., Haines, C.J., Feng, H.L., 2007. Role(s) of the serine/threonine protein phosphatase 1 on mammalian sperm motility. Arch. Androl. 53, 169–177. Hardy, D.M., Oda, M.N., Friend, D.S., Huang, T.T.F., 1991. A mechanism for differential release of acrosomal enzymes during the acrosome reaction. Biochem. J. 272, 759–766. Harrison, R.A., 2004. Rapid PKA-catalysed phosphorylation of boar sperm proteins induced by the capacitating agent bicarbonate. Mol. Reprod. Dev. 67, 337–352.

G. Jagan Mohanarao, S.K. Atreja / Animal Reproduction Science 123 (2011) 40–47 Hemachand, T., Gopalakrishnan, B., Salunke, D.M., Totey, S.M., Shaha, C., 2002. Sperm plasma-membrane-associated glutathione Stransferases as gamete recognition molecules. J. Cell Sci. 115, 2053–2065. Hinsch, K.D., Asmarinah, Hinsch, E., Konrad, L., 2001. Vdac2 (porin-2) expression pattern and localization in the bovine testis. Biochim. Biophys. Acta 1518, 329–333. Hinsch, K.D., De Pinto, V., Aires, V.A., Schneider, X., Messina, A., Hinsch, E., 2004. Voltage-dependent anion selective channels VDAC2 and VDAC3 are abundant proteins in bovine outer dense fibers, a cytoskeletal component of the sperm flagellum. J. Biol. Chem. 279, 15281–15288. Ho, H.C., Suarez, S.S., 2000. Hyperactivation of mammalian spermatozoa: function and regulation. Reproduction 122, 519–526. Hunter, T., 2000. Signaling—2000 and beyond. Cell 100, 113–127. Jha, K.N., Shivaji, S., 2002. Identification of the major tyrosine phosphorylated protein of capacitated hamster spermatozoa as a homologue of mammalian sperm a kinase anchoring protein. Mol. Reprod. Dev. 61, 258–270. Johnson, L.R., Foster, J.A., Haig-Ladewig, L., VanScoy, H., Rubin, C.S., Moss, S.B., Gerton, G.L., 1997. Assembly of AKAP82, a protein kinase A anchor protein, into the fibrous sheath of mouse sperm. Dev. Biol. 192, 340–350. Kalab, P., Peknicova, J., Geussova, G., Moos, J., 1998. Regulation of protein tyrosine phosphorylation in boar sperm through a cAMP dependent pathway. Mol. Reprod. Dev. 51, 304–314. Kota, V., Dhople, V.M., Shivaji, S., 2009. Tyrosine phosphoproteome of hamster spermatozoa: role of glycerol-3-phosphate dehydrogenase 2 in sperm capacitation. Proteomics 9, 1809–1826. Krapf, D., Arcelay, E., Wertheimer, E.V., Sanjay, A., Pilder, S.H., Salicioni, A.M., Visconti, P.E., 2010. Inhibition of ser/thr phosphatases induces capacitation-associated signaling in the presence of src kinase inhibitors. J. Biol. Chem. 285 (11), 7977–7985. Kumar, V., Rangaraj, N., Shivaji, S., 2006. Activity of pyruvate dehydrogenase a (pdha) in hamster spermatozoa correlates positively with hyperactivation and is associated with sperm capacitation. Biol. Reprod. 75, 767–777. Leclerc, P., de Lamirande, E., Gagnon, C., 1996. Cyclic adenosine 3 ,5 -monophosphate-dependent regulation of protein tyrosine phosphorylation in relation to human sperm capacitation and motility. Biol. Reprod. 55, 684–695. Luconi, M., Porazzi, I., Feruzzi, P., Marchiani, S., Forti, G., Baldi, E., 2005. Tyrosine phosphorylation of the A kinase anchoring protein 3 (AKAP3) and soluble adenylate cyclase are involved in the increase of human sperm motility by bicarbonate. Biol. Reprod. 72, 22–32. Mandal, A., Naaby-Hansen, S., Wolkowicz, M.J., 1999. FSP95, a testisspecific 95-kilodalton fibrous sheath antigen that undergoes tyrosine phosphorylation in capacitated human spermatozoa. Biol. Reprod. 61, 1184–1197. Mariappa, D., Aladakatti, R.H., Dasari, S.K., Sreekumar, A., Wolkowicz, M., Hoorn, F.V.D., Seshagiri, P.B., 2010. Inhibition of tyrosine phosphorylation of sperm flagellar proteins, outer dense fiber protein-2 and tektin-2, is associated with impaired motility during capacitation of hamster spermatozoa. Mol. Reprod. Dev. 77, 182–193. Miki, K., Willis, W.D., Brown, P.R., Goulding, E.H., Fulcher, K.D., Eddy, E.M., 2002. Targeted disruption of Akap4 gene causes defects in sperm flagellum and motility. Dev. Biol. 248, 331–342. Miller, D.J., Winer, M.A., Ax, R.L., 1990. Heparin-binding proteins from seminal plasma bind to bovine spermatozoa and modulate capacitation by heparin. Biol. Reprod. 42, 899–915. Mitra, K., Shivaji, S., 2004. Novel tyrosine-phosphorylated post-pyruvate metabolic enzyme, dihydrolipoamide dehydrogenase, involved in capacitation of hamster spermatozoa. Biol. Reprod. 70, 887–899. Mitra, K., Rangaraj, N., Shivaji, S., 2005. Novelty of the pyruvate metabolic enzyme dihydrolipoamide dehydrogenase in spermatozoa. J. Biol. Chem. 280, 25743–25753. Moss, S.B., Turner, R.M.O., Burkert, K.L., Butt, H.V., Gerton, G.L., 1999. Conservation and function of a bovine sperm A kinase anchor protein homologous to mouse AKAP82. Biol. Reprod. 61, 335–342. Osheroff, J.E., Visconti, P.E., Valenzuela, J.P., Travis, A.J., Alvarez, J., Kopf, G.S., 1999. Regulation of human sperm capacitation by a cholesterol efflux-stimulated signal transduction pathway leading to protein kinase A-mediated up-regulation of protein tyrosine phosphorylation. Mol. Hum. Reprod. 5, 1017–1026. Otter, T., King, S.M., Witman, G.B., 1987. A two-step procedure for efficient transfer of both high-molecular-weight (>400,000) and lowmolecular-weight (<20,000) proteins. Anal. Biochem. 162, 370–377. Parrish, J.J., Susko-Parrish, J., Winer, M., First, N., 1988. Capacitation of bovine sperm by heparin. Biol. Reprod. 38, 1171–1180.

47

Parrish, J.J., Susko-Parrish, J.L., Uguz, C., First, N.L., 1994. Differences in the role of cyclic adenosine 3 ,5 -monophosphate during capacitation of bovine sperm by heparin or oviduct fluid. Biol. Reprod. 51, 1099–1108. Pawson, T., 2004. Specificity in signal transduction: from phosphotyrosine–SH2 domain interactions to complex cellular systems. Cell 116, 191–203. Roy, A., Lin, Y.N., Agno, J.E., De Mayo, F.J., Matzuk, M.M., 2009. Tektin 3 is required for progressive sperm motility in mice. Mol. Hum. Reprod. 76, 453–459. Roy, S.C., Atreja, S.K., 2007. Tyrosine phosphorylation in a 38-kDa capacitation associated buffalo (Bubalus bubalis) sperm proteins is induced by l-arginine and regulated through a cAMP/PKA independent pathway. Int. J. Androl. 31, 12–24. Roy, S.C., Atreja, S.K., 2008a. Production of superoxide anion and hydrogen peroxide by capacitating buffalo (Bubalus bubalis) spermatozoa. Anim. Reprod. Sci. 103, 260–270. Roy, S.C., Atreja, S.K., 2008b. Effect of reactive oxygen species (ROS) on capacitation associated protein tyrosine phosphorylation in buffalo (Bubalus bubalis) spermatozoa. Anim. Reprod. Sci. 107, 68–84. Roy, S.C., Atreja, S.K., 2009. Capacitation-associated protein tyrosine phosphorylation starts early in buffalo (Bubalus bubalis) spermatozoa as compared to cattle. Anim. Reprod. Sci. 110, 319–325. Salinovich, O., Montelaro, R.C., 1986. Reversible staining and peptide mapping of proteins transferred to nitrocellulose after separation by sodium dodecylsulfate-polyacrylamide gel electrophoresis. Anal. Biochem. 156, 341–347. Shi, Q., Roldan, E., 1995. Bicarbonate/CO2 is not required for zona pellucida or progesterone-induced acrosomal exocytosis of mouse spermatozoa but is essential for capacitation. Biol. Reprod. 52, 540–546. Sidhu, K.S., Dhindsa, J.S., Guraya, S.S., 1992. A simple staining procedure for detecting the true acrosome reaction in buffalo (Bubalus bubalis) spermatozoa. Biotech. Histochem. 67 (1), 35–39. Smith, G.D., Wolf, D.P., Trautman, K.C., da Cruz e Silva, E.F., Greengard, P., Vijayaraghavan, S., 1996. Primate sperm contain protein phosphatase 1, a biochemical mediator of motility. Biol. Reprod. 3, 719–727. Smith, G.D., Wolf, D.P., Trautman, K., Vijayaraghavan, S., 1999. Motility potential of macaque epididymal sperm: the role of protein phosphatase and glycogen synthase kinase-3 activities. J. Androl. 1, 47–53. Therien, I., Bleau, G., Manjunath, P., 1995. Phosphatidylcholine-binding proteins of bovine seminal plasma modulate capacitation of spermatozoa by heparin. Biol. Reprod. 52, 1372–1379. Therien, I., Manjunath, P., 2003. Effect of progesterone on bovine sperm capacitation and acrosome reaction. Biol. Reprod. 69, 1408–1415. Tomar, N.S., 1997. Artificial Insemination and Reproduction of Cattle and Buffaloes, 4th ed. Saroj Prakashan, Allahabad, India. Vijayaraghavan, S., Stephens, D.T., Trautman, K., Smith, G.D., Khatra, B., da Cruz e Silva, E.F., Greengard, P., 1996. Sperm motility development in the epididymis is associated with decreased glycogen synthase kinase-3 and protein phosphatase 1 activity. Biol. Reprod. 54, 709–718. Vijayaraghavan, S., Liberty, G.A., Mohan, J., Winfrey, V.P., Olson, G.E., Carr, D.W., 1999. Isolation and molecular characterization of AKAP110. A novel, sperm-specific protein kinase A-anchoring protein. Mol. Endocrinol. 13, 705–717. Visconti, P., Bailey, J., Moore, G., Pan, D., Olds-Clark, P., Kopf, G., 1995a. Capacitation of mouse spermatozoa. I. Correlation between the capacitation state and protein tyrosine phosphorylation. Development 121, 1129–1137. Visconti, P.E., Moore, G.D., Bailey, J.L., Leclerc, P., Connors, S.A., Pan, D., Olds-Clarke, P., Kopf, G.S., 1995b. Capacitation in mouse spermatozoa. II. Protein tyrosine phosphorylation and capacitation are regulated by a cAMP-dependent pathway. Development 121, 1139–1150. Visconti, P.E., Galantino-Homer, H., Moore, G.D., Bailey, J.L., Ning, X., Fornes, M., Kopf, G.S., 1998. The molecular basis of sperm capacitation. J. Androl. 19, 242–248. Ward, C.R., Storey, B.T., 1984. Determination of the time course of capacitation in mouse spermatozoa using a chlortetracycline fluorescence assay. Dev. Biol. 104, 287–296. Yanagimachi, R., 1970. The movement of golden hamster spermatozoa before and after capacitation. J. Reprod. Fertil. 23, 193–196. Yanagimachi, R., 1994. Mammalian fertilization. In: Knobil, E., Neill, J.D. (Eds.), The Physiology of Reproduction. Raven Press Ltd., New York, pp. 189–317. Yi, L.S.H., Runion, C.M., Polakoski, K.L., 1992. Demonstration of a boar testicular protein band that is immunoreactive to proacrosin and proacrosin binding protein antibodies. Biochem. Biophys. Res. Commun. 184, 760–764.