Ovastacin, a cortical granule protease, cleaves ZP2 in the zona pellucida to prevent polyspermy

Laboratory of Cellular and Developmental Biology, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, MD 20892, USA.
The Journal of Cell Biology (Impact Factor: 9.83). 04/2012; 197(1):37-44. DOI: 10.1083/jcb.201112094
Source: PubMed


The mouse zona pellucida is composed of three glycoproteins (ZP1, ZP2, and ZP3), of which ZP2 is proteolytically cleaved after gamete fusion to prevent polyspermy. This cleavage is associated with exocytosis of cortical granules that are peripherally located subcellular organelles unique to ovulated eggs. Based on the cleavage site of ZP2, ovastacin was selected as a candidate protease. Encoded by the single-copy Astl gene, ovastacin is an oocyte-specific member of the astacin family of metalloendoproteases. Using specific antiserum, ovastacin was detected in cortical granules before, but not after, fertilization. Recombinant ovastacin cleaved ZP2 in native zonae pellucidae, documenting that ZP2 was a direct substrate of this metalloendoprotease. Female mice lacking ovastacin did not cleave ZP2 after fertilization, and mouse sperm bound as well to Astl-null two-cell embryos as they did to normal eggs. Ovastacin is a pioneer component of mouse cortical granules and plays a definitive role in the postfertilization block to sperm binding that ensures monospermic fertilization and successful development.

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    • "Mammalian fertilization requires the successful completion of a number of steps in a compulsory order [1] [2]. Previous studies have reported that the important players involved in fertilization, i.e., the sperm cell, the oocyte, and its surrounding cumulus cells, all contain and/or secrete several proteases [3] [4] [5] [6] [7] [8] [9]. The family of serine proteases, named after the serine residue in the active site of the enzyme, is the largest family of proteases and based on the current knowledge also the most represented on gametes [5]. "
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    ABSTRACT: Serine proteases are involved in mammalian fertilization. Inhibitors of serine proteases can be applied to investigate at which point these enzymes exert their action. We selected two serine protease inhibitors, 4-(2-aminoethyl)benzenesulfonyl fluoride hydrochloride (AEBSF, 100 μM) and soybean trypsin inhibitor (STI, 5 μM) from Glycine max, via previous dose-response IVF experiments and sperm toxicity tests. In the present study, we evaluated how these inhibitors affect porcine fertilization in vitro as calculated on total fertilization rate, polyspermy rate, and the sperm number per fertilized oocyte of cumulus-intact, cumulus-free, and zona-free oocytes. In the control group (no inhibitor), these parameters were 86%, 49%, and 2.2 for cumulus-intact oocytes and 77%, 43%, and 2.2 for cumulus-free oocytes (6-hour gamete incubation period, 1.25 × 10(5) spermatozoa/mL). 4-(2-Aminoethyl)benzenesulfonyl fluoride hydrochloride and STI significantly reduced total fertilization and polyspermy rate in cumulus-intact and cumulus-free oocytes (P < 0.05). Total fertilization rates were respectively 65% and 53% (AEBSF) and 36% and 17% (STI). Inhibition rates were higher in cumulus-free oocytes than in cumulus-intact oocytes, indicating that inhibitors exerted their action after sperm passage through the cumulus. 4-(2-Aminoethyl)benzenesulfonyl fluoride hydrochloride but not STI reduced sperm binding to the ZP. The acrosome reaction was significantly inhibited by both inhibitors. Only 40.4% (AEBSF) and 11.4% (STI) of spermatozoa completed a calcium-induced acrosome reaction compared to 86.7% of spermatozoa in the control group. There was no effect on sperm binding or fertilization parameters in zona-free oocytes. In conclusion, sperm-zona binding and acrosome reaction were inhibited by serine protease inhibitors during porcine IVF. Copyright © 2015 Elsevier Inc. All rights reserved.
    Theriogenology 08/2015; 84(8). DOI:10.1016/j.theriogenology.2015.07.022 · 1.80 Impact Factor
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    • "Thus, zona pellucida hardening (ZPH) is important for a second, 'slow' block against poly spermy, which would be incompatible with embryonic development in most mammalian species. Moreover, studies in ovastacin null mice showed that the cleavage of ZP2 also prevents binding of additional sperm cells to the zygote, indicating that ZP2 is a binding structure for spermatozoa at the zona pellucida (Burkart et al., 2012; Avella et al., 2013). "
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    ABSTRACT: Abstract The zona pellucida, a glycoprotein matrix surrounding the mammalian oocyte, hardens after intrusion of the first spermatozoon, thus protecting the embryo until implantation and preventing multiple fertilizations (polyspermy). Definitive zona hardening is mediated by the metalloprotease ovastacin, which is released from cortical granules of the oocyte upon sperm penetration. However, traces of ovastacin seep from unfertilized eggs to cause zona hardening even in the absence of sperm. These small amounts of protease are inactivated by the plasma protein fetuin-B, thus keeping eggs fertilizable. Once a sperm has penetrated the egg, ovastacin from cortical vesicles overrides fetuin-B and initiates zona hardening.
    Biological Chemistry 10/2014; 395(10):1195-9. DOI:10.1515/hsz-2014-0189 · 3.27 Impact Factor
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    • "Mut eggs serve as positive controls and normal 2-cell embryos as negative controls. Modified from refs Gahlay et al. (2010) and Burkart et al. (2012). "

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