Capacitation, acrosome reaction, zona binding

After a sperm leaves the male reproductive tract and enters the female reproductive tract it still has a long way to travel and many obstacles to overcome before it can fertilize the egg. When sperm first enter the female reproductive tract they are not capable of fertilization, but require a maturation step called capacitation. Capacitation has not been completely defined but it is thought to involve cell surface and metabolic changes. As a result of capacitation the sperm have an altered pattern of motility (called hyperactivation) and are capable of undergoing an acrosome reaction.

Acrosome reaction
The acrosome reaction is a Ca2+-stimulated exocytosis involving reorganization of the membranes in the head of the spermatozoan. Multiple fusions occur between the outer region of the acrosomal membrane and the plasma membrane that overlies the acrosome. The hybrid vesicles which result are released into the surrounding environment along with the fluid contents of the acrosome. Loss of these anterior portions of membrane reveals the inner acrosomal membrane which, together with the original posterior head plasma membrane, form the new head cell membrane of the acrosome-reacted sperm (Fig. 1). There are specific movements of proteins from one membrane region to another during the reorganization, although mixing of the membrane components is incomplete. The acrosomal contents are a rich source of enzymes, including hyaluronidase and the protease, acrosin, that may function in penetration of the sperm through the egg investments. The hybrid vesicles released during the acrosome reaction could also carry such enzymes on their surfaces. The newly exposed inner acrosomal membrane represents a further source of molecules that could be involved in digestion of a pathway for the sperm though the egg investments or in binding sperm to the zona pellucida.

Where the sperm are when they acrosome react and what signal(s) causes them to acrosome react are not completely clear. Binding of sperm to the zona pellucida (an extracellular coat surrounding the oocyte) can induce the acrosome reaction, but molecules that the sperm contacts earlier in its progress through the female reproductive tract may also facilitate or induce the acrosome reaction. These include molecules in oviductal and follicular fluids (e.g. progesterone) and also components of the matrix surrounding cells of the cumulus oophorus (see below). It is possible that subpopulations of sperm acrosome react at different sites on their passage to the egg ( Fig. 2).

Cumulus penetration
When sperm reach the egg (more correctly referred to as an oocyte because it has not yet completed meiosis) they first encounter a mass of cells, the cumulus oophorus, that surrounds the egg (Fig. 2). The cumulus cells are follicular cells that encase the oocyte and are ovulated along with the egg. Sperm swim between these cells to reach the egg, apparently dissolving the extracellular matrix that holds the cells together. The sperm carry with them in the acrosomal contents and in the plasma membrane the enzyme hyaluronidase that is required for the penetration of sperm through this cell layer rich in hyaluronic acid. Depending on whether sperm acrosome react in the cumulus mass or remain intact, either pool of hyaluronidase could be used for cumulus penetration.

Zona pellucida
When sperm reach the zona pellucida they recognize it and bind to it. Although there is not a strict species specificity in terms of which sperm will bind to a particular zona pellucida, there is often a strong preference for binding between sperm and zona of the same species.

In mouse, the zona pellucida is composed of three glycoproteins called ZP1, ZP2 and ZP3. Numerous studies have indicated that acrosome-intact mouse sperm initially bind to the carbohydrate region of ZP3. The identity of the partner molecule on the sperm surface is not yet firmly established; however, there is excellent evidence that a galactosyl transferase on the sperm surface binds to one of its substrates (N-acetylglucosamine) on the zona and, because the second substrate (UDP galactose) is missing, the sperm remain bound. Other candidates exist that could operate instead of, or in addition to, galactosyl transferase. If sperm are acrosome-intact when they bind to the zona, they are induced to undergo the acrosome reaction as a result of binding. The acrosome reaction has been induced experimentally by the clustering of sperm surface molecules using the multivalent zona protein ZP3, or using antibodies that recognize specific sperm membrane molecules.

Acrosome-reacted sperm are also able to bind to the zona pellucida. It has been shown in guinea pig that acrosome-reacted sperm can initiate binding to the zona pellucida as effectively as acrosome-intact sperm, and this may also be true of rabbit and human sperm. In all species it is presumed that, after the acrosome reaction, the sperm must bind or rebind at least until zona penetration has begun so that they will not be lost from the zona surface. In mouse there is some evidence that the binding of acrosome-reacted sperm occurs to ZP2. The identity of the binding partner(s) on acrosome reacted sperm is still being researched.

In order for sperm to reach the egg plasma membrane they must penetrate through the zona pellucida (Fig. 2). This may involve digestion of a path through the zona and could require enzymes either released by acrosome reacting sperm, or associated with the sperm surface, including the newly inserted inner acrosomal membrane. Only acrosome-reacted sperm have been observed to penetrate through the zona. Motility is maintained during penetration and the narrow penetration slit in the zona that the sperm move through may also be created, in part, by mechanical forces.

When sperm penetrate the zona they come to lie in the narrow space between the inner boundary of the zona and the egg plasma membrane, the perivitelline space (Fig. 2). At this stage the sperm will first bind to the egg plasma membrane and then fuse with it. There may be more than one mechanism that allows sperm to bind, because sperm from heterologous species or acrosome-intact sperm can bind without being able to fuse. The binding that is required for fusion may involve a sperm membrane protein called fertilin (or PH-30) which probably has an egg membrane integrin as an adhesion partner. If this binding step is blocked, then fusion is also blocked. One region of the fertilin/PH-30 protein that may participate in the fusion of the two lipid bilayers contains a sequence that resembles the fusion peptide of viral fusion proteins.

Fusion results in confluency between the sperm and egg membranes as well as the sperm and egg cytoplasms. At the time of fertilization, the egg receives an unknown signal that results in a rise in intracellular free Ca2+ and thereby activates the egg to initiate development of the new embryo. One of the consequences of activation is completion of meiosis, including production of the second polar body, and the initiation of mitotic divisions.

Suggested Reading

Florman HM, Babcock DF. Progress toward understanding the molecular basis of capacitation. In: Wassarman PM, ed. Elements of Mammalian Fertilization. Boca Raton: CRC Press; 1991:105-203.

Kopf GS, Gerton GL. The mammalian sperm acrosome and the acrosome reaction. In: Wassarman PM, ed. Elements of Mammalian Fertilization. Boca Raton: CRC Press; 1991:153-203.

Myles DG. Molecular mechanisms of sperm-egg membrane binding and fusion in mammals. Dev Biol 1993; I58:35-45.

Ward C, Kopf G. Molecular events mediating sperm activation. Dev Biol 1993;158: 9-34.

Yanagimachi R. Mammalian Fertilization. In: Knobil E, Neill J, eds. The Physiology of Reproduction. New York: Raven Press, Ltd.; 1988:135-185.