ISI Impact Factor (2004): 1.096

Editor-in-Chief
Prof. Yi-Fei WANG,

Carbohydrates mediate sperm-ovum adhesion and triggering of the acrosome reaction

Daulat R.P. Tulsiani

Departments of Obstetrics & Gynecology and Cell Biology, Vanderbilt University School of Medicine, Room D-3243 MCN, Nashville, Tennessee 37232-2633, USA

Asian J Androl  2000 Jun; 2:  87-97

Keywords: sperm capacitation; sperm-ovum interaction; acrosome reaction; carbohydrates; fertilization

Abstract

Figure 1. Progressive stages of sperm-ovum interaction in the mouse: 1, the acrosome-intact spermatozoon passes through cumulus cells and the innermost layer, the corona radiata; 2, sperm receptor(s) bind to glycan moiety(ies) of mZP3 and starts a signal transduction cascade resulting in acrosomal exocytosis; 3, the acrosome-reacted spermatozoon penetrate the ZP; and 4, the spermatozoon passes through the perivitelline space and fuses with the ovum plasma membrane.

1.1 Sperm capacitation

Mammalian spermatozoa released from the seminiferous tubules and those present within the proximal regions of the epididymis are non-motile and unable to bind to the ZP and undergo the acrosome reaction. They acquire progressive motility during transit through epididymis, a process termed epididymal maturation^[1]. During this passage, the epididymis provides a specific intraluminal environment in which functionally immature sperm cells undergo morphological and biochemical alterations necessary to produce competent spermatozoa capable of undergoing capacitation and successful binding to the ZP^[1]. During epididymal maturation, sperm plasma membrane, a vital component in the early events of fertilization undergoes extensive biochemical changes and remodeling^[5-7]. These modifications are an essential part of the maturation process resulting in the production of a self-propelled, functionally-competent spermatozoon.

In addition to maturation in the epididymis, spermatozoa must undergo functional changes between the events of mating and fertilization^[1,2]. During residence in the female reproductive tract, they undergo a series of biochemical and functional modifications collectively referred to as capacitation^[1]. The net change during this process is a combined effect of multiple molecular modifications in the sperm plasma membrane proteins/glycoproteins and lipid components that are thought to modify the ion channels in the plasmalemma^[8]. The preparatory alterations include removal of seminal plasma molecules adsorbed to the surface of ejaculated spermatozoa as well as modification and/or reorganization of sperm surface components^[2]. The adsorbed seminal plasma factors are believed to prevent spermatozoa from undergoing premature acrosome reaction before completing their migratory journey through the female reproductive tract.  As a result of these alterations, capacitated spermatozoa become acrosomally responsive by yet unknown mechanisms. Experimental evidence from several studies strongly suggests that sperm loses cholesterol before becoming acrosomally responsive^[9].

Although the significance of capacitation has been known for nearly 40 years^[10,11], the molecular mechanisms responsible for the functional changes are not fully understood.  Most researchers agree that capacitation results from multiple molecular changes in sperm plasma membrane proteins/glycoproteins and lipid components that likely modify ion channels. The reported changes in cholesterol and phospholipid during  in vivo capacitation is consistent with this possibility^[12].  As capacitation proceeds, a number of biochemical changes occur on spermatozoa. These modifications allow the transmembrane flux of ions that are believed to be important in initiating the events of capacitation, hyperactivation and acrosome reaction^[2]. The former two events take place before sperm-ovum (zona) interaction, and the latter event, at least in the mouse and several other species including man, is initiated after sperm-ovum binding. It is, therefore, reasonable to assume that the three events are independent and involve region-specific alterations in sperm plasma membrane.

In most mammalian species studied, spermatozoa become hyperactive in the isthmus region of the oviduct, and the hyperactivated spermatozoa move to the ampullary environment, the site of in vivo fertilization^[13-15]. A possible functional significance of hyperactivation could be that the hyperactivated beat pattern of a spermatozoon will definitely enhance its thrust (physical force) at the site of its binding to ZP and undergoing the acrosome reaction^[16]. The hydrolytic action of glycohydrolases and proteases released at the site of sperm-zona binding, along with the enhanced thrust generated by the hyperactivated beat pattern of the bound spermatozoon, are important in regulating the penetration of ZP and fusion with the ovum plasma membrane. 

All mammalian spermatozoa studied thus far undergo capacitation after residing in the female genital tract for a certain period of time. Similarly, spermatozoa recovered from the female genital tract after mating, or cauda epididymal spermatozoa following incubation with oviductal secretions, are able to fertilize an ovum in vitro^[17,18]. The precise site of capacitation may be different in different species; however, several studies suggest that capacitation is most efficient when the spermatozoa pass through the uterus and oviduct. The oviductal secretions collected from the estrous females have been demonstrated to be most efficient in rendering the functional changes in spermatozoa in vitro^[1,18]. 

Mammalian sperm can also be capacitated in vitro in a chemically defined medium supplemented with energy substrates, such as glucose and pyruvate, bovine serum albumin, and appropriate buffer^[1,19]. It is noteworthy that albumin is the major protein in the female genital tract secretions and is an important component for in vitro and in vivo capacitation^[19,20] protein is believed to facilitate capacitation by efflux of fatty acid/cholesterol/phospholipids from sperm plasma membrane. The precise mechanism as to how the loss of cholesterol/phospholipids regulates the functional changes is not yet known; however, it is generally believed that the loss of fatty acids/cholesterol increases fluidity and permeability of the sperm plasma membrane and initiate the events of capacitation and the acrosome reaction.

As capacitation proceeds, a number of intracellular and cell surface changes occur on spermatozoa. The known changes include a) increased adenylate cyclase and cAMP^[21,22], b) increase in intracellular pH^[23], c) calcium influx^[24], d) loss of sperm surface components^[25], e) modification/alteration of sperm plasma membrane^[26], and f) tyrosine phosphorylation of a subset of sperm proteins^[27,28]. Recent studies suggest that the tyrosine phosphorylation is dependent on increased cAMP, a result consistent with the suggestion of a relationship between cAMP-dependent protein kinase A and tyrosine kinases and/or phosphatases^[27-29]. Whether any of the above changes are necessary for capacitation is not yet known. The molecular mechanism(s) as to how these changes affect the functional state of spermatozoa is currently under intensive investigation.

One important surface alteration that happens following capacitation is a change in the lectin-binding properties of spermatozoa^[30-33], a result indicative that the terminally exposed carbohydrate moieties are altered during this process. A possible explanation for these changes in vivo could be the association of an oviductal glycoprotein secreted from oviductal epithelial cells, and reported to bind to the posterior region of the spermhead, midpiece, and flagellum^[2,34]. A second possibility is that the existing sperm plasma membrane glycoproteins are modified in vivo by glycosyltransferases present in the uterus and oviduct secretions, and which display a temporal surge in the genital tract during the esterous cycle^[35]. The plausible modification of the endogenous sperm surface glycoproteins by addition of sugar residues to the terminally exposed glycan moieties will be expected to alter the lectin-binding properties of capacitated spermatozoa. Alternatively, the glycohydrolases present within the sperm acrosome of noncapacitated spermatozoa could translocate (redistribute) on the sperm surface of capacitated sperm as has been reported for hyaluronidase^[36]. These enzymes not only hydrolyze glycosyl residues from glycoproteins, they themselves are glycoproteins^[6]. The appearance of intra-acrosomal glycohydrolases (glycoproteins) on the sperm surface following capacitation is expected to alter the lectin-binding properties of spermatozoa.

Finally, several studies have provided evidence suggesting a role for proteoglycans and glycosaminoglycans in altering the functional state of spermatozoa. Heparin, the most effective glycosaminoglycan, binds to spermatozoa from several species, including man^[37]. The binding is said to be sensitive to pH, ionic strength, and temperature, and thus has characteristics of a typical receptor-ligand interaction.  However, the receptor for glycosaminoglycan has not been identified, and the molecular mechanisms responsible for these interactions are not fully understood. 

2.1 Zona pellucida glycoconjugates

ZP is the extracellular glycocalyx which surrounds all mammalian ovums. The ZP in all species studied is a relatively simple structure composed of three families of glycoproteins, designated as ZP1, ZP2, and ZP3, and the pig has a fourth form as well (Table 1). The main functions of ZP in fertilization have been generally accepted as the mediation of the relative species specificity of sperm binding, induction of the sperm acrosome reaction, secondary binding events, blocking of polyspermy, and protection of growing embryo from fertilization to implantation^[1]. It is now clear that the proteins which make up the zona pellucida are highly conserved among mammalian species, suggesting that their organization may also be conserved.  In the mouse, the three glycoproteins are synthesized and secreted by the growing oocytes. Two of the glycoproteins, mouse ZP2 (mZP2) and mZP3, interact noncovalently to form filaments of structural repeats that are interconnected by mZP1 forming a three-dimensional network of cross-linked filaments^[40].  Such a structure may explain the elasticity of ZP and the relative ease of its penetration by the hyperactivated acrosome-reacted spermatozoa.

Table 1. Characteristics of mammalian ZP glycoproteins.