Sarvamangala V. Prasad

Evaluating zona pellucida structure and function using antibodies to rabbit 55 kDa ZP protein expressed in baculovirus expression system.

A cDNA encoding the rabbit 55 kDa ZP protein was expressed using a baculovirus expression system and was evaluated for its ability to elicit antibodies which may interfere with sperm‐ZP interaction. The expressed glycosylated protein, BV55, was purified by wheat germ agglutinin lectin affinity chromatography. Antisera made in guinea pigs immunized with BV55 (GP‐α‐BV55) is specific for the 55 kDa rabbit ZP protein. Indirect immunofluorescence studies indicate that GP‐α‐BV55 localizes to a filamentous meshwork on the surface of the ZP of isolated rabbit eggs. Immunohistochemical analysis of rabbit ovaries demonstrated that this antigen is localized within the ZP of primary and more advanced stage ovarian follicles but is not detected in primordial follicles. In addition, the 55 kDa antigen was detected in the granulosa cells of secondary stage follicles but not in the oocyte. GP‐α‐BV55 effectively blocked the binding of rabbit sperm to rabbit eggs in vitro. However, Fab fragments generated from GP‐α‐BV55 failed to block sperm binding, suggesting that the inhibitory effect of GP‐α‐BV55 was due to stearic hindrance rather than specific blocking of a sperm receptor site. Although the Fab fragment did not inhibit sperm binding, additional studies demonstrated that biotinylated BV55 protein bound to rabbit sperm in the acrosomal region in a manner consistent with ligand activity in the sperm‐ZP interaction, and that BV55 bound to rabbit sperm in a dose‐dependent manner. These studies therefore demonstrate that antibodies against recombinant ZP proteins recognize the native intact ZP and inhibit sperm‐ZP interaction. They also provide evidence that the rabbit 55 kDa ZP protein, which is the homolog of the pig ZP3α sperm receptor protein, has sperm receptor activity.

Comparative Structure and Function of Mammalian Zonae Pellucidae

The process of fertilization, like other aspects of mammalian reproduction, varies markedly among different species. Of the many complex events that accompany this process, the interaction between the sperm and the zona pellucida (ZP) is one of the most significant. The mammalian ZP is an extracellular glycoprotein structure that is formed around the oocyte during the early stages of ovarian follicular development. This matrix is important in the initial stages of fertilization because the sperm must bind to and penetrate it before fusing with the oocyte plasma membrane (Austin and Braden, 1956; Austin, 1975). The ZP is also involved in the block to polyspermy in many species and further serves to protect the embryo after fertilization until implantation in the uterine wall. Species differences have been reported for several steps in the fertilization process, including sperm capacitation (the changes spermatozoa undergo in the female reproductive tract in order to develop the capacity to fertilize the egg) and sperm binding to and penetration of the ZP. Many of these differences can be attributed to morphological and biochemical variations of the gametes themselves (Yanagimachi, 1977; Bedford, 1974; other chapters in this text).

Zona pellucida antigens: targets for contraceptive vaccines.

The mammalian ovary is unique among secretory organs in its ability both to produce the female gamete and to provide the necessary endocrine support for its orderly development. This set of functions utilizes complex feedback mechanisms involving the hypothalamus and the anterior pituitary over the reproductive life of the individual. The late stages of follicular development just prior to ovulation have been the focus of most ovarian studies. However, it is necessary to investigate the earliest stages of follicular development in order to begin to understand the foundations of immunologically based interference with ovarian function.

Molecular Analysis of a Carbohydrate Antigen Involved in the Structure and Function of Zona Pellucida Glycoproteins

Abstract A lactosaminoglycan-associated antigen is associated with a carbohydrate moiety of all three zona pellucida (ZP) glycoproteins of pig and rabbit but is absent in the mouse and rat. A monoclonal antibody (PS1) recognizing this determinant was obtained by immunizing mice with a porcine ZP glycoprotein isoform purified by two-dimensional polyacrylamide gel electrophoresis. Conditions known to remove O-linked or sialic acid carbohydrate moieties (alkaline reduction; O-glycanase or neuraminidase enzymatic cleavage) did not remove the carbohydrate epitope. However, treatment with endo-β-glycosidase, endoglycosidase F, or combinations of neuraminidase plus β-galactosidase, totally removed the determinant, indicating that it is associated with a poly-N-acetyllactosaminoglycan structure present on an N-linked oligosaccharide. Molecular morphology studies using immunofluorescence and confocal microscopy techniques demonstrate that the PS1 antigen is localized at the surface of the ZP. Confirmation of this localization was obtained through studies that show that this antibody will inhibit homologous sperm binding to the pig ZP. Additional analyses using modular contrast microscopy and immunocytochemistry demonstrate that this carbohydrate-associated antigen is localized in discrete layers throughout the ZP matrix. These studies are the first to demonstrate the presence of a lactosaminoglycan type carbohydrate moiety in all three ZP proteins using a monoclonal antibody that appears to be involved in sperm recognition and structural organization.

Structure and Function of Mammalian Zonae Pellucidae

The zona pellucida (ZP) is the unique extracellular matrix surrounding the mammalian oocyte. During the fertilization process, sperm must first bind to and penetrate the zona before it comes into contact with its plasma membrane and fuses with the oocyte. While the association of the sperm with the ZP is thought to be a species-specific event in some species, this specificity has been shown to be limited in other mammalian species. After binding, the sperm must penetrate the ZP matrix through limited proteolytic digestion by sperm enzymes. The ZP of some species has also been shown to be modified following fertilization resulting in a block to polyspermy, a process referred to as the “zona reaction”. The zona reaction has been attributed to chemical modifications of the ZP presumably due to the release of components from cortical granules initiated by fertilization (1). The ZP matrix remains intact after fertilization in order to support the cleaving cells of the blastocyst. It has also been thought to aid in the movement of embryo in the oviduct, and to ensure proper embryonic development as well as to prevent embryo fusion in the oviduct.