Zona pellucida protein function

For fertiliation to occur, sperm has to reach the the egg plasma membrane, which, in vertebrates involves binding and penetration of a thick extracellular coat that surrounds the egg. This laver, called zona pellucida in mammals and perivitelline membrane in birds, is responsible not only for the initial sperm binding, but also for species specificitv, and participates in the induction of the acrosome reaction and the block to polyspermy. In the best- studied model, the mouse, it is composed of these major proteins, ZP l, ZPZ and ZP3. The study of these proteins in fertilization has been difficult because of the small amounts available and the necessity to sacrifce a large number of animals. The current proposal avoids these problems by examining the chicken perivitelline membrane (pvm) and ist constituents. Central to it is our capability in purifying large quantities of both chicken ZP1 and chicken ZP3, the two major components of the pvm. We will use these native proteins to examine their interaction with each other as well as with sperm and will define the respective receptors an both sperm and the pvm. The biosynthesis and the control of experssion of these important molecules will be examined. and post-translational processes as well as degradation of the pvm will be elucidated. Because of the highly conserved nature of the composition of the zona pellucida, we expect our findings to he of importance to other organisms as well. In addition, the existence of zona pellucida proteins not found in the mature egg envelope both in the chicken and in humans will be investigated with respect to the function of these proteins.

The eggs of all vertebrates are surrounded by a layer of extracellular material, which is called zona pellucida (zp) in humans and mammals, and perivitelline membrane (pvm) in birds. The first contact of sperm with the egg is made through this structure. Because of the obvious difficulties in experimenting with human oocytes, model animals such as mice are commonly used. However, in this particular case, results from mice are hard to interpret, because their zp is made of different molecules than the human zp. We have studied chicken pvm, which has several advantages: the eggs are large, so that large amounts of materials can be obtained, and for many experiments, the animals do not have to be killed, because laid eggs can serve as a source of the pvm. We have shown that the pvm of the chicken is produced in two steps. First, an early pvm is laid down, made by the oocyte itself. Then, when the egg begins to grow rapidly, the cells that surround it as well as the liver synthesize the molecules needed for the mature zpm. These synthetic processes are hormone-driven. All the proteins involved in these events share a common motif, the so-called zp domain, which is known to be involved in protein-protein interactions. In the course of this study, we have now established that the chicken ovary contains at least 8 different zp proteins (2 were known when we started). The liver-derived component, called ZP1, is released into the bloodstream and travels so to the ovary. At the same time, in male chickens, another zp protein, called ZP4, is being deposited on the tip of the sperm head. ZP1 on the egg can interact with ZP4 on the sperm and participate in sperm-egg recognition. After binding of sperm, ZP1 is rapidly degraded, which manifests itself as the appearance of holes in the pvm. In addition to ZP1, another zp protein is made in the liver and not in the ovary: ZPAY. It has a close relative, however, that is produced only in the ovary, and which is called ZPAX. Just like ZP1, ZPAY synthesis is dependent on sufficient amounts of estrogen and the protein is secreted into the bloodstream. Interestingly, it cannot be detected in the ovary at all, but rather in the tubular structures found in the kidney, in arteries and in blood vessels in the brain. This might indicate a special reinforcement need for these tubules in the chicken, where in females large quantities of lipoproteins are being transported in the blood to produce the yolk necessary for proper development of the chick.