- Email: [email protected]
Signal transduction processes leading to acrosomal exocytosis in mammalian spermatozoa
Huhtaniemi I, Warren DW, Catt KJ: Regulation of infant and developing rat testicular gonadotropin and prolactin receptors and steroidogenesis by treatments with human chorionic gonadotropin, gonadotropin-releasing hormone analogs, bromocriptine, prolactin and estrogens. Biol Reprod 1985; 32:721. Huhtaniemi IT, Dunkel L, Perheentupa J: The transient increase in postnatal testicular activity is not revealed by longitudinal measurements of salivary testosterone. Pediatr Res 1986a; 20: 1324. Huhtaniemi I, Nevo N, 2: Effect of postnatal nadotropin-releasing on sexual maturation prod 1986b; 35:SOl.
Amsterdam A, Naor treatment with a gohormone antagonist of male rats. Biol Re-
Huhtaniemi IT, Yamamoto M, Ranta T, Jalkanen J, Jaffe RB: Follicle-stimulating hormone receptors appear earlier in the primate fetal testis than in the ovary. J Clin Endocrinol Metab 1987; 65:1210. Hutson JM, Metcalfe SA, MacLaughlin et al.: Mullerian inhibiting substance. Burger H, de Kretser D, eds. The Testis, ed. New York, Raven, 1989, p 143.
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Jost A, Vigier B, Prepin J, Perchellet JP: Studies on sex differentiation in mammals. Recent Prog Horm Res 1973; 29: 1. Kaplan SL, Grumbach MM, Aubert ML: The ontogenesis of pituitary hormones and hypothalamic factors in the human fetus: maturation of central nervous system regulation of anterior pituitary function. Recent Prog Horm Res 1976; 32:161. Kolho K-L, Nikula H, Huhtaniemi I: Sexual maturation of male rats treated postnatally with a gonadotrophin-releasing hormone antagonist. J Endocrinol 1988; 116:241. Meidan R, Lim P, McAllister JM, Hsueh AJW: Hormonal regulation of androgen biosynthesis by primary cultures of testicular cells from neonatal rats. Endocrinology 1985; 116:2473. Mulchaney JJ, DiBlasio AM, Martin MC, Blumenfeld Z, Jaffe RB: Hormone production and peptide regulation of the human fetal pituitary gland. Endocr Rev 1987; 8:406. Pakarinen P, Huhtaniemi I: Gonadal and sex steroid feedback regulation of pituitary gonadotropin mRNA levels and secretion in neonatal male and female rats. J Mol Endocrinol 1989; 3:139. Pelliniemi LJ, Dym M: The fetal gonad and sex differentiation. In Tulchinsky D, Ryan KJ, eds. Maternal-Fetal Endocrinology. Philadelphia, WB Saunders, 1980, p 252. Reyes FI, Winter JSD, Faiman C: Endocrinology of the fetal testis. In Burger H, de Kretser D, eds. The Testis, 2nd ed. New York, Raven, 1989, p 119. Rivier C, Cajander S, Vaughan J, Hsueh AJW, Vale W: Age-dependent changes in physiological action, content, and immunostaining of inhibin in male rats. Endocrinology 1988; 123:120.
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Schwanzel-Fukuda M, Pfaff DW: Origin teinizing hormone-releasing hormone rons. Nature 1989; 338:161.
01 luneu-
Takagi S, Yoshida T, Tsubata K, ct al.: Sex differences in fetal gonadotropins and androgens. J Steroid Biochem 1977; 8:609. Tapanainen J, Koivisto M, Vihko R, Huhtaniemi I: Enhanced activity of the pituitary-gonadal axis in premature human infants. J Clin Endocrinol Metab 1981; 52:235. Vigier B, Forest MG, Eychenne B, et al.: Antimtillerian hormone produced endocrine sex reversal of fetal ovaries. Proc Nat1 Acad Sci USA 1989; 86:3684. Voutilainen R, Eramaa M: Hormonally regulated inhibin gene expression in human fetal adrenals and testes (Abstract). Third Joint Meeting of the ESPE/LWPES, Jerusalem, October 1989, no. 57.
Warren
DW: Development
ol the inhibitor.!
guanine nucleotide-binding rcgulator~ protein in the rat testis. Biol Reprod 1989; 40: 1208. Warren DW, Dufau ML, Catt KJ: Hormonal regulation of gonadotropin receptors and steroidogenesis in cultured fetal rat tcstcs. Science 1982; 218:375. Warren DW, Pasupuleti V, Platler B, Lu Y, Horton R: Tumor necrosis factor and interleukin-1 inhibit testosterone secretion in cultured fetal rat testis cells but stimulate testosterone production in adult testis cells (Abstract). The Endocrine Society Annual Meeting, Seattle, WA, June 1989, no. 1159. Word RA, George FW, Wilson JD, Carr BR: Testosterone synthesis and adenylate cyclase activity in the early human fetal testis appear to be independent of human chorionic gonadotropin control. J Clin EndocriTEM nol Metab 1989; 69:204.
Signal Transduction Processes Leading to Acrosomal Exocytosis in Mammalian Spermatozoa Gregory S. Kopf and Mary W. Wilde Many components of intercellular signaling involved in species-specific sperm binding to the egg’s extracellular matrix, the zona pellucida, and the induction of acrosomal exocytosis, an absolute prerequisite to successful fertilization, have properties similar to intercellular signaling mechanisms controlling somatic cell function. Sperm-associated receptors for zona pellucida glycoproteins have been postulated to serve as the initial components of signal transduction cascades leading to the stimulation of cellular effector systems that modulate sperm function. Such receptor-effector systems appear to be coupled through guanine nucleotide-binding regulatory proteins (G proteins).
??
ment, These activation
Background
Intercellular communication between male and female gametes of all species leads to the subsequent cellular activation of the respective gametes, a process essential to both successful fertilization and initiation of early embryo developGregory S. Kopf and Mary W. Wilde are at the Division of Reproductive Biology, Department of Obstetrics and Gynecology, University of Pennsylvania School of Medicine, Philadelphia, PA 19104-6080, USA.
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processes
can be
either egg-induced categorized as sperm activation events or sperm-induced egg activation events. Sperm-female reproductive tract interaction and sperm-egg interaction prior to spermegg fusion occur at different sites in vivo and are the culmination of a number of integrated processes designed to deliver sperm with optimal fertilizing potential to the site of fertilization, e.g., the ampullary region of the oviduct (Katz et al. 1989).
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Sperm from all invertebrate
and ver-
tebrate stimuli
species respond to external in their environment with
changes
in cellular
function
that
a
f$?oO+_ OaQ,Q
PS
Cumulus Oopohorus
are
prerequisites to successful sperm-egg interaction and fertilization (Kopf 1988; Kopf and Gerton 1990). Such stimuli are elaborated by the egg itself, the cellular investments surrounding the egg, and/or factors associated with the female reproductive tract. In mammals, these stimuli may be responsible for modulating such functions as sperm motility and metabolism (essential for the delivery of sperm to the site of fertil-
Cortical Granule Plasma Membrane
y WZona
p
Perivitelline Space Pellucida
ization), capacitation (a poorly understood process that confers upon the mammalian sperm the ability to fertilize an egg), and the acrosome reaction (an exocytotic process that is essential for the sperm to penetrate the zona pellucida of the egg). The molecular identities of these stimuli in various species are only starting to be elucidated. At present, most of the information regarding ligand-effector systems regulating mammalian sperm function prior to fertilization has been discerned from studies of those processes regulating sperm-zona pellucida interaction and acrosomal exocytosis in the mouse. Consequently, this review will focus, for the most part, on studies carried out in this species. Studies of this system have revealed that sperm perceive and process signals from the zona pellucida in a fashion analogous to the way somatic cell function is regulated by humoral, autocrine, or paracrine factors. A more comprehensive treatment of this subject can be found in additional reviews (Wassarman 1987 and 1988; Yanagimachi 1988; Garbers 1989; Saling 1989; Kopf and Gerton 1990).
?? The
Zona Pellucida, an Egg-Associated Extracellular Matrix, Functions as a Regulatory Ligand for Sperm
The zona pellucida is an oocyte-derived acellular coat that surrounds the egg and can be considered an extracellular matrix (Figure 1). This structure plays an important role both prior to and after fertilization, as well as during subsequent preimplantation development. The zonae pellucidae from all mammals appear to be composed of 2-4 glycoproteins, the number of which appears to be species dependent (Was-
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Figure 1. Diagrammatic representation of the events comprising sperm-zona pellucida interaction in the mouse both prior to (a and b) and after (c and d) sperm-egg fusion. (A) An ovulated egg surrounded by the zona pellucida which is synthesized by the growing oocyte. The zona pellucida-enclosed egg is surrounded by cells comprising the cumuZus oophorus. Cortical granules are shown immediately beneath the plasma membrane of the egg. These cortical granules have not undergone exocytosis, as shown in the figure by the filled circles. Both acrosome-intact (filled heads) and acrosome-reacted (open heads) sperm are present. Acrosome-reacted sperm do not bind to the zona pellucida, as shown by the presence of theX. PB, polar body. (b) The sequence of events of sperm binding to the zona pellucida of the ovulated egg. Acrosome-intact sperm establish primary binding to the zona pellucida (via ZP3 and sperm-associated receptors for ZP3) (I) and, having undergone acrosomal exocytosis, establish secondary binding interactions with the zona pellucida (via ZP2 and spermassociated receptors for ZP2) (2). Once the acrosome reaction has occurred, sperm are then able to penetrate the zona pellucida (3). (c) Fusion of the egg plasma membrane and the plasma membrane of acrosome-reacted sperm results in an elevation of intracellular free Ca2+within the egg, the subsequent exocytosis of the cortical granules (as shown by the open circles), and the intercalation of the cortical granule membrane into the egg plasma membrane (as shown by the absence of the circles). This Ca2+-induced exocytosis proceeds as a centripetal wave over the entire egg (double-headed arrow) originating from the point of sperm-egg fusion. Accompanying cortical granule exocytosis is a modification of the zona pellucida such that the biochemical and biological properties of this extracellular matrix are now altered (as shown by the stippling of the zona pellucida). (d) As a consequence of these egg-induced modifications of the zona pellucida, the ability of acrosome-intact sperm to bind to ZP3 (I) and the ability of bound acrosome-intact sperm to undergo ZP3-induced acrosomal exocytosis (2) is lost (X) due to the conversion of ZP3 to ZP3t. Those acrosome-reacted sperm that are bound to the zona pellucida (3) lose their ability to penetrate the zona since they cannot establish secondary binding interactions with ZP2t (X). These egg-induced modifications of the zona pellucida constitute the zona pellucida block to polyspermy. This figure is for illustrative purposes only and is not drawn to scale.
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sarman
1988;
Kopf and Gerton
1990).
Sperm-egg recognition and binding, sperm activation (i.e., acrosomal exocytosis), and an egg-induced block to polyspermy in the mouse (and in a growing number of other species) all appear to be mediated by this extracellular matrix. The zona pellucida of the mouse egg is composed of three sulfated glycoproteins designated as ZPl, ZP2, and ZP3, which are present in molar ratios of 1: 10: 10, respectively (Wassarman 1988). These glycoproteins are truly egg-associated products, as they are synthesized and secreted throughout the period of oocyte growth. ZPl has been proposed to maintain the three-dimensional structure of the zona pellucida by virtue of its ability to cross-link filaments composed of repeating ZP2iZP3 heterodimers. ZP2 may function to mediate the binding of acrosome-reacted sperm to the zona pellucida. Upon fertilization, the egg effects a modification of ZP2 to a form called ZP2t by the action of a protease(s), which is most likely from the egg as a consequence
secreted of corti-
cal granule exocytosis (Moller and Wassarman 1989). The biological consequence of the ZP2 to ZP2t conversion is that ZP2t no longer retains the capacity to bind acrosome-reacted sperm. ZP3 accounts for both the sperm-binding and the acrosome reaction-inducing activities of the zona pellucida of unfertilized eggs. Fertilization results in a loss of both of these activities of the ZP3 molecule, although it is not clear at present whether these modifications occur in response to the same protease that modifies ZP2. The properties of these three zona pellucida glycoproteins from both unfertilized and fertilized eggs provide a framework with which to formulate a model to explain the interaction of sperm with this specialized extracellular matrix (Figure 1). Since only acrosome-intact mouse sperm bind to the zona pellucida (Saling et al. 1979), specific receptor(s) for ZP3 on the plasma membrane overlying the sperm acrosome would mediate sperm binding and induction of the acrosome reaction. Subsequent to ZP3-mediated binding and acrosomal exocytosis, acrosome-reacted sperm would then maintain their interaction with the zona pellucida through secondary interactions with sperm-associated receptor(s) for ZP2 lo-
364
cated
on the
inner
acrosomal
mem-
fact that its site and mode 01 action arc’
brane. Upon penetration of the zona pellucida by acrosome-reacted sperm, these cells traverse the perivitelline space, bind to, and then fuse with the plasma membrane of the egg. Spermegg fusion occurs at discrete regions in
restricted to events associated with fcrtilization and preimplantation development. Little amino acid sequence homology exists between ZP3 and an!’ other known proteins or glycoprotcins thus far examined (Ringuette ct al.
the postacrosomal region of the sperm head (Yanagimachi 1988). Subsequent to sperm-egg fusion, the egg undergoes the cortical granule reaction, which results in the release of cortical granule associated enzymes (Wassarman 1987). These enzymes convert ZP2 to ZP2r and
1988), suggesting that it serves a unique function. ZP3 is an immobilized ligand that acts at a localized and restricted distance, owing to its presence in the zona pellucida as a ZPZlZP3 heterodimer cross-linked in an orderly fashion by ZPl (Wassarman 1988). ZP3 is biologically potent since both the spermbinding and acrosome reaction-inducing activities of this molecule are observed in the nanomolar range (Florman and Wassarman 1985; Bleil and
modify ZP3 (to ZP3t), so that acrosome-intact sperm no longer bind to the zona pellucida (through ZP3) and acrosome-reacted sperm that are bound to the zona pellucida (through ZP2) no longer interact and penetrate this extracellular matrix, since they are unable to establish secondary binding interactions with ZP2t. Such egg-induced modifications of the zona pellucida constitute the zona pellucida block to polyspermy. Inherent in such a model is the highly specific and coordinated nature of the interactions of acrosome-intact and acrosome-reacted sperm with ZP3 and ZP2, respectively. These interactions would presumably be mediated by specific sperm-associated receptors for these zona pellucida glycoproteins.
Several observations support the contention that sperm-zona pellucida interaction in the mouse (and probably in other mammalian species) represents a process akin to ligand-receptor interactions in somatic cells. ZP3, for example, possesses a variety of properties that make it ideally suited as a ligand to mediate both initial steps of sperm-egg interaction proper (e.g., sperm binding) and subsequent sperm activation (e.g., induction of acrosomal exocytosis). ZP3 is an egg-associated product that is synthesized only by the growing oocyte (Wassarman 1988), consistent with the
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Wassarman 1986). Acrosome-intact mouse sperm appear to possess complementary binding sites (receptors?) for ZP3 that are localized to the acrosomal cap region and are present in numbers (lO,OOO-50,000 binding sites/cell) similar to those observed for receptor numbers present in many hormone-responsive cells (Bleil and Wassarman 1986). Acrosome-intact mouse sperm bind to ZP3 immobilized onto silica beads via the sperm head (Vazquez et al. 1989), further supporting the idea that the sperm-associated ZP3 binding moiety(ies) is associated with the plasma membrane overlying the acrosomal region, that region of initial contact where sperm-zona pellucida interaction first occurs. Therefore, although conserved at the genomic level in a number of species (Ringuette et al. 1988), ZP3 subserves extremely specific functions as a component of the egg-associated extracellular matrix known as the zona pellucida (Wassarman 1987 and 1988). Similarly, the specific binding of ZP2 to the inner acrosomal membrane of acrosome-reacted, but not acrosome-intact, mouse sperm suggests that this membrane may possess specific receptors for this particular zona pellucida glycoprotein (Bleil et al. 1988).
??
Sperm-Associated Zona Pellucida
Receptors
for the
Although sea urchin sperm remain the sole experimental system in which receptor-mediated signal transduction has been unequivocally demonstrated (Garbers 1989), studies in the mammal
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are beginning to yield information regarding the nature of sperm-associated receptors for components of the zona pellucida. Two different approaches have been utilized to describe putative sperm-associated receptors for the zona pellucida. The first, and more direct, approach involves the elucidation of components of the sperm surface that interact directly with the individual zona pellucida glycoproteins. Utilizing this approach, Bleil and Wassarman examined the specific binding of 1251-ZP3 to mouse sperm by whole mount autoradiography and demonstrated that binding is associated solely with the plasma membrane overlying the acrosomal region of the sperm and is not observed on acrosome-reacted sperm (Bleil and Wassarman 1986). Binding of this radiolabeled ligand is competed by unlabeled ZP3 but not ZP2, and ZP3 binding does not occur on somatic cells. Similar localize
techniques have been used to binding sites for ZP2 to the in-
ner acrosomal membranes of acrosomereacted, but not acrosome-intact, sperm (Bleil et al. 1988). Although the methodology utilized in these studies has limitations with regard to the characterization of the sperm-associated moiety(ies) involved in ZP3 and ZP2 binding, these results suggest that specific sperm-associated binding sites for these ligands exist in discrete domains of this highly differentiated cell. Reinvestigators demoncently, these strated
that
either
purified
ZP3
or
ZP3 possessing glycopeptides of sperm-binding activity can be specifically cross-linked to a M, = 56,000 protein of acrosome-intact mouse sperm (Bleil and Wassarman 1989). This protein interacts specifically with ZP3, but not ZP2, affinity columns. Whole mount autoradiography utilizing radiolabeled, cross-linked ZP3 glycopeptides that possess sperm-binding activity demonstrates a localization to the head region of acrosome-intact sperm. This experimental approach may provide a great deal of promise with regard to establishing the molecular identity of such sperm-associated receptors for zona pellucida glycoproteins. Recently Leyton and Saling (1989a) demonstrated that antiphosphotyrosine antibodies react with mouse sperm plasma membrane proteins of M, = 52,000,75,000, and 95,000, and that this positive immunoreactivity is localized
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to the acrosomal
region
of the sperm
head. 1251-ZP3 binds, on nitrocellulose blots, to a M, = 95,000 protein, and these workers conclude that this M, = 95,000 protein is the protein that also reacts with the antiphosphotyrosine antibodies. Although it is suggested that this M, = 95,000 protein may be a receptor for ZP3 that acts either as a substrate for tyrosine phosphorylation or possesses tyrosine kinase activity itself, additional studies will be required to clearly demonstrate the specificity of the ZP3 binding activity of this protein. The second, more indirect, approach involves the delineation of sperm-associated binding sites for the zona pellucida glycoproteins by examining the ability of specific agents to interfere with sperm-zona pellucida interaction. Sperm surface-associated protease inhibitor-sensitive sites, galactosyltransferase activity, and fucosyltransferase activity have all been implicated in the binding of sperm to the zona pellucida, presumably through ZP3 (Saling 1989; Kopf and Gerton 1990, and references therein). However, the relationship of these sites to one another and to those sites defined by ZP3 binding (as described above) is not clear at this time. Although little is known regarding the identity of the sperm-associated receptor for ZP3, recent studies have shed some light on the potential nature of the interaction of the ZP3 molecule with the sperm surface to mediate both sperm binding and acrosomal exocytosis. Studies from three independent laboratories using different approaches have provided evidence that the interaction of ZP3 with the sperm surface may occur in a multivalent (or cooperative) fashion, and that multiple interactions followed by possible receptor aggregation may ultimately lead to signal transduction and acrosomal exocytosis (Bleil and Wassarman 1983; Endo et al. 1987b; Kopf et al. 1989; Leyton and Saling 1989b). The idea that receptor aggregation is required to elicit ligand-dependent signal transduction and a cellular response is not unique (Yarden and Schlessinger 1987). It has been recently demonstrated that extracellular matrices from a variety of tissues contain domains that aggregate acetylcholine receptors (Godfrey et al. 1988). It is certainly possible that the zona pellucida, itself an extracellular matrix, also
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possesses the ability to aggregate tors on the sperm surface.
??
recep-
Signal Transduction Mechanisms Mediating Sperm-Zona Pellucida Interactions
The unique structure of ZP3, its biological potency, and the probable existence on the plasma membrane of acrosomeintact sperm of a complementary ZP3 receptor(s) satisfy a number of criteria required for a means to control specific cell-cell recognition events in a receptor-mediated fashion. The consequence of these specific interactions is that sperm undergo an exocytotic event, the acrosome reaction, which is an absolute prerequisite to successful penetration of the zona pellucida and subsequent fusion of the sperm and egg plasma membranes. The mechanism by which ZP3 effects informational flow across the sperm plasma membrane as a consequence of binding to its putative receptor has been recently examined.
As in many somatic cells, guanine nucleotide-binding regulatory proteins (G proteins) appear to play a critical role(s) as signal-transducing elements coupling zona pellucida-sperm interaction to the generation of intracellular second messengers that ultimately regulate acrosomal exocytosis. Spermatozoa from all species studied thus far (invertebrate as well as mammalian) contain G proteins, as assessed by bacterial toxin-catalyzed ADP-ribosylation, GTPy3%-binding, and immunoblotting techniques (Kopf et al. 1986; Bentley et al. 1986). Mammalian sperm contain a G protein with properties similar to Gi; the exact nature of
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al. 1989), indicating
that a G,-like pro-
this G protein has yet to be determined. Consistent with observations made by other investigators, sperm do not appear to contain a G protein with properties similar to G, (Kopf and Gerton 1990). Immunocytochemical studies have demonstrated that G proteins in bovine (Garty et al. 1988) as well as mouse and guinea pig (Glassner et al.
Recently, it has been demonstrated that solubilized zonae pellucidae from mouse eggs stimulate a high-affinity GTPase activity associated with the G,like protein of mouse sperm (Wilde and Kopf 1989). Ligand-dependent stimulation of this activity reflects the termination of a G protein activation cycle (Gilman 1987). Zonae pellucidae stimu-
1989) sperm are present in the acrosoma1 region of these highly differentiated cells. The physiological role of the sperm G,-like protein in the zona pellucidamediated acrosome reaction has thus far been examined in both the murine and bovine systems. Functional inactivation of the mouse sperm Gi-like protein by pertussis toxin treatment of the
late this activity in a concentration-dependent manner and the stimulatory efof fect occurs as a consequence increased GTP turnover. These data support the idea that a component(s) of the zona pellucida (perhaps ZP3) binds to a sperm-associated receptor that is G protein coupled, and that receptor-mediated G protein activation results in the activation of specific intracellular
tein may regulate such ionic changes in response to this egg-associated cxtracellular matrix. Studies from other laboratories have suggested that alterations in phospholipid metabolism and/or cyclic nucleotide metabolism may play important intermediary roles in the sperm acrosome reaction (Kopf and Gerton 1990). Lee et al. (1987) demonstrated that biologically active phorbol diesters and diacylglycerols alter the kinetics of the zona pellucida-mediated acrosome reaction in mouse sperm, thus providing evidence for the role of protein kinase C in regulating this exocytotic event. The products of phospholipase C turnover (e.g., inositol 1,4,5trisphosphate and
cells does not affect the ability of sperm to interact with the zona pellucida, but inhibits the bound sperm from under-
effector systems required for the induction of acrosomal exocytosis.
1,2 diacylglycerol), as well as the role of other phospholipases (A* or D), have not
Intracellular Effecters Modulating Zona Pellucida-Mediated Acrosomal Exocytosis
yet been examined in sperm challenged with zonae pellucidae or ZP3. Noland et al. (1988) have reported that solubilized zonae pellucidae from mouse eggs induce transient elevations in mouse sperm CAMP concentrations that are dependent on the presence of extracellular Ca*+. These CAMP elevations precede and are correlated with the induction of the acrosome reaction by the zona pellucida, suggesting that CAMP may be a potential participant in the signaling pathway leading to acrosomal exocytosis. It will be of interest to determine whether such intracellular signaling systems are coupled to the sperm G,-like protein, since these second messenger systems are coupled in a receptor-mediated fashion to G proteins in other cell types.
going acrosomal exocytosis (Endo et al. 1987a). The inhibitory effect of this bacterial toxin on this event is strictly confined to acrosomal exocytosis induced by the physiologically relevant component of the zona pellucida, ZP3, whereas exocytosis that occurs either spontaneously in a small population of cells or in response to a nonspecific agent such as a divalent cation ionophore (A-23187) is insensitive to this treatment (Endo et al. 1988). Pertussis toxin similarly inhibits the zona pellucida-induced acrosome reaction in bovine sperm (Florman et al. 1989). If one considers the zona pellucida (ZP3)-induced acrosome reaction as an example of stimulus-secretion coupling that occurs in a receptor-mediated fashion, it would be anticipated that receptor-G-protein interaction subsequently leads to the generation of intracellular second messengers and/or to the modulation of ionic changes within the sperm. Two criteria would have to be met in order to establish the signal transducing function of a G protein in such a system. First, occupation of putative sperm-associated receptors for ZP3 should result in G protein activation in a manner described for other ligand-receptor-G-protein interactions. Second, resultant G protein activation should then modulate intracellular effector systems (e.g., second messengers; ionic changes) that ultimately regulate acrosomal exocytosis. There is evidence to support both of these criteria.
366
??
Although the sperm-associated Gi-like protein appears to play an important intermediary role in the ZP3-induced acrosome reaction, the intracellular signaling systems to which this particular signal-transducing protein is coupled are not known at this time. Moreover, the nature of the intracellular signal pathways activated in response to the sperm-zona pellucida (or ZP3) interaction are only starting to be investigated. Studies in both mouse and bull sperm have revealed that elevations in intracellular Ca2+, as well as intracellular pH, represent some of the earliest responses of sperm incubated with zonae pellucidae (Lee and Storey 1985 and 1989; Florman et al. 1989). Pertussis toxin inhibits the zona pellucida (and ZP3)-induced pH changes in mouse sperm (Endo et al. 1988), as well as the zona pellucida-induced pH and Ca*+ changes in bull sperm (Florman et
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??
Conclusions
and Future
Directions
We are only starting to understand and to appreciate some of the complexities involved in species-specific mammalian gamete recognition and subsequent
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gamete activation ter fertilization.
both prior to and afIt is clear that many of
these events are controlled by the interaction of specific components on the sperm surface with the extracellular matrix surrounding the egg (the zona pellucida). The specificity and uniqueness of these components suggest that these interactions occur in a receptormediated fashion. The identity and characterization of the sperm-associated receptor(s) for the zona pellucida glycoproteins will represent a significant step toward the elucidation of the signal transduction and effector mechanisms controlling sperm functions such as acrosomal exocytosis. The presence of G proteins in mammalian sperm and their important intermediary role in the ZP3-mediated acrosome reaction suggests that regulatory elements involved in Iigand-receptor-G-proteinsecond-messenger systems of somatic cells may regulate sperm function. These similarities will be important when one considers the nature of the ZP3 receptor. For example, does the ZP3 receptor possess domains that are characteristic of many other G proteincoupled receptors? The specific function of the Gi-like protein in the ZP3-induced acrosome reaction is not known at this time, although possible roles in regulating ionic movements, cyclic nucleotide metabolism, and phospholipid turnover are possible candidates for regulation. Studies directed at the biochemical identity of the sperm Gi-like protein may also aid in defining the intracellular effector system(s) that this regulatory protein may modulate. Studies of zona pellucida-mediated signal transduction in mammalian sperm may also contribute to our knowledge of intercellular signaling processes in other systems. For example, sperm cells are an excellent model to study exocytosis. Similarly, spermzona pellucida interaction represents an excellent model for the study of cell interaction with extracellular matrices.
References
Bentley JK, Garbers DL, Domino SE, Noland TD, VanDop C: Spermatozoa contain a guanine nucleotide binding protein ADPribosylated by pertussis toxin. Biochem Biophys Res Commun 1986; 138:728.
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Bleil JD, Wassarman PM: Sperm-egg interactions in the mouse: sequence of events and induction of the acrosome reaction by a zona pellucida glycoprotein. Dev Biol 1983; 951317.
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Regulation of sperm function by nucleotide-binding regulatory proteins (G-proteins). In Haseltine F, First N, eds. Meiotic Inhibition: Molecular Control of Meiosis. New York, Alan Liss, 1988, p 357.
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1986; of a ZP3.
Bleil JD, Greve JM, Wassarman PM: Identification of a secondary sperm receptor in the mouse egg zona pellucida: role in maintenance of binding of acrosome-reacted sperm to eggs. Dev Biol 1988; 1281376. Endo Y, Lee MA, Kopf GS: Evidence for the role of a guanine nucleotide-binding regulatory protein in the zona pellucida-induced mouse sperm acrosome reaction. Dev Biol 1987a; 119:210. Endo Y, Mattei P, Kopf GS, Schultz RM: Effects of a phorbol ester on mouse eggs: dissociation of sperm receptor activity from acrosome reaction-inducing activity of the mouse zona pellucida protein, ZP3. Dev Biol 1987b; 123:574. Endo Y, Lee MA, Kopf GS: Characterization of an islet activating protein-sensitive site in mouse sperm that is involved in the zona pellucida-induced acrosome reaction. Dev Biol 1988; 129:12. Florman HM, Wassarman PM: O-linked oligosaccharides of mouse egg ZP3 account for its sperm receptor activity. Cell 1985; 41:313. Florman HM, Tombes RM, First NL, Babcock DF: An adhesion-associated agonist from the zona pellucida activates G protein-promoted elevations of internal CaZ+ and pH that mediate mammalian sperm acrosomal exocytosis. Dev Biol 1989; 135:133. Garbers DL: Molecular basis of fertilization. Annu Rev Biochem 1989; 58:719. Garty NB, Galiani D, Aharonheim A, et al.: G-proteins in mammalian gametes: an immunocytochemical study. J Cell Sci 1988; 91:21. Gilman AG: G proteins: transducers of receptor-generated signals. Annu Rev Biochem 1987; 56:615.
Kopf GS, Gerton GL: The Mammalian Sperm Acrosome and the Acrosome Reaction. In Wassarman PM, ed. The Biology and Chemistry of Mammalian Fertilization. Boca Raton, FL, CRC Uniscience Series, CRC, 1990 (in press). Kopf GS, Woolkalis MJ, Gerton GL: Evidence for a guanine nucleotide-binding regulatory protein in invertebrate and mammalian sperm: identification by isletactivating protein-catalyzed ADP-ribosylation and immunochemical methods. J Biol Chem 1986; 261:7327. Kopf GS, Endo Y, Mattei P, Kurasawa S, Schultz RM: Egg-induced modifications of In Nuccitelli the murine zona pellucida. RL, Clark WH, Cherr GN, eds. Mechanisms of Egg Activation. New York, Plenum, 1989, p 249. Lee MA, Storey BT: Evidence for plasma membrane impermeability to small ions in acrosome-intact mouse spermatozoa bound to mouse zonae pellucidae, using an aminoacridine fluorescent probe: time course of the zona-induced acrosome reaction monitored by both chlortetracycline and pH probe fluorescence. Biol Reprod 1985; 33:235. Lee MA, Storey BT: Endpoint of first stage of zona pellucida-induced acrosome reaction in mouse spermatozoa characterized by acrosomal H+ and Ca*+ permeability: population and single cell kinetics. Gamete Res 1989; 24:303. Lee MA, Kopf GS, Storey BT: Effects of phorbol esters and a diacylglycerol on the mouse sperm acrosome reaction induced by the zona pellucida. Biol Reprod 1987; 36~617. Leyton L, Saling PM: 95 kD sperm proteins bind ZP3 and serve as tyrosine kinase substrates in response to zona binding. Cell 1989a; 57:123. Leyton L, Saling PM: Evidence that aggregation of mouse sperm receptors by ZP3 triggers the acrosome reaction. J Cell Biol 1989b; 108:2163.
Glassner M, Abisogun AO, Kligman I, Woolkalis MJ, Gerton GL, Kopf GS: Immunocytochemical and biochemical analysis of guanine nucleotide-binding regulatory proteins (G proteins) in mammalian spermatozoa. J Cell Biol 1989; 109:250a.
Moller CC, Wassarman PM: Characterization of a proteinase that cleaves zona pellucida glycoprotein ZP2 following activation of mouse eggs. Dev Biol 1989; 132:103.
Godfrey EW, Dietz ME, Morstad AL, Wallskog PA, Yorde DE: Acetylcholine receptor-aggregating proteins are associated with the extracellular matrix of many tissues in Torpedo. J Cell Biol 1988; 106:1263.
Noland TD, Garbers DL, Kopf GS: An elevation in cyclic AMP concentration precedes the zona pellucida-induced acrosome reaction of mouse spermatozoa. Biol Reprod 1988; 38(suppl):94.
Katz DF, Drobnis EZ, Overstreet JW: Factors regulating mammalian sperm migration
Ringuette Sobieski
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Co., Inc.,
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MJ, Chamberlin ME, Baur AW, DA, Dean J: Molecular analysis of
367
cDNA coding for ZP3, a sperm binding protein of the mouse zona pellucida. De\; Biol
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127:287.
Saling PM: Mammalian sperm interaction with extracellular matrices of the egg. Ox Rev Reprod Biol 1989; I 1:339. Saling PM, Sowinski J, Storey BT: An ultrastructural study of epididymal mouse spermatozoa binding to zonae pellucidae in vitro: sequential relationship to the acrosome reaction. J Exp Zoo1 1979; 209:229.
TEM
FOR
ciated
Vazquez MH, Phillips DM, Wassarman PM: Interaction of mouse sperm with purified sperm receptors covalently linked to silica beads. J Cell Sci 1989; 92:713. Wassarman PM: Early fertilization. Annu 3:lOY.
MW, Kopf
events in mammalian Rev Cell Biol 1987;
1989;
the lona
pcllu-
109:251a.
Yarden Y, Schlessinger J: Epidcrmal growth factor induces rapid, reversible aggregation of the purified epidermal growth factor receptor. Biochemistry 1987; 26:1443.
of a G prosperm by an egg-asso-
GS: Activation
tein in mammalian
matrix.
J Cell Biol
Yanagimachi R: Mammalian tertilization. In Knobil E, Neil1 JD, et al., eds. The Physiology of Reproduction. New York, Raven, 1988, p 13.5.
Wassarman PM: Zona pellucida glycoproteins. Annu Rev Biochem 1988; 57:415. Wilde
extracellular
cida.
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Amsterdam A, Naor treatment with a gohormone antagonist of male rats. Biol Re-
Huhtaniemi IT, Yamamoto M, Ranta T, Jalkanen J, Jaffe RB: Follicle-stimulating hormone receptors appear earlier in the primate fetal testis than in the ovary. J Clin Endocrinol Metab 1987; 65:1210. Hutson JM, Metcalfe SA, MacLaughlin et al.: Mullerian inhibiting substance. Burger H, de Kretser D, eds. The Testis, ed. New York, Raven, 1989, p 143.
DT, In 2nd
Jost A, Vigier B, Prepin J, Perchellet JP: Studies on sex differentiation in mammals. Recent Prog Horm Res 1973; 29: 1. Kaplan SL, Grumbach MM, Aubert ML: The ontogenesis of pituitary hormones and hypothalamic factors in the human fetus: maturation of central nervous system regulation of anterior pituitary function. Recent Prog Horm Res 1976; 32:161. Kolho K-L, Nikula H, Huhtaniemi I: Sexual maturation of male rats treated postnatally with a gonadotrophin-releasing hormone antagonist. J Endocrinol 1988; 116:241. Meidan R, Lim P, McAllister JM, Hsueh AJW: Hormonal regulation of androgen biosynthesis by primary cultures of testicular cells from neonatal rats. Endocrinology 1985; 116:2473. Mulchaney JJ, DiBlasio AM, Martin MC, Blumenfeld Z, Jaffe RB: Hormone production and peptide regulation of the human fetal pituitary gland. Endocr Rev 1987; 8:406. Pakarinen P, Huhtaniemi I: Gonadal and sex steroid feedback regulation of pituitary gonadotropin mRNA levels and secretion in neonatal male and female rats. J Mol Endocrinol 1989; 3:139. Pelliniemi LJ, Dym M: The fetal gonad and sex differentiation. In Tulchinsky D, Ryan KJ, eds. Maternal-Fetal Endocrinology. Philadelphia, WB Saunders, 1980, p 252. Reyes FI, Winter JSD, Faiman C: Endocrinology of the fetal testis. In Burger H, de Kretser D, eds. The Testis, 2nd ed. New York, Raven, 1989, p 119. Rivier C, Cajander S, Vaughan J, Hsueh AJW, Vale W: Age-dependent changes in physiological action, content, and immunostaining of inhibin in male rats. Endocrinology 1988; 123:120.
362
Schwanzel-Fukuda M, Pfaff DW: Origin teinizing hormone-releasing hormone rons. Nature 1989; 338:161.
01 luneu-
Takagi S, Yoshida T, Tsubata K, ct al.: Sex differences in fetal gonadotropins and androgens. J Steroid Biochem 1977; 8:609. Tapanainen J, Koivisto M, Vihko R, Huhtaniemi I: Enhanced activity of the pituitary-gonadal axis in premature human infants. J Clin Endocrinol Metab 1981; 52:235. Vigier B, Forest MG, Eychenne B, et al.: Antimtillerian hormone produced endocrine sex reversal of fetal ovaries. Proc Nat1 Acad Sci USA 1989; 86:3684. Voutilainen R, Eramaa M: Hormonally regulated inhibin gene expression in human fetal adrenals and testes (Abstract). Third Joint Meeting of the ESPE/LWPES, Jerusalem, October 1989, no. 57.
Warren
DW: Development
ol the inhibitor.!
guanine nucleotide-binding rcgulator~ protein in the rat testis. Biol Reprod 1989; 40: 1208. Warren DW, Dufau ML, Catt KJ: Hormonal regulation of gonadotropin receptors and steroidogenesis in cultured fetal rat tcstcs. Science 1982; 218:375. Warren DW, Pasupuleti V, Platler B, Lu Y, Horton R: Tumor necrosis factor and interleukin-1 inhibit testosterone secretion in cultured fetal rat testis cells but stimulate testosterone production in adult testis cells (Abstract). The Endocrine Society Annual Meeting, Seattle, WA, June 1989, no. 1159. Word RA, George FW, Wilson JD, Carr BR: Testosterone synthesis and adenylate cyclase activity in the early human fetal testis appear to be independent of human chorionic gonadotropin control. J Clin EndocriTEM nol Metab 1989; 69:204.
Signal Transduction Processes Leading to Acrosomal Exocytosis in Mammalian Spermatozoa Gregory S. Kopf and Mary W. Wilde Many components of intercellular signaling involved in species-specific sperm binding to the egg’s extracellular matrix, the zona pellucida, and the induction of acrosomal exocytosis, an absolute prerequisite to successful fertilization, have properties similar to intercellular signaling mechanisms controlling somatic cell function. Sperm-associated receptors for zona pellucida glycoproteins have been postulated to serve as the initial components of signal transduction cascades leading to the stimulation of cellular effector systems that modulate sperm function. Such receptor-effector systems appear to be coupled through guanine nucleotide-binding regulatory proteins (G proteins).
??
ment, These activation
Background
Intercellular communication between male and female gametes of all species leads to the subsequent cellular activation of the respective gametes, a process essential to both successful fertilization and initiation of early embryo developGregory S. Kopf and Mary W. Wilde are at the Division of Reproductive Biology, Department of Obstetrics and Gynecology, University of Pennsylvania School of Medicine, Philadelphia, PA 19104-6080, USA.
0 1990, Elsevirr Science Publishing
processes
can be
either egg-induced categorized as sperm activation events or sperm-induced egg activation events. Sperm-female reproductive tract interaction and sperm-egg interaction prior to spermegg fusion occur at different sites in vivo and are the culmination of a number of integrated processes designed to deliver sperm with optimal fertilizing potential to the site of fertilization, e.g., the ampullary region of the oviduct (Katz et al. 1989).
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Sperm from all invertebrate
and ver-
tebrate stimuli
species respond to external in their environment with
changes
in cellular
function
that
a
f$?oO+_ OaQ,Q
PS
Cumulus Oopohorus
are
prerequisites to successful sperm-egg interaction and fertilization (Kopf 1988; Kopf and Gerton 1990). Such stimuli are elaborated by the egg itself, the cellular investments surrounding the egg, and/or factors associated with the female reproductive tract. In mammals, these stimuli may be responsible for modulating such functions as sperm motility and metabolism (essential for the delivery of sperm to the site of fertil-
Cortical Granule Plasma Membrane
y WZona
p
Perivitelline Space Pellucida
ization), capacitation (a poorly understood process that confers upon the mammalian sperm the ability to fertilize an egg), and the acrosome reaction (an exocytotic process that is essential for the sperm to penetrate the zona pellucida of the egg). The molecular identities of these stimuli in various species are only starting to be elucidated. At present, most of the information regarding ligand-effector systems regulating mammalian sperm function prior to fertilization has been discerned from studies of those processes regulating sperm-zona pellucida interaction and acrosomal exocytosis in the mouse. Consequently, this review will focus, for the most part, on studies carried out in this species. Studies of this system have revealed that sperm perceive and process signals from the zona pellucida in a fashion analogous to the way somatic cell function is regulated by humoral, autocrine, or paracrine factors. A more comprehensive treatment of this subject can be found in additional reviews (Wassarman 1987 and 1988; Yanagimachi 1988; Garbers 1989; Saling 1989; Kopf and Gerton 1990).
?? The
Zona Pellucida, an Egg-Associated Extracellular Matrix, Functions as a Regulatory Ligand for Sperm
The zona pellucida is an oocyte-derived acellular coat that surrounds the egg and can be considered an extracellular matrix (Figure 1). This structure plays an important role both prior to and after fertilization, as well as during subsequent preimplantation development. The zonae pellucidae from all mammals appear to be composed of 2-4 glycoproteins, the number of which appears to be species dependent (Was-
TEA4 SeptemberlOctober
Figure 1. Diagrammatic representation of the events comprising sperm-zona pellucida interaction in the mouse both prior to (a and b) and after (c and d) sperm-egg fusion. (A) An ovulated egg surrounded by the zona pellucida which is synthesized by the growing oocyte. The zona pellucida-enclosed egg is surrounded by cells comprising the cumuZus oophorus. Cortical granules are shown immediately beneath the plasma membrane of the egg. These cortical granules have not undergone exocytosis, as shown in the figure by the filled circles. Both acrosome-intact (filled heads) and acrosome-reacted (open heads) sperm are present. Acrosome-reacted sperm do not bind to the zona pellucida, as shown by the presence of theX. PB, polar body. (b) The sequence of events of sperm binding to the zona pellucida of the ovulated egg. Acrosome-intact sperm establish primary binding to the zona pellucida (via ZP3 and sperm-associated receptors for ZP3) (I) and, having undergone acrosomal exocytosis, establish secondary binding interactions with the zona pellucida (via ZP2 and spermassociated receptors for ZP2) (2). Once the acrosome reaction has occurred, sperm are then able to penetrate the zona pellucida (3). (c) Fusion of the egg plasma membrane and the plasma membrane of acrosome-reacted sperm results in an elevation of intracellular free Ca2+within the egg, the subsequent exocytosis of the cortical granules (as shown by the open circles), and the intercalation of the cortical granule membrane into the egg plasma membrane (as shown by the absence of the circles). This Ca2+-induced exocytosis proceeds as a centripetal wave over the entire egg (double-headed arrow) originating from the point of sperm-egg fusion. Accompanying cortical granule exocytosis is a modification of the zona pellucida such that the biochemical and biological properties of this extracellular matrix are now altered (as shown by the stippling of the zona pellucida). (d) As a consequence of these egg-induced modifications of the zona pellucida, the ability of acrosome-intact sperm to bind to ZP3 (I) and the ability of bound acrosome-intact sperm to undergo ZP3-induced acrosomal exocytosis (2) is lost (X) due to the conversion of ZP3 to ZP3t. Those acrosome-reacted sperm that are bound to the zona pellucida (3) lose their ability to penetrate the zona since they cannot establish secondary binding interactions with ZP2t (X). These egg-induced modifications of the zona pellucida constitute the zona pellucida block to polyspermy. This figure is for illustrative purposes only and is not drawn to scale.
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sarman
1988;
Kopf and Gerton
1990).
Sperm-egg recognition and binding, sperm activation (i.e., acrosomal exocytosis), and an egg-induced block to polyspermy in the mouse (and in a growing number of other species) all appear to be mediated by this extracellular matrix. The zona pellucida of the mouse egg is composed of three sulfated glycoproteins designated as ZPl, ZP2, and ZP3, which are present in molar ratios of 1: 10: 10, respectively (Wassarman 1988). These glycoproteins are truly egg-associated products, as they are synthesized and secreted throughout the period of oocyte growth. ZPl has been proposed to maintain the three-dimensional structure of the zona pellucida by virtue of its ability to cross-link filaments composed of repeating ZP2iZP3 heterodimers. ZP2 may function to mediate the binding of acrosome-reacted sperm to the zona pellucida. Upon fertilization, the egg effects a modification of ZP2 to a form called ZP2t by the action of a protease(s), which is most likely from the egg as a consequence
secreted of corti-
cal granule exocytosis (Moller and Wassarman 1989). The biological consequence of the ZP2 to ZP2t conversion is that ZP2t no longer retains the capacity to bind acrosome-reacted sperm. ZP3 accounts for both the sperm-binding and the acrosome reaction-inducing activities of the zona pellucida of unfertilized eggs. Fertilization results in a loss of both of these activities of the ZP3 molecule, although it is not clear at present whether these modifications occur in response to the same protease that modifies ZP2. The properties of these three zona pellucida glycoproteins from both unfertilized and fertilized eggs provide a framework with which to formulate a model to explain the interaction of sperm with this specialized extracellular matrix (Figure 1). Since only acrosome-intact mouse sperm bind to the zona pellucida (Saling et al. 1979), specific receptor(s) for ZP3 on the plasma membrane overlying the sperm acrosome would mediate sperm binding and induction of the acrosome reaction. Subsequent to ZP3-mediated binding and acrosomal exocytosis, acrosome-reacted sperm would then maintain their interaction with the zona pellucida through secondary interactions with sperm-associated receptor(s) for ZP2 lo-
364
cated
on the
inner
acrosomal
mem-
fact that its site and mode 01 action arc’
brane. Upon penetration of the zona pellucida by acrosome-reacted sperm, these cells traverse the perivitelline space, bind to, and then fuse with the plasma membrane of the egg. Spermegg fusion occurs at discrete regions in
restricted to events associated with fcrtilization and preimplantation development. Little amino acid sequence homology exists between ZP3 and an!’ other known proteins or glycoprotcins thus far examined (Ringuette ct al.
the postacrosomal region of the sperm head (Yanagimachi 1988). Subsequent to sperm-egg fusion, the egg undergoes the cortical granule reaction, which results in the release of cortical granule associated enzymes (Wassarman 1987). These enzymes convert ZP2 to ZP2r and
1988), suggesting that it serves a unique function. ZP3 is an immobilized ligand that acts at a localized and restricted distance, owing to its presence in the zona pellucida as a ZPZlZP3 heterodimer cross-linked in an orderly fashion by ZPl (Wassarman 1988). ZP3 is biologically potent since both the spermbinding and acrosome reaction-inducing activities of this molecule are observed in the nanomolar range (Florman and Wassarman 1985; Bleil and
modify ZP3 (to ZP3t), so that acrosome-intact sperm no longer bind to the zona pellucida (through ZP3) and acrosome-reacted sperm that are bound to the zona pellucida (through ZP2) no longer interact and penetrate this extracellular matrix, since they are unable to establish secondary binding interactions with ZP2t. Such egg-induced modifications of the zona pellucida constitute the zona pellucida block to polyspermy. Inherent in such a model is the highly specific and coordinated nature of the interactions of acrosome-intact and acrosome-reacted sperm with ZP3 and ZP2, respectively. These interactions would presumably be mediated by specific sperm-associated receptors for these zona pellucida glycoproteins.
Several observations support the contention that sperm-zona pellucida interaction in the mouse (and probably in other mammalian species) represents a process akin to ligand-receptor interactions in somatic cells. ZP3, for example, possesses a variety of properties that make it ideally suited as a ligand to mediate both initial steps of sperm-egg interaction proper (e.g., sperm binding) and subsequent sperm activation (e.g., induction of acrosomal exocytosis). ZP3 is an egg-associated product that is synthesized only by the growing oocyte (Wassarman 1988), consistent with the
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Wassarman 1986). Acrosome-intact mouse sperm appear to possess complementary binding sites (receptors?) for ZP3 that are localized to the acrosomal cap region and are present in numbers (lO,OOO-50,000 binding sites/cell) similar to those observed for receptor numbers present in many hormone-responsive cells (Bleil and Wassarman 1986). Acrosome-intact mouse sperm bind to ZP3 immobilized onto silica beads via the sperm head (Vazquez et al. 1989), further supporting the idea that the sperm-associated ZP3 binding moiety(ies) is associated with the plasma membrane overlying the acrosomal region, that region of initial contact where sperm-zona pellucida interaction first occurs. Therefore, although conserved at the genomic level in a number of species (Ringuette et al. 1988), ZP3 subserves extremely specific functions as a component of the egg-associated extracellular matrix known as the zona pellucida (Wassarman 1987 and 1988). Similarly, the specific binding of ZP2 to the inner acrosomal membrane of acrosome-reacted, but not acrosome-intact, mouse sperm suggests that this membrane may possess specific receptors for this particular zona pellucida glycoprotein (Bleil et al. 1988).
??
Sperm-Associated Zona Pellucida
Receptors
for the
Although sea urchin sperm remain the sole experimental system in which receptor-mediated signal transduction has been unequivocally demonstrated (Garbers 1989), studies in the mammal
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are beginning to yield information regarding the nature of sperm-associated receptors for components of the zona pellucida. Two different approaches have been utilized to describe putative sperm-associated receptors for the zona pellucida. The first, and more direct, approach involves the elucidation of components of the sperm surface that interact directly with the individual zona pellucida glycoproteins. Utilizing this approach, Bleil and Wassarman examined the specific binding of 1251-ZP3 to mouse sperm by whole mount autoradiography and demonstrated that binding is associated solely with the plasma membrane overlying the acrosomal region of the sperm and is not observed on acrosome-reacted sperm (Bleil and Wassarman 1986). Binding of this radiolabeled ligand is competed by unlabeled ZP3 but not ZP2, and ZP3 binding does not occur on somatic cells. Similar localize
techniques have been used to binding sites for ZP2 to the in-
ner acrosomal membranes of acrosomereacted, but not acrosome-intact, sperm (Bleil et al. 1988). Although the methodology utilized in these studies has limitations with regard to the characterization of the sperm-associated moiety(ies) involved in ZP3 and ZP2 binding, these results suggest that specific sperm-associated binding sites for these ligands exist in discrete domains of this highly differentiated cell. Reinvestigators demoncently, these strated
that
either
purified
ZP3
or
ZP3 possessing glycopeptides of sperm-binding activity can be specifically cross-linked to a M, = 56,000 protein of acrosome-intact mouse sperm (Bleil and Wassarman 1989). This protein interacts specifically with ZP3, but not ZP2, affinity columns. Whole mount autoradiography utilizing radiolabeled, cross-linked ZP3 glycopeptides that possess sperm-binding activity demonstrates a localization to the head region of acrosome-intact sperm. This experimental approach may provide a great deal of promise with regard to establishing the molecular identity of such sperm-associated receptors for zona pellucida glycoproteins. Recently Leyton and Saling (1989a) demonstrated that antiphosphotyrosine antibodies react with mouse sperm plasma membrane proteins of M, = 52,000,75,000, and 95,000, and that this positive immunoreactivity is localized
TEM SeptemberiOctobev
0
to the acrosomal
region
of the sperm
head. 1251-ZP3 binds, on nitrocellulose blots, to a M, = 95,000 protein, and these workers conclude that this M, = 95,000 protein is the protein that also reacts with the antiphosphotyrosine antibodies. Although it is suggested that this M, = 95,000 protein may be a receptor for ZP3 that acts either as a substrate for tyrosine phosphorylation or possesses tyrosine kinase activity itself, additional studies will be required to clearly demonstrate the specificity of the ZP3 binding activity of this protein. The second, more indirect, approach involves the delineation of sperm-associated binding sites for the zona pellucida glycoproteins by examining the ability of specific agents to interfere with sperm-zona pellucida interaction. Sperm surface-associated protease inhibitor-sensitive sites, galactosyltransferase activity, and fucosyltransferase activity have all been implicated in the binding of sperm to the zona pellucida, presumably through ZP3 (Saling 1989; Kopf and Gerton 1990, and references therein). However, the relationship of these sites to one another and to those sites defined by ZP3 binding (as described above) is not clear at this time. Although little is known regarding the identity of the sperm-associated receptor for ZP3, recent studies have shed some light on the potential nature of the interaction of the ZP3 molecule with the sperm surface to mediate both sperm binding and acrosomal exocytosis. Studies from three independent laboratories using different approaches have provided evidence that the interaction of ZP3 with the sperm surface may occur in a multivalent (or cooperative) fashion, and that multiple interactions followed by possible receptor aggregation may ultimately lead to signal transduction and acrosomal exocytosis (Bleil and Wassarman 1983; Endo et al. 1987b; Kopf et al. 1989; Leyton and Saling 1989b). The idea that receptor aggregation is required to elicit ligand-dependent signal transduction and a cellular response is not unique (Yarden and Schlessinger 1987). It has been recently demonstrated that extracellular matrices from a variety of tissues contain domains that aggregate acetylcholine receptors (Godfrey et al. 1988). It is certainly possible that the zona pellucida, itself an extracellular matrix, also
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possesses the ability to aggregate tors on the sperm surface.
??
recep-
Signal Transduction Mechanisms Mediating Sperm-Zona Pellucida Interactions
The unique structure of ZP3, its biological potency, and the probable existence on the plasma membrane of acrosomeintact sperm of a complementary ZP3 receptor(s) satisfy a number of criteria required for a means to control specific cell-cell recognition events in a receptor-mediated fashion. The consequence of these specific interactions is that sperm undergo an exocytotic event, the acrosome reaction, which is an absolute prerequisite to successful penetration of the zona pellucida and subsequent fusion of the sperm and egg plasma membranes. The mechanism by which ZP3 effects informational flow across the sperm plasma membrane as a consequence of binding to its putative receptor has been recently examined.
As in many somatic cells, guanine nucleotide-binding regulatory proteins (G proteins) appear to play a critical role(s) as signal-transducing elements coupling zona pellucida-sperm interaction to the generation of intracellular second messengers that ultimately regulate acrosomal exocytosis. Spermatozoa from all species studied thus far (invertebrate as well as mammalian) contain G proteins, as assessed by bacterial toxin-catalyzed ADP-ribosylation, GTPy3%-binding, and immunoblotting techniques (Kopf et al. 1986; Bentley et al. 1986). Mammalian sperm contain a G protein with properties similar to Gi; the exact nature of
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365
al. 1989), indicating
that a G,-like pro-
this G protein has yet to be determined. Consistent with observations made by other investigators, sperm do not appear to contain a G protein with properties similar to G, (Kopf and Gerton 1990). Immunocytochemical studies have demonstrated that G proteins in bovine (Garty et al. 1988) as well as mouse and guinea pig (Glassner et al.
Recently, it has been demonstrated that solubilized zonae pellucidae from mouse eggs stimulate a high-affinity GTPase activity associated with the G,like protein of mouse sperm (Wilde and Kopf 1989). Ligand-dependent stimulation of this activity reflects the termination of a G protein activation cycle (Gilman 1987). Zonae pellucidae stimu-
1989) sperm are present in the acrosoma1 region of these highly differentiated cells. The physiological role of the sperm G,-like protein in the zona pellucidamediated acrosome reaction has thus far been examined in both the murine and bovine systems. Functional inactivation of the mouse sperm Gi-like protein by pertussis toxin treatment of the
late this activity in a concentration-dependent manner and the stimulatory efof fect occurs as a consequence increased GTP turnover. These data support the idea that a component(s) of the zona pellucida (perhaps ZP3) binds to a sperm-associated receptor that is G protein coupled, and that receptor-mediated G protein activation results in the activation of specific intracellular
tein may regulate such ionic changes in response to this egg-associated cxtracellular matrix. Studies from other laboratories have suggested that alterations in phospholipid metabolism and/or cyclic nucleotide metabolism may play important intermediary roles in the sperm acrosome reaction (Kopf and Gerton 1990). Lee et al. (1987) demonstrated that biologically active phorbol diesters and diacylglycerols alter the kinetics of the zona pellucida-mediated acrosome reaction in mouse sperm, thus providing evidence for the role of protein kinase C in regulating this exocytotic event. The products of phospholipase C turnover (e.g., inositol 1,4,5trisphosphate and
cells does not affect the ability of sperm to interact with the zona pellucida, but inhibits the bound sperm from under-
effector systems required for the induction of acrosomal exocytosis.
1,2 diacylglycerol), as well as the role of other phospholipases (A* or D), have not
Intracellular Effecters Modulating Zona Pellucida-Mediated Acrosomal Exocytosis
yet been examined in sperm challenged with zonae pellucidae or ZP3. Noland et al. (1988) have reported that solubilized zonae pellucidae from mouse eggs induce transient elevations in mouse sperm CAMP concentrations that are dependent on the presence of extracellular Ca*+. These CAMP elevations precede and are correlated with the induction of the acrosome reaction by the zona pellucida, suggesting that CAMP may be a potential participant in the signaling pathway leading to acrosomal exocytosis. It will be of interest to determine whether such intracellular signaling systems are coupled to the sperm G,-like protein, since these second messenger systems are coupled in a receptor-mediated fashion to G proteins in other cell types.
going acrosomal exocytosis (Endo et al. 1987a). The inhibitory effect of this bacterial toxin on this event is strictly confined to acrosomal exocytosis induced by the physiologically relevant component of the zona pellucida, ZP3, whereas exocytosis that occurs either spontaneously in a small population of cells or in response to a nonspecific agent such as a divalent cation ionophore (A-23187) is insensitive to this treatment (Endo et al. 1988). Pertussis toxin similarly inhibits the zona pellucida-induced acrosome reaction in bovine sperm (Florman et al. 1989). If one considers the zona pellucida (ZP3)-induced acrosome reaction as an example of stimulus-secretion coupling that occurs in a receptor-mediated fashion, it would be anticipated that receptor-G-protein interaction subsequently leads to the generation of intracellular second messengers and/or to the modulation of ionic changes within the sperm. Two criteria would have to be met in order to establish the signal transducing function of a G protein in such a system. First, occupation of putative sperm-associated receptors for ZP3 should result in G protein activation in a manner described for other ligand-receptor-G-protein interactions. Second, resultant G protein activation should then modulate intracellular effector systems (e.g., second messengers; ionic changes) that ultimately regulate acrosomal exocytosis. There is evidence to support both of these criteria.
366
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Although the sperm-associated Gi-like protein appears to play an important intermediary role in the ZP3-induced acrosome reaction, the intracellular signaling systems to which this particular signal-transducing protein is coupled are not known at this time. Moreover, the nature of the intracellular signal pathways activated in response to the sperm-zona pellucida (or ZP3) interaction are only starting to be investigated. Studies in both mouse and bull sperm have revealed that elevations in intracellular Ca2+, as well as intracellular pH, represent some of the earliest responses of sperm incubated with zonae pellucidae (Lee and Storey 1985 and 1989; Florman et al. 1989). Pertussis toxin inhibits the zona pellucida (and ZP3)-induced pH changes in mouse sperm (Endo et al. 1988), as well as the zona pellucida-induced pH and Ca*+ changes in bull sperm (Florman et
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gamete activation ter fertilization.
both prior to and afIt is clear that many of
these events are controlled by the interaction of specific components on the sperm surface with the extracellular matrix surrounding the egg (the zona pellucida). The specificity and uniqueness of these components suggest that these interactions occur in a receptormediated fashion. The identity and characterization of the sperm-associated receptor(s) for the zona pellucida glycoproteins will represent a significant step toward the elucidation of the signal transduction and effector mechanisms controlling sperm functions such as acrosomal exocytosis. The presence of G proteins in mammalian sperm and their important intermediary role in the ZP3-mediated acrosome reaction suggests that regulatory elements involved in Iigand-receptor-G-proteinsecond-messenger systems of somatic cells may regulate sperm function. These similarities will be important when one considers the nature of the ZP3 receptor. For example, does the ZP3 receptor possess domains that are characteristic of many other G proteincoupled receptors? The specific function of the Gi-like protein in the ZP3-induced acrosome reaction is not known at this time, although possible roles in regulating ionic movements, cyclic nucleotide metabolism, and phospholipid turnover are possible candidates for regulation. Studies directed at the biochemical identity of the sperm Gi-like protein may also aid in defining the intracellular effector system(s) that this regulatory protein may modulate. Studies of zona pellucida-mediated signal transduction in mammalian sperm may also contribute to our knowledge of intercellular signaling processes in other systems. For example, sperm cells are an excellent model to study exocytosis. Similarly, spermzona pellucida interaction represents an excellent model for the study of cell interaction with extracellular matrices.
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Bleil JD, Greve JM, Wassarman PM: Identification of a secondary sperm receptor in the mouse egg zona pellucida: role in maintenance of binding of acrosome-reacted sperm to eggs. Dev Biol 1988; 1281376. Endo Y, Lee MA, Kopf GS: Evidence for the role of a guanine nucleotide-binding regulatory protein in the zona pellucida-induced mouse sperm acrosome reaction. Dev Biol 1987a; 119:210. Endo Y, Mattei P, Kopf GS, Schultz RM: Effects of a phorbol ester on mouse eggs: dissociation of sperm receptor activity from acrosome reaction-inducing activity of the mouse zona pellucida protein, ZP3. Dev Biol 1987b; 123:574. Endo Y, Lee MA, Kopf GS: Characterization of an islet activating protein-sensitive site in mouse sperm that is involved in the zona pellucida-induced acrosome reaction. Dev Biol 1988; 129:12. Florman HM, Wassarman PM: O-linked oligosaccharides of mouse egg ZP3 account for its sperm receptor activity. Cell 1985; 41:313. Florman HM, Tombes RM, First NL, Babcock DF: An adhesion-associated agonist from the zona pellucida activates G protein-promoted elevations of internal CaZ+ and pH that mediate mammalian sperm acrosomal exocytosis. Dev Biol 1989; 135:133. Garbers DL: Molecular basis of fertilization. Annu Rev Biochem 1989; 58:719. Garty NB, Galiani D, Aharonheim A, et al.: G-proteins in mammalian gametes: an immunocytochemical study. J Cell Sci 1988; 91:21. Gilman AG: G proteins: transducers of receptor-generated signals. Annu Rev Biochem 1987; 56:615.
Kopf GS, Gerton GL: The Mammalian Sperm Acrosome and the Acrosome Reaction. In Wassarman PM, ed. The Biology and Chemistry of Mammalian Fertilization. Boca Raton, FL, CRC Uniscience Series, CRC, 1990 (in press). Kopf GS, Woolkalis MJ, Gerton GL: Evidence for a guanine nucleotide-binding regulatory protein in invertebrate and mammalian sperm: identification by isletactivating protein-catalyzed ADP-ribosylation and immunochemical methods. J Biol Chem 1986; 261:7327. Kopf GS, Endo Y, Mattei P, Kurasawa S, Schultz RM: Egg-induced modifications of In Nuccitelli the murine zona pellucida. RL, Clark WH, Cherr GN, eds. Mechanisms of Egg Activation. New York, Plenum, 1989, p 249. Lee MA, Storey BT: Evidence for plasma membrane impermeability to small ions in acrosome-intact mouse spermatozoa bound to mouse zonae pellucidae, using an aminoacridine fluorescent probe: time course of the zona-induced acrosome reaction monitored by both chlortetracycline and pH probe fluorescence. Biol Reprod 1985; 33:235. Lee MA, Storey BT: Endpoint of first stage of zona pellucida-induced acrosome reaction in mouse spermatozoa characterized by acrosomal H+ and Ca*+ permeability: population and single cell kinetics. Gamete Res 1989; 24:303. Lee MA, Kopf GS, Storey BT: Effects of phorbol esters and a diacylglycerol on the mouse sperm acrosome reaction induced by the zona pellucida. Biol Reprod 1987; 36~617. Leyton L, Saling PM: 95 kD sperm proteins bind ZP3 and serve as tyrosine kinase substrates in response to zona binding. Cell 1989a; 57:123. Leyton L, Saling PM: Evidence that aggregation of mouse sperm receptors by ZP3 triggers the acrosome reaction. J Cell Biol 1989b; 108:2163.
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