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Biochemistry of Fertilization in Mammals

A. Cheng, D. Stout, N. Kresge, H. Fox,* J. Bleil

* Department of Neuropharmacology, TSRI

The first step in mammalian fertilization is recognition between the sperm head and the zona pellucida, an extracellular glycoprotein coat surrounding the egg. Fertilizing mouse sperm bind to ZP3, 1 of 3 glycoproteins in the zona pellucida. After binding, sperm undergo the acrosome reaction, which involves fusion between the plasma membrane overlying the sperm head and the apposed membrane of the acrosome, a lysosomal vesicle surrounding the nucleus. Acrosomal proteases allow the sperm to bore through the zona pellucida and fertilize the egg.

We have identified the mouse egg recognition protein (mERP), a homo-octameric 490-kD polypeptide with high and specific affinity for ZP3. In mice, mERP is expressed exclusively on sperm and in spermatogenic cells. The homo-octamer is assembled from pro-sp56 monomers, by formation of intermolecular disulfides, in the endoplasmic reticulum of early, round spermatids. Native mERP is transported to the Golgi complex of late-stage round spermatids and accumulates on the dorsal anterior surface of the heads of differentiated sperm. The mERP is tethered to the sperm head by an anchoring protein, which may play a role in signal transduction and the acrosome reaction (Fig. 1).

Freshly isolated mouse sperm bear mERP on their surface but do not recognize and bind to the zona pellucida. This lack of binding is largely due to the presence of molecules that accumulate on sperm during the transit of sperm through the epididymis, "coat" the surface of the sperm head, and apparently block mERP association with ZP3. Within 30 minutes in tissue culture, sperm lose this coat and acquire the ability to bind to the zona pellucida and undergo the acrosome reaction, a process known as "capacitation." In collaboration with H. Fox, Department of Neuropharmacology, we recently identified 2 transient sperm antigens that may function as part of the sperm coat. These antigens were identified on the basis of cross-reactivity with affinity-purified antibodies directed against a mouse brain protein, mBEC. The antigens accumulate on the head of epididymal sperm, precisely where mERP is located, and elute from sperm during capacitation. We propose to isolate and characterize these newly identified antigens and to test whether, by association with mERP, they prevent binding of sperm to the egg before completion of capacitation.

Structural studies on mERP, in collaboration with D. Stout, Department of Molecular Biology, have been enabled by heterologous expression of the protein from baculovirus. The expressed protein is secreted by insect cells as a biologically active homo-octamer.