Cryobiology 70 (2015) 211–216

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Review

The protein tyrosine phosphorylation during in vitro capacitation and cryopreservation of mammalian spermatozoa q Sai Naresh, Suresh Kumar Atreja ⇑ Reproductive Biochemistry Laboratory, Animal Biochemistry Division, National Dairy Research Institute, Karnal, Haryana 132001, India

a r t i c l e

i n f o

Article history: Received 18 January 2015 Accepted 21 March 2015 Available online 28 March 2015 Keywords: Spermatozoa Capacitation Protein tyrosine phosphorylation Cryopreservation Cryo-capacitation

a b s t r a c t Before the process of fertilization, spermatozoa necessitate a period of residence in the female reproductive environment, and undergo a sequence of physiological and biochemical changes collectively referred to as capacitation. Accumulated evidences from several laboratories indicated that the protein tyrosine phosphorylation (PTP) is one of the most important intracellular signaling events regulating sperm function, and is a meaningful indicator of capacitation. Different factors that affect PTP are cholesterol efflux, 2+ influx of HCO 3 , increased intracellular Ca , cAMP and reactive oxygen species (ROS). cAMP/PKA and extracellular signal regulated kinases (ERKs) are the known important signaling pathways primarily involved in PTP. Advanced proteomics approaches have revealed several proteins that undergo tyrosine phosphorylation during capacitation. Semen cryopreservation subjects spermatozoa to frequent stressors, which result in capacitation like changes (cryo-capacitation). The cryo-capacitated spermatozoa usually show different patterns of PTP than the normal in vitro capacitated spermatozoa. In the current manuscript, we have summarized some information about the proteins undergoing tyrosine phosphorylation during capacitation and the effect of cryopreservation on PTP as well as the possibilities to reduce the changes associated with cryopreservation process. Ó 2015 Elsevier Inc. All rights reserved.

Introduction Mammalian haploid spermatozoa are different from the somatic cells in morphology and function. The metabolic pathways that occur in mature spermatozoa facilitate them to exist in two dissimilar kinds of environment, i.e. the male and the female reproductive tract. The residence time of the spermatozoa in the female reproductive tract is a very crucial period in the life cycle of spermatozoa. During this resident time, spermatozoa undergo multiple biochemical and physiological changes leading to an unique phenomenon called capacitation [1,36]. With the discovery of capacitation [8,21] many scientists became aware of the requirement of molecular interaction between the male and female gametes which later leads to the path towards in vitro fertilization (IVF). The changes that occur during capacitation are not fully

Abbreviations: PTP, protein tyrosine phosphorylation; ROS, reactive oxygen species; AR, acrosome reaction; IVF, in vitro fertilization. q Statement of funding: This study was funded by Indian Council of Agriculture Research, New Delhi, India and National Dairy Research Institute, Karnal, India. ⇑ Corresponding author. E-mail addresses: [email protected] (S. Naresh), [email protected] (S.K. Atreja).

http://dx.doi.org/10.1016/j.cryobiol.2015.03.008 0011-2240/Ó 2015 Elsevier Inc. All rights reserved.

understood till date, nevertheless, some molecular changes seem to affect the membrane polarization, changes in membrane lipid composition, intracellular alkalinization, an increase in intra2+ cellular concentrations of HCO 3 , Ca , cAMP, generation of reactive oxygen species (ROS) and ATP [34,35,77,104,105]. These alterations promote essential intracellular signaling events such as PTP in a group of proteins on different regions of spermatozoa [5,48,49,98]. Some major physiological changes like hyperactivation and acrosome reaction (AR). Hyper activation enhances the sperm motility, which might be helpful in release of sperm from the oviduct reservoirs and to penetrate the extracellular matrix of the egg [19,99]. Acrosome reaction (AR) comprises the exocytosis of the acrosomal contents that facilitate penetration of the zona pellucida and expose the membrane components required for gamete fusion [33,85]. Sperm cryopreservation (dilution, cooling and freezing/thawing) is an invaluable technique for artificial insemination (AI). It is also an useful therapeutic alternative in the management of infertility [3,44,76]. On the other hand, spermatozoa during cryopreservation are exposed to oxidative and osmotic stresses that dramatically alter the membrane lipid composition, sperm motility, viability and acrosome status [75,82,94,101]. Some of these modifications are reversible and others are lethal [39]. It has been

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accepted that the cryopreservation process induce capacitationlike changes to spermatozoa [83,100]. Some similar changes observed between in vitro capacitation and cryo-capacitation are plasma membrane reorganization, increase in intracellular Ca2+ concentration and occurrence of PTP [54,81,96]. Though, some of these changes seems to be similar (membrane changes and intra cellular Ca2+ concentration) there are striking difference in pattern of PTP and it should not be considered as true capacitation [37]. These pre-maturation processes, reduce the longevity of cryopreserved sperm in the female reproductive tract [10,100]. In the last few years, countless experiments have been guaranteed to realize the function of tyrosine phosphorylation during capacitation and cryopreservation. In this article, we have overviewed the data from our testing ground and other laboratories to identify the proteins undergoing tyrosine phosphorylation during in vitro capacitation and cryopreservation, their role in sperm function as well as the possibilities to reduce the changes associated with cryopreservation process. Protein tyrosine phosphorylation (PTP) Capacitation is principally dependent on the post-translational modifications like phosphorylation of pre-existing sperm proteins, as the terminally differentiated spermatozoa are transcriptionally and translationally silent [38,86]. Phosphorylation of proteins is one of the regulatory mechanisms to control diverse cellular processes [47,80]. In sperm both serine/threonine and tyrosine residues undergo phosphorylation [48,49,74]. However, the phosphorylation at tyrosine residue is the consistent indicator of sperm capacitation as many studies demonstrated that inhibition of tyrosine phosphorylation block the sperm capacitation, AR and IVF

[84,102,104]. Some important factors and signaling pathways that have an effect on the PTP are illustrated in Fig. 1. Immunocytochemistry and proteomics studies in diverse species have revealed that a different sequence of PTP was observed in different compartments of spermatozoa during capacitation and upon binding to the zona pellucida [88,102]. The flagellum seems to be the major component of sperm cell that undergoes tyrosine phosphorylation in most species suggesting a major role during hyperactivation, which in turn is needed for penetration of spermatozoa into zona pellucida [62,90]. Capacitation-associated redistribution of proteins undergo tyrosine phosphorylation to the neck and acrosome regions upon binding to zona pellucida have high affinity to the sperm–zona binding and/or fusion events [14,92]. Recently, using flow cytometry, it has been proved that all the spermatozoa do not undergo tyrosine phosphorylation in response to capacitation and cryopreservation conditions and there are sub populations of spermatozoa that exhibit different susceptibility to tyrosine phosphorylation [59]. Thus, these evidences indicate that the tyrosine phosphorylation of protein sub population on different regions of spermatozoa is very crucial for capacitation and fertilization processes. Identified tyrosine phosphorylated proteins in in vitro capacitated spermatozoa Sperm capacitation has been correlated with the increase in tyrosine phosphorylation of a sub population of proteins [34,87]. Although PTP is established as an important factor in capacitation, the defined relationship between the phosphorylation status of mammalian sperm and their capacity to fertilize has not been clarified till date. However, it is well established that the tyrosine

Fig. 1. A schematic representation of the molecular mechanism of tyrosine phosphorylation during sperm capacitation. The removal of cholesterol from the plasma 2+ membrane by cholesterol acceptors increase the membrane fluidity (1), that results influx of HCO ions through Naþ =HCO 3 and Ca 3 -co transporter and calcium channels (2). +2 The increased intracellular concentration of HCO , Ca and ROS, activates SACY/PKA pathway (3). PKA phosphorylation is depending on phosphatase inactivation by 3 phosphorylation at the C-terminal end of PP2A by Src family kinases (4). Onset of TK activation (5) followed by protein tyrosine phosphorylation (6). Growth factor and growth factor receptor (EGF-EGFR) binding activates the ERK pathway which increase PTP (7). NO can activates the ERK pathway intermediate Ras/Rho protein (8). GLUTs transport glucose, fructose into the sperm cell (9), which are useful in ATP generation by glycolysis and OXPR (10). Then ATP is used for sperm hyperactivation motility and PTP (11). High NO can also up regulate the cGMP/PKG pathway (12). These sequential steps like PTP and hyper activation further lead to sperm capacitation (13). SACY, soluble adenylyl cyclase; PKA, proteinkinase A; TK, tyrosine kinase; NO, nitric oxide; GLUTs, glucose transporters; OXPR, oxidative phosphorylation; ERK, extracellular regulated kinase; PKG, protein kinase G.

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phosphorylation mediated capacitation prepares the sperm to undergo the AR and fertilization. During this period, some significant alterations such as changes in protein localization and modification of protein–protein interactions in sperm protein machinery has also been reported [6,9]. Two dimensional (2D) poly acrylamide gel electrophoresis (PAGE) followed by tandem mass spectrometry (MS/MS) facilitates the identification of tyrosine phosphorylated proteins and the proteins involved in cell signaling during capacitation [48,49,57]. This strategy has been reported for the identification of tyrosine phosphorylated proteins in sperm cells of mouse [5], human [34], boar [11], hamster [55], rat [13], cattle and buffalo [48,49,57]. The first proteins to be identified as substrates for tyrosine phosphorylation were A-kinase anchor proteins (AKAP3 and AKAP4) in mouse [72], human [31] and hamster [50,53] spermatozoa. An aggregate of eight tyrosine phosphorylation sites was mapped in human spermatozoa for these proteins [34]. AKAP4 protein is present only in spermatogenic cells and it is the predominant protein in the fibrous sheath of the sperm flagellum [12]. The AKAPs can regulate spermatozoa motility due to its association with PKA regulatory subunits [18,65]. Targeted deletion in the Akap4 gene results in the shorter sperm flagellum and lack of fibrous sheath formation which causes a loss in motility and ultimately leads to infertility [69]. Additionally, the CABYR (calciumbinding and tyrosine phosphorylation regulated) protein, localized to the sperm fibrous sheath, binds to calcium during capacitation following tyrosine phosphorylation and it is likely to play a role in hyperactivation [61,73]. Various groups have shown that the chaperones such as heat shock protein-90 (HSP-90), HSP-70, HSP-60 and endoplasmin (erp99) undergo tyrosine phosphorylation during capacitation and cryopreservation in mammalian spermatozoa. The expression and phosphorylation of these HSPs are increased under stress conditions and are necessary components of a variety of signaling pathways. The tyrosine phosphorylation of chaperone proteins facilitated conformational changes on the sperm head surface that result in zona recognition [7,23,29,30,70]. Another important protein known to be tyrosine phosphorylated in mammalian spermatozoa is the valosin-containing protein (VCP/p97), which could possess a role as a link between capacitation and AR. This protein can act as a chaperone, bringing relevant membrane fusion proteins to the site of the AR [31]. The post-pyruvate metabolic enzymes pyruvate dehydrogenase (PDH) and dihydrolipoamide dehydrogenase (DLD) are tyrosine phosphorylated in hamster and buffalo spermatozoa and localized to the principal piece of the flagella and the acrosome regions of spermatozoa during capacitation [48,49,71,79]. Some cytoskeletal proteins phosphorylated at tyrosine residue include spectrin [28], various tubulins [51], dynein [30] and actin [49]. Tyrosine phosphorylation induced the polymerization of globular (G)-actin to filamentous (F)-actin during capacitation, and the inhibition of tyrosine phosphorylation prevents actin polymerization and capacitation [17]. In mammalian sperm the regions reported to contain actin include the equatorial, post acrosomal and the tail regions, which indicate its potential role in sperm motility and AR [16,17]. Mariappa et al. [67] reported the critical importance of flagellar proteins tyrosine phosphorylation in sperm motility where inhibition of tyrosine kinase reduced the tyrosine phosphorylation status of ODF-2 and tektin-2 resulting in circular motility of spermatozoa. It has likewise been reported that male null mice for TEKT2 gene produce sperm with reduced motility and increased flagellar structural defects [48,89]. Serine/threonine protein phosphatases (PP1a, PP1c1 and PP1c2) undergo tyrosine phosphorylation during capacitation of bovine spermatozoa, and are localized to the posterior, equatorial regions of head and entire flagellum including a mid piece of bovine

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spermatozoa. This increased phosphorylation of PP1c2 is necessary for motility, AR and signaling events during capacitation [45,46,48,49]. Recently it was found that a voltage-dependent anion channels (VDAC), also known as mitochondrial porins, are tyrosine phosphorylated in human, mouse, bovine and buffalo spermatozoa in the process of capacitation [5,31,48,49,64,68]. The VDAC2 and VDAC3 isoforms are abundant in outer dense fiber (ODF) of bovine sperm flagellum [43]. Mice lacking VDAC gene were infertile and had poor spermatozoa motility [22,91]. Tyrosine phosphorylation of glutathione-S-transferase (GST) isoforms such as GSTmu5 has been reported in mouse [5] and hamster [6] sperm capacitation, whereas tyrosine phosphorylation of GSTmu3 was observed in buffalo [48,49] and human [55] sperm capacitation. The GST is located in principle piece and end piece of the flagellum. It is involved in maintaining the oxidative balance and detoxification during sperm capacitation. Tyrosine phosphorylation level of GST increased following cryopreservation which signified its role in protection from oxidative stress during cryopreservation [57]. The differential cAMP/PKA pathway dependent tyrosine phosphorylation of eight isoforms of GST in in vitro capacitated and five isoforms in cryopreserved buffalo spermatozoa [57]. The tyrosine phosphorylated GST showed higher activity as compared to their dephosphorylated forms [57]. Proacrosin binding protein/p32 tyrosine phosphorylation during capacitation has been reported in boar [27,41], human [55,95], mouse [5] and buffalo [48,49] spermatozoa. It is localized to the acrosomal cap and mid piece of boar spermatozoa, suggesting a role in capacitation and AR [27]. Other tyrosine phosphoproteins are the tyrosine kinase c-yes [60], fibrous sheath CABYR binding protein (FSCB) [63], alpha-enolase, succinate dehydrogenase and glutamine synthase [48,49]. In summation, this information, contributes only a concise impression of tyrosine phosphorylated proteins identified to date and also stands for the importance of tyrosine phosphorylation in sperm in vitro capacitation and IVF. Effect of cryopreservation on protein tyrosine phosphorylation The molecular mechanism of tyrosine phosphorylation during cryopreservation is not completely understood, but the process of cooling-freeze/thawing has distinct effects on the sperm structure and function. Cryopreservation results in temperature fluctuation and cell dehydration that induces reordering of membrane lipid components, loss of polyunsaturated fatty acids and cholesterol [20,26,66]. This initial damage is enough to generate capacitation like changes in spermatozoa. Membrane reordering increases the permeability of the sperm surface to water, Ca2+ and cryoprotectants [40,78]. Ca2+ influx activates adenylyl cyclase with increased generation of cAMP and 1,2-Diacyl glycerol (DAG) resulting in sperm capacitation through PTP [58,59,96] (see Fig. 2). These early capacitation like changes during coolingfreeze/thawing of spermatozoa makes them unfit to fertilize even though they are viable [106]. Previous studies have been reported that cryopreservation induces protein degradation in boar [32], human [25], and buffalo [57] spermatozoa. Moreover, cryopreservation induces PTP in spermatozoa [54,56]. In boar 33 and 32 kDa proteins were not tyrosine phosphorylated in fresh spermatozoa, but PTP of these proteins appeared after cooling the semen to 5 °C. The protein of 32 kDa was found to be tyrosine phosphorylated throughout the cryopreservation process [37,58], indicating the effect of the cooling process on PTP in boar spermatozoa. It was suggested that the method of freezing significantly influences the degree of tyrosine phosphorylation in post thaw spermatozoa [58]. Kumar and Atreja [54] noted an increase of PTP from two (P72 and P86) proteins in fresh semen, to nine proteins (p20, p30, p32, p38, p49, p56, p59, p72 and p86) following cryopreservation of buffalo

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Fig. 2. Diagram illustrating the capacitation like changes upon cryopreservation. Cooling-freeze/thawing generates cold shock and osmotic stress on sperm plasma membrane that result in membrane damage and increased membrane permeability. Altered membrane permeability cause influx of calcium that elevates intracellular concentration of cAMP and 1,2-Diacylglycerol (DAG) which ultimately leads to protein tyrosine phosphorylation. The generation of cold shock during cryopreservation is associated with high amount of reactive oxygen species (ROS) also lead to oxidative stress and PTP. ?; unknown pathway.

spermatozoa due to increased levels of intracellular signaling molecules like cAMP, calcium and DAG as a result of cryo-damage [96]. The cryopreservation induced PTP significantly reduced the binding ability of sperm to the zona pellucida as compared to in vitro capacitated spermatozoa in bovine [24], buffalo [52] and human [2]. These findings clearly indicated that the PTP during cryopreservation could be one of the causes for poor fertility of cryopreserved sperm. Some important tyrosine phosphorylated proteins in capacitated and cryopreserved spermatozoa were compared in Table 1. Recently we have reported that the addition of trehalose (100 mM) or taurine (50 mM) to the freezing extender improved the sperm quality and reduced PTP signals (p20, p30, p32, p38, p49, p56, p59, p72 and p86) of frozen-thawed buffalo spermatozoa [56]. Similarly, these additives also reduced the concentration of intracellular signaling molecules like Ca2+, cAMP and DAG, which were responsible for PTP [96]. The addition of seminal plasma to spermatozoa after post-thaw reduced PTP in cryopreserved equine and porcine spermatozoa [4,103]. Addition of a-tocopherol to freezing extender also reduced the tyrosine phosphorylation of 32 kDa protein in boar spermatozoa [93]. The newly developed plant based soya milk extender improved the quality and reduced the capacitation like changes of cryopreserved buffalo spermatozoa [97]. These data indicate that the supplementation of additives to cryopreservation extender could reduce the capacitation like changes in cryopreserved spermatozoa. Conclusion This paper covers the tyrosine phosphorylated proteins identified to date and the importance of PTP during mammalian sperm capacitation and cryopreservation. Tyrosine phosphorylation in a group of proteins on different regions of spermatozoa is crucial for sperm capacitation and fertilization. The process of freezing/ thawing induces capacitation like changes which are responsible for poor fertility of cryopreserved spermatozoa. The pattern of PTP occur during cryopreservation is different from the induced in vitro capacitation. Supplementation of additives in

Table 1 Some important proteins undergoing tyrosine phosphorylation during in vitro capacitation and cooling-freeze/thawing. Tyrosine phosphorylated proteins In vitro capacitation A-kinase anchor proteins [29,46,49,68] CABYR (calcium-binding and tyrosine phosphorylation regulated protein) [57,69] Heat shock proteins (HSP-90), HSP-70, HSP-60 and endoplasmin (erp99) [7,22,27,28,66] Valosin-containing protein (VCP/ p97)[29] Pyruvate dehydrogenase (PDH) Dihydrolipoamide dehydrogenase (DLD) [44,45,67,74] Spectrin [26] Tubulins [47] Dynein [28] Actin [45] Outer dense fibers (ODF-2) Tektin-2 [44,84] Serine/threonine protein phosphatases (PP1a, PP1c1 and PP1c2) [41,42,44,45] Voltage-dependent anion channels (VDAC) [5,29,44,45,61,64] Glutathione-S-transferase (GSTmu5 and GSTmu3) [44,45,51] Proacrosin binding protein/p32 [37,44,45] Tyrosine kinase c-yes [56], fibrous sheath CABYR binding protein (FSCB) [60] alpha-enolase, Succinate dehydrogenase, Glutamine synthase [44,45].

Cooling-freeze/thawing Buffalo 20, 30, 32, 38, 49, 56, 59, 72 and 86 kDa [50] Glutathione-S-transferase (GST) mu3 [53] Boar 33 kDa [37] 32 kDa [15] 19, 32, 41and 48 kDa [54] Equine 100 kDa, 65–75 kDa, 44– 52 kDa and 35–40 kDa [93] Bull 56 pp (56000 Mr) [23] Japanese black bull 35, 41 kDa [38] Rhesus macaque HSP-70 and HSP-90 [22]

cryopreservation media can partly reduce PTP and capacitation like changes in cryopreserved spermatozoa. However, additional improvement in this area is needed to get clarification on the differences between the in vitro sperm capacitation and the capacitation like changes during cryopreservation. Further, investigation

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of differential sperm proteins undergoing tyrosine phosphorylation during capacitation and cryopreservation could lead to better understanding of differential pathways undergoing during capacitation and cryopreservation process. Substantial improvement in formulating effective cryopreservation strategies are required to reduce the cryodamages.

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