And you thought you knew all there was to know about fertilization… The enormous population growth and high incidence of infertility in humans demand a fuller appreciation of the mechanisms that govern fertilization. But although the principle cellular events of fertilization have been known for many years, we are frequently reminded how little we really understand of the underlying biochemical mechanisms. Looks can be deceiving. The remarkable transformation of round, diploid spermatogonia to highly polarized haploid spermatozoa occurs in the seminiferous tubules of the testis. Further biochemical differentiation occurs as sperm travel through the epididymis. Although epididymal sperm appear morphologically mature, they are unable to fertilize an egg unless they undergo capacitation - a complex and poorly understood process that occurs in the female reproductive tract. Capacitation is initiated by bicarbonate and other uterine effectors that modulate sperm intracellular second messengers, notably cAMP. Elevated cAMP levels lead to changes in sperm motility and reorganization of membrane components, changes that are essential for sperm binding and penetration of the egg coat, as well as for subsequent fusion with the egg plasma membrane. Why so many sperm? Although the ejaculate contains millions of sperm, only one is needed to produce a viable offspring. In fact, more than one fertilizing sperm is lethal. In species such as marine invertebrates that deposit their gametes into open seawater, it seems reasonable that enormous numbers of sperm are necessary to counter the effects of dilution and fluid dynamics. Egg secretions serve as sperm chemoattractants to further increase the likelihood of fertilization. In internally fertilizing mammals, excess sperm are held in storage crypts within the female reproductive tract, enabling the timed release of additional aliquots of capacitated sperm, each of which has an independent opportunity to fertilize the egg. As in marine invertebrates, secretions from the ovulated mammalian egg may serve to attract sperm up the oviduct. So many barriers, so little time. Given the importance of fertilization to the continuation of the species, one would think that nature would make it easy for gametes to bind and fuse with one another. But once the sperm has finally reached the vicinity of the ovulated egg, it must overcome a variety of barriers that impede its access to the egg. First, the cumulus layer. Sperm initially confront the cumulus cells that nursed the egg during oogenesis and which remain adherent to the egg coat at ovulation. Sperm employ a variety of strategies, both enzymatic and mechanical, to clear a path through the cumulus cells. It is unclear, though, whether the pioneering sperm is the one to fertilize the egg, or a sacrificial sperm that clears a path for subsequent sperm. Then, the egg coat. After they trek across the cumulus layer, sperm encounter the zona pellucida, a thick extracellular coat that protects the egg. It is here that species-specific gamete binding occurs (Figure 1). This not only insures union of homologous gametes, but also serves as a point of speciation, as naturally occurring polymorphisms in the relevant receptors may result in new binding specificities that become segregated from the original parental species.

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Figure 1. Mouse sperm labeled with two fluorescent dyes bound to the egg coat.

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The structure of the mammalian zona pellucida is relatively simple, being composed of only three glycoproteins. One of these, ZP3, has carbohydrate groups thought to provide binding sites for protein receptors on the sperm surface. The best-studied candidate receptor is β1,4-galactosyl-transferase, a carbohydrate-binding protein usually confined to intracellular membranes, but which also functions as a cell surface receptor on selected cell types. Such protein-carbohydrate complementarity appears to be a generalized mechanism to facilitate sperm-egg binding in a wide range of species. Things are never so simple. ZP3 carbohydrates were thought to be sufficient to account for species-specific sperm-egg binding, but recent studies cast doubt on whether they are the primary site of sperm binding. Sperm with reduced ability to bind ZP3 still adhere to the egg coat, despite their poor ability to penetrate the coat. Similarly, mouse sperm still bind and fertilize eggs in which murine ZP3 has been replaced with human ZP3. These and other studies indicate that sperm-egg binding likely involves a multiplicity of interacting receptors. Some have recently been identified, such as SED1, a multidomain sperm protein that mediates binding to the egg coat, and a novel component of the egg coat that facilitates sperm adhesion. This reliance upon multiple cell adhesion mechanisms is reminiscent of lymphocyte binding to the vascular endothelium. Cells that exist in body fluids and are subject to high shear forces, such as lymphocytes and gametes, may require the concerted action of low-affinity and high-affinity receptor-ligand interactions to ensure successful binding. Still not there. Although we do not yet appreciate the full repertoire of receptors that mediate sperm binding to the egg coat, such binding leads to activation of sperm heterotrimeric G-proteins and selective ion channels that trigger exocytosis of the acrosome. Hydrolytic enzymes released from the acrosome vesicle digest a path through the egg coat; however, the precise acrosomal enzymes responsible for sperm penetration remain obscure. Finally, the egg membrane. After traversing through the cumulus layer and the zona pellucida, the pioneering sperm finds itself facing the egg plasma membrane - the last barrier to fertilization. A novel class of cell adhesion molecules, the ADAMs, some of which are localized on sperm, were initially thought to bind integrin receptors on the egg membrane, thus facilitating membrane binding and fusion. But as before, nothing is as straightforward as it initially appeared, and the contribution of sperm ADAMS and their egg integrin receptors is still actively debated. Recent evidence has implicated a role for tetraspanin and GPI-linked proteins of the egg membrane, as well as a cysteine-rich secretory protein on sperm, as mediators of gamete fusion. Only one sperm please. One responsibility of the fertilizing sperm is to prevent entry of additional sperm, which would be lethal to the new zygote. This block to polyspermy is achieved by changes in the egg membrane that prevent entry of additional sperm, and by enzymatic modifications to the zona pellucida by exudates of egg cortical vesicles that are released following fertilization. Ultimately, the fertilizing sperm activates development via receptor mediated signal transduction cascades and/or by introduction of critical sperm effectors that initiate metabolic activation.

And you thought you knew all there was to know about fertilization… The enormous population growth and high incidence of infertility in humans demand a fuller appreciation of the mechanisms that govern fertilization. But although the principle cellular events of fertilization have been known for many years, we are frequently reminded how little we really understand of the underlying biochemical mechanisms. Looks can be deceiving. The remarkable transformation of round, diploid spermatogonia to highly polarized haploid spermatozoa occurs in the seminiferous tubules of the testis. Further biochemical differentiation occurs as sperm travel through the epididymis. Although epididymal sperm appear morphologically mature, they are unable to fertilize an egg unless they undergo capacitation - a complex and poorly understood process that occurs in the female reproductive tract. Capacitation is initiated by bicarbonate and other uterine effectors that modulate sperm intracellular second messengers, notably cAMP. Elevated cAMP levels lead to changes in sperm motility and reorganization of membrane components, changes that are essential for sperm binding and penetration of the egg coat, as well as for subsequent fusion with the egg plasma membrane. Why so many sperm? Although the ejaculate contains millions of sperm, only one is needed to produce a viable offspring. In fact, more than one fertilizing sperm is lethal. In species such as marine invertebrates that deposit their gametes into open seawater, it seems reasonable that enormous numbers of sperm are necessary to counter the effects of dilution and fluid dynamics. Egg secretions serve as sperm chemoattractants to further increase the likelihood of fertilization. In internally fertilizing mammals, excess sperm are held in storage crypts within the female reproductive tract, enabling the timed release of additional aliquots of capacitated sperm, each of which has an independent opportunity to fertilize the egg. As in marine invertebrates, secretions from the ovulated mammalian egg may serve to attract sperm up the oviduct. So many barriers, so little time. Given the importance of fertilization to the continuation of the species, one would think that nature would make it easy for gametes to bind and fuse with one another. But once the sperm has finally reached the vicinity of the ovulated egg, it must overcome a variety of barriers that impede its access to the egg. First, the cumulus layer. Sperm initially confront the cumulus cells that nursed the egg during oogenesis and which remain adherent to the egg coat at ovulation. Sperm employ a variety of strategies, both enzymatic and mechanical, to clear a path through the cumulus cells. It is unclear, though, whether the pioneering sperm is the one to fertilize the egg, or a sacrificial sperm that clears a path for subsequent sperm. Then, the egg coat. After they trek across the cumulus layer, sperm encounter the zona pellucida, a thick extracellular coat that protects the egg. It is here that species-specific gamete binding occurs (Figure 1). This not only insures union of homologous gametes, but also serves as a point of speciation, as naturally occurring polymorphisms in the relevant receptors may result in new binding specificities that become segregated from the original parental species.

Full-size image (60K)

Figure 1. Mouse sperm labeled with two fluorescent dyes bound to the egg coat.

View Within Article

The structure of the mammalian zona pellucida is relatively simple, being composed of only three glycoproteins. One of these, ZP3, has carbohydrate groups thought to provide binding sites for protein receptors on the sperm surface. The best-studied candidate receptor is β1,4-galactosyl-transferase, a carbohydrate-binding protein usually confined to intracellular membranes, but which also functions as a cell surface receptor on selected cell types. Such protein-carbohydrate complementarity appears to be a generalized mechanism to facilitate sperm-egg binding in a wide range of species. Things are never so simple. ZP3 carbohydrates were thought to be sufficient to account for species-specific sperm-egg binding, but recent studies cast doubt on whether they are the primary site of sperm binding. Sperm with reduced ability to bind ZP3 still adhere to the egg coat, despite their poor ability to penetrate the coat. Similarly, mouse sperm still bind and fertilize eggs in which murine ZP3 has been replaced with human ZP3. These and other studies indicate that sperm-egg binding likely involves a multiplicity of interacting receptors. Some have recently been identified, such as SED1, a multidomain sperm protein that mediates binding to the egg coat, and a novel component of the egg coat that facilitates sperm adhesion. This reliance upon multiple cell adhesion mechanisms is reminiscent of lymphocyte binding to the vascular endothelium. Cells that exist in body fluids and are subject to high shear forces, such as lymphocytes and gametes, may require the concerted action of low-affinity and high-affinity receptor-ligand interactions to ensure successful binding. Still not there. Although we do not yet appreciate the full repertoire of receptors that mediate sperm binding to the egg coat, such binding leads to activation of sperm heterotrimeric G-proteins and selective ion channels that trigger exocytosis of the acrosome. Hydrolytic enzymes released from the acrosome vesicle digest a path through the egg coat; however, the precise acrosomal enzymes responsible for sperm penetration remain obscure. Finally, the egg membrane. After traversing through the cumulus layer and the zona pellucida, the pioneering sperm finds itself facing the egg plasma membrane - the last barrier to fertilization. A novel class of cell adhesion molecules, the ADAMs, some of which are localized on sperm, were initially thought to bind integrin receptors on the egg membrane, thus facilitating membrane binding and fusion. But as before, nothing is as straightforward as it initially appeared, and the contribution of sperm ADAMS and their egg integrin receptors is still actively debated. Recent evidence has implicated a role for tetraspanin and GPI-linked proteins of the egg membrane, as well as a cysteine-rich secretory protein on sperm, as mediators of gamete fusion. Only one sperm please. One responsibility of the fertilizing sperm is to prevent entry of additional sperm, which would be lethal to the new zygote. This block to polyspermy is achieved by changes in the egg membrane that prevent entry of additional sperm, and by enzymatic modifications to the zona pellucida by exudates of egg cortical vesicles that are released following fertilization. Ultimately, the fertilizing sperm activates development via receptor mediated signal transduction cascades and/or by introduction of critical sperm effectors that initiate metabolic activation.