Molecules on the sperm's route to fertilization.

In eutherian mammals billions of sperm are deposited at ejaculation in the female reproductive tract, but only a few thousand enter the oviduct. A few reach the ampulla at the time of fertilization and only one sperm fertilizes the egg. In most mammalian species the lower isthmus of the fallopian tubes has taken over the function of a reservoir in which sperm are stored under conditions that save sperm energy by suppressing motility and increase viability. Close to the time when the egg is ovulated into the ampulla, the sperm undergo a complex sequence of processes, named capacitation. Capacitation is a prerequisite for fertilization, enabling the sperm to recognize the egg and to respond to the egg signals in the appropriate manner. Sperm bind to the egg extracellular matrix, the zona pellucida, and upon binding undergo the acrosome reaction, followed by the passage of the zona pellucida and binding to and fusion with the egg oolemma, thus triggering the embryonic developmental program. The oviduct and the egg itself appear to coordinate sperm function to ensure that two functional competent gametes will meet, leading to fertilization. For the communication between sperm and somatic cells as well as between both gametes the information potential of carbohydrates is utilized, and this event probably prepares the next level of interactions, e.g., capacitation, acrosome reaction, egg binding, and fusion. The current perspective focuses on the role of molecules possibly implicated in sperm-oviduct and sperm-egg interactions. J. Exp. Zool. (Mol. Dev. Evol.) 285:259-266, 1999.     

Sperm penetration through cumulus mass and zona pellucida

Mammalian fertilization requires sperm to penetrate the cumulus mass and egg zona pellucida prior to fusion with the egg. Although sperm penetration through these physical barriers is essential, the molecular mechanism has not yet been completely elucidated. In addition to sperm motility, hyaluronan-hydrolyzing and proteolytic enzymes of sperm have been suggested to participate in the penetration events. Here we focus on the functional roles of hyaluronidase and protease in sperm passage through the cumulus mass and zona pellucida.     

Sperm penetration through oocyte investments in mammals

Literature on the interactions between eutherian gametes is reviewed. The oocyte cumulus complex of the female is surrounded by a zona pellucida, corona radiata, and cumulus layer. Sperm undergo an acrosome reaction before penetrating the zona pellucida. The morphological consequences of this reaction and its possible role(s) in penetration of the oocyte cumulus complex are considered. The acrosomal enzyme, hyaluronidase, has been thought to aid sperm in penetrating the cumulus layer and corona radiata. Several recent investigations, including one that shows that motile cells lacking hyaluronidase can penetrate to the zona surface, do not support this idea. Other possible roles of this enzyme in fertilization are discussed. The development of in vitro fertilization systems that employ physiological numbers (1-100) of sperm will be valuable in studying the mechanisms used by sperm to penetrate the oocyte cumulus complex.     

Molecular models for murine sperm-egg binding

Murine sperm initiate fertilization by binding to the specialized extracellular matrix of mouse eggs, known as the zona pellucida. Over the past decade, powerful genetic, biophysical, and biochemical techniques have been employed to gain new insights into this interaction. Evidence from these studies does not support either of two major models for binding first proposed over two decades ago. Two more recently established models suggest that protein-protein interactions predominate during this initial stage of fertilization. Another model proposes that about 75-80% of the murine sperm bound to zona pellucida under well defined in vitro conditions is carbohydrate dependent, with the remaining sperm bound via protein-protein interactions. Mounting evidence suggests that the carbohydrate sequences coating the murine egg could be employed as specific immune recognition markers. Continued investigation of this system may resolve many of these controversial findings and reveal novel functions for murine zona pellucida glycoproteins.     

Regulation of sperm storage and movement in the mammalian oviduct

The oviduct plays a vital role in ensuring successful fertilization and normal early embryonic development. The male inseminates many thousands or even millions of sperm, but this alone does not ensure that fertilization will be successful. The female tract, particularly the oviduct, provides filters that select for normal vigorously motile sperm. In conjunction with molecules in the seminal plasma and on sperm, the female tract regulates how and when sperm pass though the tract to reach the site of fertilization. Various regulatory processes control sperm passage into and through the oviduct. In some species, the uterotubal junction opens and closes to regulate when sperm may enter; furthermore, passage through the junction requires certain proteins on the sperm surface. Most of the sperm that manage to enter the oviduct soon become trapped and held in a reservoir. In marsupials and insectivores, this involves trapping sperm in mucosal crypts; while in most other mammalian species, this involves binding sperm to the oviductal epithelium. As the time of ovulation approaches, the sperm in the reservoir undergo capacitation, including motility hyperactivation. Capacitating sperm shed proteins that bind them to the mucosal epithelium, while hyperactivation assists the sperm in pulling off of the epithelium and escaping out of mucosal pockets. The process of sperm release is gradual, reducing chances of polyspermic fertilization. Released sperm may be guided towards the oocyte by secretions of the oviduct, cumulus cells, or oocyte. Hyperactivation likely assists sperm in penetrating the cumulus matrix and is absolutely required for penetrating the oocyte zona pellucida and achieving fertilization.     

The interaction of boar sperm proacrosin with its natural substrate, the zona pellucida, and with polysulfated polysaccharides

Boar sperm acrosin is an acrosomal protease with trypsin-like specificity, and it functions in fertilization by assisting sperm passage through the zona pellucida by limited hydrolysis of this extracellular matrix. In addition to a proteolytic active site domain, acrosin binds the zona pellucida at a separate binding domain that is lost during proacrosin autolysis. In this study, we quantitate the binding of proacrosin to the physiological substrate for acrosin, the zona pellucida, and to a non-substrate, the polysulfated polysaccharide fucoidan. Binding was analogous to sea urchin sperm bindin that binds egg jelly fucan and the vitelline envelope of sea urchin eggs. Proacrosin was found to bind to fucoidan and to the zona pellucida with binding affinities similar to bindin interaction with egg jelly fucan. These interactions were competitively inhibited by similar relative molecular mass polysulfated polymers. Since bindin and proacrosin have distinctly different amino acid sequences, their interaction with acidic sulfate esters demonstrates an example of convergent evolution wherein different macromolecules localized in analogous sperm compartments have the same biological function. From cDNA sequence analysis of proacrosin, this binding may be mediated through a consensus sequence for binding sulfated glycoconjugates. Proacrosin binding to the zona pellucida may serve as both a recognition or primary sperm receptor, as well as maintaining the sperm on the zona pellucida once the acrosome reaction has occurred.     

Identification of a secondary sperm receptor in the mouse egg zona pellucida: role in maintenance of binding of acrosome-reacted sperm to eggs

During fertilization in mice, acrosome-intact sperm bind via plasma membrane overlying their head to a glycoprotein, called ZP3, present in the egg extracellular coat or zona pellucida. Bound sperm then undergo the acrosome reaction, which results in exposure of inner acrosomal membrane, penetrate through the zona pellucida, and fuse with egg plasma membrane. Thus, in the normal course of events, acrosome-reacted sperm must remain bound to eggs, despite loss of plasma membrane from the anterior region of the head and exposure of inner acrosomal membrane. Here, we examined maintenance of binding of sperm to the zona pellucida following the acrosome reaction. We found that polyclonal antisera and monoclonal antibodies directed against ZP2, another zona pellucida glycoprotein, did not affect initial binding of sperm to eggs, but inhibited maintenance of binding of sperm that had undergone the acrosome reaction on the zona pellucida. On the other hand, polyclonal antisera and monoclonal antibodies directed against ZP3 did not affect either initial binding of acrosome-intact sperm to eggs or maintenance of binding following the acrosome reaction. We also found that soybean trypsin inhibitor, a protein reported to prevent binding of mouse sperm to eggs, did not affect initial binding of sperm to eggs, but, like antibodies directed against ZP2, inhibited maintenance of binding of sperm that had undergone the acrosome reaction on the zona pellucida. These and other observations suggest that ZP2 serves as a secondary receptor for sperm during the fertilization process in mice and that maintenance of binding of acrosome-reacted sperm to eggs may involve a sperm, trypsin-like proteinase.     

Essential role of the nonreducing terminal alpha-mannosyl residues of the N-linked carbohydrate chain of bovine zona pellucida glycoproteins in sperm-egg binding

It has been proposed that mammalian sperm bind species-specifically to carbohydrate chains of zona pellucida glycoproteins at fertilization. Although the sperm ligand carbohydrate chains have been characterized in mice and pigs, the existence of the ligands of other mammals remains unclear. In order to explore the bovine sperm ligand, two in vitro competition assay methods were applied. As a result, a high-mannose-type carbohydrate chain, Manalpha1-6(Manalpha1-3)Manalpha1-6(Manalpha1-3)Manbeta1-4GlcNAcbeta1-4GlcNAc, which is the major neutral chain in bovine egg zona glycoproteins, was shown to possess bovine sperm ligand activity. When nonreducing terminal alpha-mannosyl residues were eliminated from the zona glycoproteins by alpha-mannosidase digestion, the ligand activity was reduced, indicating that the alpha-mannosyl residues play an essential role in bovine sperm-egg binding. The number of sperm binding to eggs was reduced to about one-half after fertilization. The ligand-active high-mannose-type chain may be buried after fertilization, since its amount remains unchanged. Pretreatment of bovine sperm with the sperm ligand-carbohydrate chain significantly inhibited penetration of the sperm into oocyte and the male pronucleus formation. Thus, a correlation between the sperm ligand activity and in vitro fertilization rate was observed.     

Capacitation dependent activation of tyrosine phosphorylation generates two sperm head plasma membrane proteins with high primary binding affinity for the zona pellucida

The recognition and binding of sperm cells to the zona pellucida (the extracellular matrix of the oocyte) are essential for fertilization and are believed to be species specific. Freshly ejaculated sperm cells do not bind to the zona pellucida. Physiologically this interaction is initiated after sperm activation in the female genital tract (capacitation) via a yet unknown mechanism, resulting in the binding of a receptor in the apical sperm plasma membrane to the zona pellucida. In order to mimic this biochemically, we isolated zona pellucida fragments from gilt ovaries to prepare an affinity column with the intact zona pellucida structure and loaded this column with solubilized apical plasma membranes of boar sperm cells before and after in vitro capacitation. With this technique we demonstrated that two plasma membrane proteins of capacitated boar sperm cells showed high affinity for zona pellucida fragments. Further analysis showed that these proteins were tyrosine phosphorylated. Plasma membrane proteins from freshly ejaculated sperm cells did not exhibit any zona pellucida binding proteins, likely because these proteins were not tyrosine phosphorylated.     

What controls polyspermy in mammals,the oviduct or the oocyte?

A block to polyspermy is required for successful fertilisation and embryo survival in mammals. A higher incidence of polyspermy is observed during in vitro fertilisation (IVF) compared with the in vivo situation in several species. Two groups of mechanisms have traditionally been proposed as contributing to the block to polyspermy in mammals: oviduct‐based mechanisms, avoiding a massive arrival of spermatozoa in the proximity of the oocyte, and egg‐based mechanisms, including changes in the membrane and zona pellucida (ZP) in reaction to the fertilising sperm. Additionally, a mechanism has been described recently which involves modifications of the ZP in the oviduct before the oocyte interacts with spermatozoa, termed “pre‐fertilisation zona pellucida hardening”. This mechanism is mediated by the oviductal‐specific glycoprotein (OVGP1) secreted by the oviductal epithelial cells around the time of ovulation, and is reinforced by heparin‐like glycosaminoglycans (S‐GAGs) present in oviductal fluid. Identification of the molecules contributing to the ZP modifications in the oviduct will improve our knowledge of the mechanisms of sperm‐egg interaction and could help to increase the success of IVF systems in domestic animals and humans.     

Hyperactivation of mammalian sperm.

Mammalian sperm commonly show hyperactivated motility just before fertilization. The movement of hyperactivated sperm appears different in fluids of different viscosity and elasticity and in different species, but basically it involves an increase in flagellar bend amplitude and, usually, beat asymmetry. Hyperactivation may be critical to the success of fertilization, because it enhances the ability of sperm to detach from the wall of the oviduct, to move around in the labyrinthine lumen of the oviduct, to penetrate mucous substances and, finally, to penetrate the zona pellucida of the oocyte. Presumably, a signal or signals exist in the oviduct to initiate hyperactivation at the appropriate time; however, none have yet been identified with certainty. While the signal transduction cascade regulating hyperactivation remains to be completely described, it is clear that calcium ions interact with the axoneme of the flagellum to switch on hyperactivation. Although hyperactivation often occurs during the process of capacitation, divergent pathways regulate the two events.     

The secretions of the cumulus-oocyte complex in relation to fertilization and early mouse embryonic development: a histochemical study

Summary A study has been made of the histochemical composition of the murine cumulus—oocyte complex and zona pellucida following treatment of immature females with exogenous gonadotrophins. Selected developmental stages were studied in detail, namely (i) the ovulated and unfertilized egg, (ii) the fertilized oocyte and (iii) the preimplantation embryo. In addition, the histochemical features observed in normal fertilized embryos have been compared with those of haploid and diploid parthenogenetic embryos at comparable stages following activation. Shortly after fertilization, glycosaminoglycans, which form a major component of the extracellular matrix surrounding the cumulus cells, become incorporated into the zona pellucida of the fertilized egg. In oocytes with few or no attendant cumulus cells, there appeared to be a diminished uptake of glycosaminoglycans and a reduced intensity of the zona staining reaction to Alcian Blue. In these oocytes, uptake of glycosaminoglycans appeared to be from the secretions lining the oviduct. There was little incorporation of the glycosaminoglycans from the extra-cellular matrix of the surrounding cumulus cells into the zona pellucida in unfertilized or parthenogenetic eggs despite the activation stimulus. After fertilization or activation, the zona pellucida became increasingly PAS-positive. Enzymic studies clearly indicate that the composition of the zona pellucida of the early embryo is histochemically different from the zona that surrounds the oocyte in the preovulatory follicle. These findings are discussed in relation to the decreased viability of embryos from oocytes which have been ovulated.The death of Mrs Carol Grainge is sadly recorded.     

An update of the regulatory factors of sperm migration from the uterus into the oviduct by genetically manipulated mice

Mammalian reproductive processes involve spermatogenesis, which occurs in the testis, and fertilization, which takes place in the female genital tract. Fertilization is a successive, multistep, and extremely complicated event that usually includes sperm survival in the uterus, sperm migration through the uterotubal junction (UTJ) and the oviduct, sperm penetration through the cumulus cell layer and the zona pellucida, and sperm–egg fusion. There may be a complex molecular mechanism to ensure that the above processes run smoothly. Previous studies have discovered essential factors for these fertilization steps through in vitro fertilization experiments. However, recent gene disruption approaches in mice have revealed that many of the factors previously described as important for fertilization are largely dispensable in gene‐knockout animals, and some previously unknown factors are emerging. As a result, the molecular mechanisms of fertilization, especially sperm migration from the uterus into the oviduct, have recently been revised by the emergence of genetically modified animals. In this review, we only focus on and update the essential genes for sperm migration through the UTJ and describe recent advances in our knowledge of the basis of mammalian sperm migration.     

Zona pellucida glycoproteins

All mammalian eggs are surrounded by a relatively thick extracellular coat, the zona pellucida, that plays vital roles during oogenesis, fertilization, and preimplantation development. The mouse zona pellucida consists of three glycoproteins that are synthesized solely by growing oocytes and assemble into long fibrils that constitute a matrix. Zona pellucida glycoproteins are responsible for species-restricted binding of sperm to unfertilized eggs, inducing sperm to undergo acrosomal exocytosis, and preventing sperm from binding to fertilized eggs. Many features of mammalian and non-mammalian egg coat polypeptides have been conserved during several hundred million years of evolution.     

Overexpressing sperm surface beta 1,4-galactosyltransferase in transgenic mice affects multiple aspects of sperm-egg interactions

Sperm surface beta 1,4-galactosyltransferase (GalTase) mediates fertilization in mice by binding to specific O-linked oligosaccharide ligands on the egg coat glycoprotein ZP3. Before binding the egg, sperm GalTase is masked by epididymally derived glycosides that are shed from the sperm surface during capacitation. After binding the egg, sperm- bound oligosaccharides on ZP3 induce the acrosome reaction by receptor aggregation, presumably involving GalTase. In this study, we asked how increasing the levels of sperm surface GalTase would affect sperm-egg interactions using transgenic mice that overexpress GalTase under the control of a heterologous promoter. GalTase expression was elevated in many tissues in adult transgenic animals, including testis. Sperm from transgenic males had approximately six times the wild-type level of surface GalTase protein, which was localized appropriately on the sperm head as revealed by indirect immunofluorescence. As expected, sperm from transgenic mice bound more radiolabeled ZP3 than did wild-type sperm. However, sperm from transgenic animals were relatively unable to bind eggs, as compared to sperm from wild-type animals. The mechanistic basis for the reduced egg-binding ability of transgenic sperm was attributed to alterations in two GalTase-dependent events. First, transgenic sperm that overexpress surface GalTase bound more epididymal glycoside substrates than did sperm from wild-type mice, thus masking GalTase and preventing it from interacting with its zona pellucida ligand. Second, those sperm from transgenic mice that were able to bind the zona pellucida were hypersensitive to ZP3, such that they underwent precocious acrosome reactions and bound to eggs more tenuously than did wild-type sperm. These results demonstrate that sperm-egg binding requires an optimal, rather than maximal, level of surface GalTase expression, since increasing this level decreases sperm reproductive efficiency both before and after egg binding. Although sperm GalTase is required for fertilization by serving as a receptor for the egg zona pellucida, excess surface GalTase is counterproductive to successful sperm-egg binding.     

Functional analysis of sperm from c-mos(-/-) mice

The c-mos protooncogene, which is expressed predominantly in male and female germ cells, is crucial for normal oocyte meiosis and female fertility in mice. Inactivation of c-mos results in abnormal oocyte development and leads to ovarian cysts and tumors in vivo. In contrast to the severe effects of c-mos ablation in females, targeted inactivation of c-mos has not been reported to affect spermatogenesis in male mice. However, previously reported studies of male c-mos(-/-) mice have been limited to histological analyses of testes and in vivo matings, both of which are relatively insensitive indicators of sperm production and function. Therefore, we assayed sperm function of c-mos(-/-) males under in vitro conditions to determine whether the absence of Mos during development affected sperm production or fertilizing ability. We found no significant differences between the number of sperm collected from c-mos(-/-) and wild type mice. Additionally, sperm from c-mos(-/-) and c-mos(+/+) males performed equally well in assays of in vitro fertilization (IVF) and fertilization-associated events including zona pellucida (ZP) penetration, sperm/egg plasma membrane fusion, and sperm chromatin remodeling. Therefore, we suggest that the function of Mos in spermatogenesis is either not related to the ultimate fertilizing potential of the sperm, or else the absence of Mos is masked by a redundant kinase.     

The zona pellucida: a coat of many colors

The zona pellucida is an extracellular coat that surrounds all mammalian eggs. It is a porous matrix of interconnected filaments that are assembled from glycoproteins synthesized and secreted by growing oocytes. The zona pellucida is responsible both for species-specific binding of sperm to unfertilized eggs and inducing bound sperm to undergo the acrosome reaction. The latter enables sperm to penetrate the extracellular coat and fertilize the egg. The zona pellucida also aids in prevention of polyspermy following fertilization and in protection of preimplantation embryos. In mice, several of these important functions can now be ascribed to specific zona pellucida glycoproteins that have been purified and characterized. Furthermore, the enzyme responsible for hatching of embryos from the zona pellucida, just prior to implantation, has been identified and characterized.     

Rendez-vous in the oviduct: implications for superovulation and embryo transfer

The meeting between the maternal and paternal gametes is dependant upon a number of complicated processes. On the maternal side it involves maturation of the oocytes under the influence on both peripheral and follicular endocrine factors. Deviations in the normal pattern of maturation will lead to ovulation of inferior oocytes. On the paternal side the transport of spermatozoa in the female genital tract following mating is an area of great importance. The establishment of the sperm reservoir in the isthmus is dependant upon a number of factors (intracellular calcium concentrations, oligo-saccharides, change in estradiol: progesterone ratio in the afferent blood supply). Alterations of the normal micro-environment may disturb both binding, release and transport as a whole. The process of fertilization occurs in the ampullar region of the oviduct and it involves several well tuned steps: binding to the zona pellucida where the acrosome reaction takes place, penetration, fusion between the oolemma and the sperm plasma membrane, activation with the release of the cortical granules, decondensation of the sperm chromatin, pronucleus formation and finally syngamy where the two pronuclei fuses. The egg will experience the first cleavage shortly thereafter. Superovulation may disturb a number of these processes including oocyte maturation (arrest at MI) and sperm and zygote transport in the oviduct caused by the deviant endocrine environment, thus leading to a higher incidence of lack of fertilization and poor embryos quality.     

A model for sperm-egg binding and fusion based on ADAMs and integrins

Once a sperm meets an egg, several events must occur in order for fertilization to proceed. Sperm must bind to the zona pellucida, undergo the acrosome reaction, penetrate the zona pellucida and then bind to and fuse with the egg plasma membrane. Shortly thereafter, the egg must be activated for zygotic development. This review focuses on mammalian sperm-egg plasma membrane binding and fusion, and in particular on the roles of two families of cell-adhesion molecules, ADAMs and integrins, in this important union.     

Proteomic analysis of sperm regions that mediate sperm-egg interactions

The sperm interacts with three oocyte-associated structures during fertilization: the cumulus cell layer surrounding the oocyte, the egg extracellular matrix (the zona pellucida), and the oocyte plasma membrane. Each of these interactions is mediated by the sperm head, probably through proteins both on the sperm surface and within the acrosome, a specialized secretory granule. In this study, we have used subcellular fractionation in order to generate a proteome of the sperm head subcellular compartments that interact with oocytes. Of the proteins we identified for which a gene knockout has been tested, a third have been shown to be essential for efficient reproduction in vivo. Many of the other presently untested proteins are likely to have a similarly important role. Twenty-five percent of the cell surface fraction proteins are previously uncharacterized. We have shown that at least two of these novel proteins are localized to the sperm head. In summary, we have identified over 100 proteins that are expressed on mature sperm at the site of sperm-oocyte interactions.