Egg cortical granule N-acetylglucosaminidase is required for the mouse zona block to polyspermy.

Abstract

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.

Male Mice That Do Not Express Group VIA Phospholipase A2 Produce Spermatozoa with Impaired Motility and Have Greatly Reduced Fertility

“…In Vitro Fertilization Assay-Female mice about 30 days old were injected with pregnant mare serum gonadotropin (7.5 IU intraperitoneally) and 48 h later were injected with human chorionic gonadotropin (hCG; 7.5 IU intraperitoneally), as discussed (24). Oviducts were collected 13 h later, and the oocyte cumulus complexes were removed.…”

“…Spermatozoa and oocytes were co-incubated (5-24 h, 37°C, 5% CO 2 ). Oocytes and zygotes were passed through a pipette to remove cumulus cells, fixed (1% paraformaldehyde), placed on a microscope slide with affixed coverslip, treated with acetic acid/ ethanol (1/3 v/v, 2 min), stained (1% lacmoid/45% acetic acid), destained (45% acetic acid), and examined for oocyte fertilization reflected by a second polar body and two pronuclei with one near a sperm tail (24).…”

“…In Vivo Fertilization Assay-Superovulation of 6 -8-week-old female mice was induced by injecting pregnant mare serum gonadotropin (10 IU, intraperitoneally; Sigma) and, after 48 h, hCG (10 IU, intraperitoneal, Sigma), as described (21)(22)(23)(24). Those mice were then mated with iPLA 2 ␤ ϩ/ϩ or iPLA 2 ␤ Ϫ/Ϫ males overnight, and, after 48 h, the mice were euthanized with pentobarbital according to a protocol approved by our Animal Studies Committee.…”

Molecular cloning and sequence analysis of an ascidian egg β‐N‐acetylhexosaminidase with a potential role in fertilization

“…Although this demonstrates that ␤-hexosaminidase interferes with sperm binding and is required for the zona block to polyspermy, the precise molecular mechanism has yet to be elucidated. Furthermore, it is generally accepted that additional modifications of the zona, most probably induced by proteases, are required for a complete block to polyspermy (Miller et al 1993a). Despite striking similarities, the ascidian egg differs from the typical mammalian egg in its lack of cortical granules but its possession of accessory cells, the test cells, within the perivitelline space.…”

“…In mammals, ␤-hexo- saminidase has been localized to egg cortical granules and has been shown to be released upon fertilization-induced egg activation (Miller et al 1993a). Inhibition of this enzyme by antibodies or by competitive inhibitors prevents the loss of spermbinding activity normally seen in activated eggs.…”