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Dubé C, Tardif S, Leclerc P, Bailey J. The importance of calcium in
the appearance of p32, a boar sperm tyrosine phosphoprotein, during in vitro
capacitation. J Androl. 2003;24:727-733.
The marker most associated with time spent in "capacitating media" and acquisition of fertilizing ability is an increase in tyrosine phosphorylation of various sperm proteins; however, it should be noted that this alone should not be deemed enough to term a sperm "capacitated." Flagellar proteins (particularly the A-kinase anchoring proteins of the fibrous sheath) are probably the most studied tyrosine phosphophorylated proteins across various species' sperm, and because of location are implicated in motility changes. Immunofluorescence data using anti-phosphotyrosine antibodies suggest that phosphorylation of tail proteins is a prerequisite for tyrosine phosphorylation occurring in the sperm head (reviewed in Urner and Sakkas, 2003), implying a linkage of the different sperm subcellular compartments that have often been considered isolated. The best-known phosphotyrosine-containing protein located in the sperm head is p95, which is phosphorylated during induction of acrosome reaction by zona proteins, and has been identified as a hexokinase (Kalab et al, 1994). An in vivo inhibitor of rat sperm, tyrosine phosphorylation has recently been found in the form of Crisp-1 (Roberts et al, 2003), which is added to sperm in the epididymis, and acts upstream of cAMP production via insertion into the plasma membrane, steadily being lost over capacitation, in line with loss of cholesterol. It is tempting to speculate that Crisp-1 and similar proteins may act as part of a timing process that maintains fertility (ie, the capacitated state) as latent, until sperm have reached the vicinity of eggs.
In this issue Dube et al further characterize a 32 kDa protein (or cohort of proteins, p32) in boar sperm that are phosphorylated concomitantly with increases in intracellular calcium ion concentration. The data demonstrating a parallel change in phosphorylation as [Ca2+]i are manipulated, implying that this phosphorylation event(s) could occur downstream of a calcium signal or elevation. Previous data demonstrating that bicarbonate is not required for this effect (Tardif et al, 2003) implies some independence of the phosphorylation from the conventional soluble adenylate cyclase-protein kinase A pathway, possibly via calcium having inhibitory effects upon phosphatase action.
Data from various labs have revealed that although enhanced levels of protein phosphorylation due to elevated [Ca2+]i are observed in capacitated mouse sperm, the converse sometimes occurs in other species (eg, Carrera et al, 1996; Luconi et al, 1996). Differing phosphotyrosine outcomes on proteins in response to calcium concentration are intriguing, presumably reflecting differences in dynamic balance of enzymatic kinase vs phosphatase action on specific proteins, potentially in combination with an increase in phosphodiesterase activity (induced by the calcium elevation) that could act to lower the cAMP pool. The role of phosphatases in observed tyrosine phosphorylation data has been somewhat overlooked. Increases in intracellular calcium induced by agonists have been demonstrated to cause a substantial increase in phosphatase activity that is detectable when kinases are inhibited (progesterone, Kirkman-Brown et al, 2002; follicular-fluid, Thundathil et al, 2002; thapsigargin, Dorval et al, 2003). A ubiquitous calcium sensor is calmodulin; recent data from Zeng and Tulsiani (2003) have revealed complex roles for calmodulin in regulating mouse sperm and implied its involvement in phosphorylation of five different proteins. Thus, protein tyrosine phosphorylation/dephosphorylation processes during capacitation may be highly dynamic and regulated by several factors. A target over the coming years is to image the rapid dynamic changes that may transpire real-time in live sperm to better understand what is occurring.
An important challenge for all workers in this field is to begin to identify the proteins that they are working with or on. Pablo Visconti and coworkers have spent many years working on sperm phosphoproteins across various animal species. The recent paper from his laboratory and collaborators (Ficarro et al, 2003) on the human capacitated sperm phosphoproteome, in which they distinguish 60 proteins, has set a benchmark by developing and enhancing techniques that can be used to identify and quantify these proteins. Future use of the techniques on sperm exposed to agonists should allow further separation of events associated with the important motility changes from those involved with ability to respond appropriately to agonists of acrosome reaction, and changes postacrosome reaction induction.
More excitingly, the identification of the various proteins and their phosphorylation site sequences should allow the identification of the kinases that are acting upon these proteins. Identification of the specific kinases and phosphatases, and effects of phosphorylation on specific sites within proteins, will also allow assessment of whether they are good pharmacological targets for assisted conception or contraception. Researchers in the cancer field are currently examining the use of tyrphostins (tyrosine kinase inhibitors) as anti-cancer agents (Levitzki, 2002), and tyrosine phosphatase activity is also becoming recognized as a pharmacological target (Hooft van Huijsduijnen et al, 2002). This drug development and clinical testing may allow for the rapid crossover of these agents into the fertility field at a later date when identity of the sperm target proteins is known.
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Chang MC. Fertilising capacity of spermatozoa deposited into the fallopian tubes. Nature. 1951; 168:697-698.[Medline]
Dorval V, Dufour M, Leclerc P. Role of protein tyrosine
phosphorylation in the thapsigargin-induced intracellular Ca2+
store depletion during human sperm acrosome reaction. Mol Hum
Reprod. 2003;9(3):125-131.
Dubé C, Tardif S, Leclerc P, Bailey J. The importance of calcium in the appearance of p32, a boar sperm tyrosine phosphoprotein, during in vitro capacitation. J Androl. 2003; 24:727-733.
Ficarro S, Chertihin O, Westbrook VA, et al. Phosphoproteome
analysis of capacitated human sperm. J Biol Chem. 2003; 278:11579-11589.
Hooft van Huijsduijnen R, Bombrun A, Swinnen D. Selecting protein tyrosine phosphatases as drug targets. Drug Discov Today. 2002;7:1013-1019.[Medline]
Kalab P, Visconti P, Leclerc P, Kopf GS. P95, the major
phosphotyrosine-containing protein in mouse spermatozoa, is a hexokinase with
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Kirkman-Brown JC, Lefievre L, Bray C, Stewart PM, Barratt CLR,
Publicover SJ. Inhibitors of receptor tyrosine kinases do not suppress
progesterone-induced [Ca2+]i signaling in human
spermatozoa. Mol Hum Reprod. 2002; 8(4):326-332.
Levitzki A. Tyrosine kinases as targets for cancer therapy. Eur J Cancer. 2002; 38(Suppl 5):S11-18.
Luconi M, Krausz C, Forti G, Baldi E. Extracellular calcium negatively modulates tyrosine phosphorylation and tyrosine kinase activity during capacitation of human spermatozoa. Biol Reprod. 1996; 55:207-216.[Abstract]
Roberts KP, Wamstad JA, Ensrud KM, Hamilton DW. Inhibition of capacitation-associated tyrosine phosphorylation signaling in rat sperm by epididymal protein Crisp-1. Biol Reprod. 2003 :April 16 epub ahead of print.
Tardif S, Dube C, Bailey J. Porcine sperm capacitation and tyrosine
kinase activity are dependent on bicarbonate and calcium but protein tyrosine
phosphorylation is only associated with calcium. Biol
Reprod. 2003;68:207-213.
Thundathil J, de Lamirande E, Gagnon C. Different signal
transduction pathways are involved during human sperm capacitation induced by
biological and pharmacological agents. Mol Hum Reprod. 2002; 8(9):811-816.
Urner F, Sakkas D. Protein phosphorylation in mammalian spermatozoa. Reproduction. 2003; 125:17-26.[Abstract]
Zeng HT, Tulsiani DR. Calmodulin antagonists differentially affect
capacitation-associated protein tyrosine phosphorylation of mouse sperm
components. J Cell Sci. 2003; 116:1981-1989.
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