Despite its importance, the molecular basis of mammalian gamete recognition has remained unclear. The enzyme beta-1,4-galactosyltransferase (Gal-transferase) has been viewed traditionally as a biosynthetic component of the Golgi complex, but is also found on the surface of many cells where it can bind its specific glycoside substrate on adjacent cell surfaces or in the extracellular matrix. In mouse it has been suggested that Gal-transferase on the sperm head mediates fertilization by binding oligosaccharide residues in the egg coat, or zona pellucida, and that the ability of the zona pellucida to bind sperm is conferred by oligosaccharides of the ZP3 glycoprotein. However, it has not been confirmed that Gal-transferase and ZP3 are in fact complementary gamete receptors whose interaction mediates sperm-egg binding. Here we show that mouse sperm Gal-transferase specifically recognizes those oligosaccharides on ZP3 that have sperm-binding activity, but does not interact with other zona pellucida glycoproteins. In contrast, all zona pellucida glycoproteins are recognized by non-sperm Gal-transferase, demonstrating a more stringent substrate specificity for the sperm enzyme. This interaction is required for sperm-egg binding because blocking or removing the binding site for Gal-transferase on ZP3 inhibits its ability to bind sperm. After the release of the sperm acrosome, the transferase relocalizes to a new membrane domain where it can no longer bind to ZP3, which is consistent with the inability of acrosome-reacted sperm to bind ZP3 or to initiate binding to the zona pellucida. Following fertilization, ZP3 is modified by egg cortical granule secretions so that it loses sperm receptor activity, which can be accounted for by a selective loss of its binding site for sperm Gal-transferase. These results show that sperm surface beta-1,4-galactosyltransferase and the egg-coat glycoprotein ZP3 are complementary adhesion molecules that mediate primary gamete binding in the mouse.

The Group VIA Phospholipase A 2 (iPLA 2 ␤) is the first recognized cytosolic Ca 2؉ -independent PLA 2 and has been proposed to participate in arachidonic acid (20:4) incorporation into glycerophosphocholine lipids, cell proliferation, exocytosis, apoptosis, and other processes. To study iPLA 2 ␤ functions, we disrupted its gene by homologous recombination to generate mice that do not express iPLA 2 ␤. Heterozygous iPLA 2 ␤ ؉/؊ breeding pairs yield a Mendelian 1:2:1 ratio of iPLA 2 ␤ ؉/؉ , iPLA 2 ␤ ؉/؊ , and iPLA 2 ␤ ؊/؊ pups and a 1:1 male:female gender distribution of iPLA 2 ␤ ؊/؊ pups. Several tissues of wild-type mice express iPLA 2 ␤ mRNA, immunoreactive protein, and activity, and testes express the highest levels. Testes or other tissues of iPLA 2 ␤ ؊/؊ mice express no iPLA 2 ␤ mRNA or protein, but iPLA 2 ␤ ؊/؊ testes are not deficient in 20:4-containing glycerophosphocholine lipids, indicating that iPLA 2 ␤ does not play an obligatory role in formation of such lipids in that tissue. Spermatozoa from iPLA 2 ␤ ؊/؊ mice have reduced motility and impaired ability to fertilize mouse oocytes in vitro and in vivo, and inhibiting iPLA 2 ␤ with a bromoenol lactone suicide substrate reduces motility of wild-type spermatozoa in a time-and concentration-dependent manner. Mating iPLA 2 ␤ ؊/؊ male mice with iPLA 2 ␤ ؉/؉ , iPLA 2 ␤ ؉/؊ , or iPLA 2 ␤ ؊/؊ female mice yields only about 7% of the number of pups produced by mating pairs with an iPLA 2 ␤ ؉/؉ or iPLA 2 ␤ ؉/؊ male, but iPLA 2 ␤ ؊/؊ female mice have nearly normal fertility. These findings indicate that iPLA 2 ␤ plays an important functional role in spermatozoa, suggest a target for developing male contraceptive drugs, and complement reports that disruption of the Group IVA PLA 2 (cPLA 2 ␣) gene impairs female reproductive ability.

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Bovine spermatozoa that have been exposed to seminal plasma possess more binding sites for heparin than sperm from the cauda epididymis that have not been exposed to accessory sex gland secretions. Seminal plasma exposure enables sperm, following incubation with heparin, to undergo zonae pellucidae-induced exocytosis of the acrosome. In this study, the regulatory role of seminal plasma heparin-binding proteins in capacitation of bovine spermatozoa by heparin was investigated. Plasma membranes from sperm exposed to seminal plasma in vivo or in vitro contained a series of acidic 15-17 kDa proteins not found in cauda epididymal sperm. Western blots of membrane proteins indicated that these 15-17 kDa proteins bound [125I]-heparin. Heparin-binding proteins were isolated by heparin affinity chromatography from seminal plasma from vasectomized bulls. Gel electrophoresis indicated that the heparin-binding peaks contained 14-18 kDa proteins with isoelectric variation, a basic 24 kDa protein, and a 31 kDa protein. Western blots probed with [125I]-heparin confirmed the ability of each of these proteins to bind heparin. Each of these proteins, as well as control proteins, bound to epididymal sperm. The seminal plasma proteins were peripherally associated with sperm since they were removed by hypertonic medium and did not segregate into the detergent phase of Triton X-114. Seminal plasma heparin-binding proteins potentiated zonae pellucidae-induced acrosome reactions in epididymal sperm. However, seminal plasma proteins that did not bind to the heparin affinity column were unable to stimulate zonae-sensitivity. Control proteins, including lysozyme--which binds to both heparin and sperm, were ineffective at enhancing zonae-induced acrosome reactions. These data provide evidence for a positive regulatory role of seminal plasma heparin-binding proteins in capacitation of bovine spermatozoa.

Abstract. The mammalian egg must be fertilized by only one sperm to prevent polyploidy. In most mammals studied to date, the primary block to polyspermy occurs at the zona pellucida, the mammalian egg coat, after exocytosis of the contents of the cortical granules into the perivitelline space. The exudate acts on the zona, causing it to lose its ability to bind sperm and to be penetrated by sperm previously bound to the zona. However, the cortical granule components responsible for the zona block have not been identified. Studies described herein demonstrate that N-acetylglucosaminidase is localized in cortical granules and is responsible for the loss in sperm-binding activity leading to the zona block to polyspermy.Before fertilization, sperm initially bind to the zona by an interaction between sperm surface GalTase and terminal N-acetylglucosamine residues on specific oligosaccharides of the zona glycoprotein ZP3 (Miller, D. J., M. B. Macek, and B. D. Shur. 1992. Nature (Lond.). 357:589-593). These GalTase-binding sites are lost from ZP3 after fertilization, an effect that can be duplicated by N-acetylglucosaminidase treatment.