• Zona Pellucida
• The Zona Pellucida is made up of three major glycoproteins
• The Acrosome Reaction I
• Sperm-Egg Recognition & Binding
• Sperm-Egg Binding: Sequence of Events
• The Acrosome Reaction II
• Calcium & the Acrosome Rx
• Sperm Binding to the Zona & the Egg Cell Membrane
• Block to Polyspermy
• The Cortical Reaction

• Protein coat surrounding egg
• Species-specific barrier to sperm binding and penetration; keeps sperm of foreign species out
• Not 100% effective; some cross-species fertilization can occur (e.g., horse & donkey = mule; some species of monkeys can cross fertilize)
• Remove the ZP and other species sperm can fertilize and egg (e.g., Hamster Test for Male Fertility; human sperm can fertilize the zona-less hamster egg)

According to Magerkurth et al. (1999), there are four types of ZP. "Type A represented a distinct net-like structure made out of numerous pores and hollows which were arranged on the surface like windows. Type B was also a net-like structure which was composed of pores and hollows. In contrast to type A they were flatter and of smaller diameter. The zonae of type C had an uneven and spongy surface with very few or no porous areas. The net-like structure had nearly disappeared. Type D was characterized by a relatively smooth exterior of the zona pellucida." Below are photomicrographs of these types of zona pellucida.

• ZP3 mediates Sperm-Specific Egg Binding
• ZP2 mediates subsequent sperm binding
• ZP1 cross-links ZP2 and ZP3 as protein meshwork; not essential for fertilization but is important for structural integrity of zona pellucida; mice that lack ZP1 produce embryos that hatch prematurely causing developmental problems (Rankin et al, 1999. Development 126: 3847-3855).

The following diagram demonstrates how ZP1, ZP2 and ZP3 interact to form the zona pellucida. Learn this structure now because we'll re-examine it when we discuss the process of sperm-egg binding and, later, the block to polyspermy in mammals. This model is derived from research on mice and may vary slightly in humans (after Wassarman, 1990. Develop. Biol. 108: 1-17). However, the relevance of the mouse model in understanding human fertilization was recently shown when mutant mice which lacked ZP3 were given the human ZP3 gene in place of their own ZP3 gene (Rankin et al, 1998. Development 125: 2415-2424). The genes encoding mouse and human ZP3 are over 2/3 identical. The mutant mice which lacked ZP3 couldn't bind the sperm to the zona pellucida and thus did not get fertilized. When their missing ZP3 gene was replaced with a human ZP3 gene, the mice made the human ZP3 and it was incorporated into their zona pellucida. With the human ZP3 in their zonas the eggs could be fertilized by mouse sperm revealing that human and mouse ZP3 can mediate the same critical events.

When it contacts the egg cell layers, the sperm has an intact acrosome. Stimulation of the sperm by agents from the cumulus oophorus and especially the corona radiata followed by binding to the zona pellucida, leads to the acrosome reaction. The following sequence of events shows how this occurs and what events are associated with the acrosome reaction.

• Sperm contacts zona pellucida
• Sperm-egg binding occurs: Receptors on sperm bind to ZP3
• ZP3 and sperm-receptors are clustered away from contact site
• Acrosin digests pathway through zona and may assist in binding via ZP2
• ZP2-sperm binding holds sperm in place as it works its way through the zona

The mechanism of mammalian sperm-egg binding is under intense scrutiny. The problem is that the data from various labs is often contradictory. For an exhaustive review of this subject see: G. F. Clark and A. Dell, 2006. Molecular Models for Murine Sperm-Egg Binding. J. Biol. Chem. 281: 13853 - 13856. The possibility likely exists that there are various fall-back mechanisms in play so that if a mutation in one pathway affects sperm-egg binding then the sperm and egg use other binding mechanisms to ensure fertilization occurs. Here we will look at only one of the classic mechanisms of sperm-egg binding that leads to the acrosome reaction.

• Each sperm has galactosyl transferase (GalTase) enzymes on its head
• Galactosyl transferase is an enzyme that transfers a galactosyl group from one molecule to another
• Egg has ZP3 in ZP
• At binding, the cortical granules are intact
• Acrosome is is also intact

• Each sperm binds to sugar (N-acetylgalactosamine, GlcNAc) residues on the ZP3 via the galactosyl transferase (GalTase) enzymes on the sperm head
• The binding doesn't result in sugar transfer because some of the components needed for the enzyme reaction are missing; so the sperm remain attached via the sugar-enzyme binding because the enzyme reaction can not go to completion
• The receptors on the sperm head cluster together as calcium levels increase in the sperm cell cytoplasm
• Calcium is a known mediator of biomembrane fusion and this increase of intracellular calcium plays a role in the fusion of the acrosomal membrane with the sperm cell membrane causing vesicles to form (see below)
• This vesiculation allows the contents of the acrosome to flow out
• The enzymes begin to digest the zona pellucida

Calcium is known to trigger many biomembrane fusion events especially those involving exocytosis (e.g., transmitter release from synaptic vesicles in nerve cells). Calcium plays a part in the exocytosis of the acrosome during fertilization as evidenced by the following results.

While the interaction of galactosyltransferase with ZP3 in the zona is well characterized, there are many other proteins which appear to be involved in sperm-egg binding. In addition to GalTase and acrosin (mentioned in the previous lecture), there are other candidate proteins that may be involved in sperm-zona binding (Frayne & Hall, 1999. BioEssays 21: 183-187). Once through the zona, the sperm then contacts the egg cell membrane. Binding of the sperm cell membrane to the egg cell membrane is a critical step that occurs prior to sperm-egg fusion and also initiates the cortical reaction (discussed below). Of several proteins that mediate these membrane interactions, fertilin b appears to be important, at least in mice. Mouse mutants that lack fertilin b have a markedly reduced fertility (Cho et al, 1988. Science 281: 1857-1859).

• Polyspermy is the penetration of the egg by more than one sperm
• Polyspermy leads to abnormal development because the chromosomal # is altered
• Normally the low number of sperm cells in the fallopian tubes in mammals reduces chance of polyspermy
• Special Blocks to Polyspermy exist: 1. Fast Block: electrical change in egg membrane; 2. Slow Block: modification of zona pellucida

Calcium not only plays a role in the acrosome reaction, it also mediates the subsequent event of cortical granule exocytosis in the egg. Again much of the following is gleaned from studies on the mouse and other mammalian species.