Sperm Proteasomes Degrade Sperm Receptor on the Egg Zona Pellucida during Mammalian Fertilization

Satish K Gupta,Peter Sutovsky,Miriam Sutovsky,David J Miller,Shawn W Zimmerman,Young-Joo Yi,John F Odhiambo,Michael D Powell,Gaurishankar Manandhar,Vladimir Uversky

doi:10.1371/journal.pone.0017256

Abstract

Despite decades of research, the mechanism by which the fertilizing spermatozoon penetrates the mammalian vitelline membrane, the zona pellucida (ZP) remains one of the unexplained fundamental events of human/mammalian development. Evidence has been accumulating in support of the 26S proteasome as a candidate for echinoderm, ascidian and mammalian egg coat lysin. Monitoring ZP protein degradation by sperm during fertilization is nearly impossible because those few spermatozoa that penetrate the ZP leave behind a virtually untraceable residue of degraded proteins. We have overcome this hurdle by designing an experimentally consistent in vitro system in which live boar spermatozoa are co-incubated with ZP-proteins (ZPP) solubilized from porcine oocytes. Using this assay, mimicking sperm-egg interactions, we demonstrate that the sperm-borne proteasomes can degrade the sperm receptor protein ZPC. Upon coincubation with motile spermatozoa, the solubilized ZPP, which appear to be ubiquitinated, adhered to sperm acrosomal caps and induced acrosomal exocytosis/formation of the acrosomal shroud. The degradation of the sperm receptor protein ZPC was assessed by Western blotting band-densitometry and proteomics. A nearly identical pattern of sperm receptor degradation, evident already within the first 5 min of coincubation, was observed when the spermatozoa were replaced with the isolated, enzymatically active, sperm-derived proteasomes. ZPC degradation was blocked by proteasomal inhibitors and accelerated by ubiquitin-aldehyde(UBAL), a modified ubiquitin protein that stimulates proteasomal proteolysis. Such a degradation pattern of ZPC is consistent with in vitro fertilization studies, in which proteasomal inhibitors completely blocked fertilization, and UBAL increased fertilization and polyspermy rates. Preincubation of intact zona-enclosed ova with isolated active sperm proteasomes caused digestion, abrasions and loosening of the exposed zonae, and significantly reduced the fertilization/polyspermy rates after IVF, accompanied by en-mass detachment of zona bound sperm. Thus, the sperm borne 26S proteasome is a candidate zona lysin in mammals. This new paradigm has implications for contraception and assisted reproductive technologies in humans, as well as animals.

Highlights

Mammalian spermatozoa become fertilization-competent during capacitation in the female oviduct, a process that alters the sperm motility pattern and primes the sperm exocytotic organelle, the sperm head acrosome, for interactions with the egg zona pellucida (ZP)[1,2]
Western blot analysis of porcine sperm receptor component ZPC was conducted on the supernatant fraction which, after coincubation, contained partially degraded ZP proteins as well as acrosomal shrouds of acrosome-reacted spermatozoa
We show here for the first time that mammalian sperm proteasomes degrade ZP proteins in a coincubation system relevant to fertilization

Summary

Introduction

Mammalian spermatozoa become fertilization-competent during capacitation in the female oviduct, a process that alters the sperm motility pattern and primes the sperm exocytotic organelle, the sperm head acrosome, for interactions with the egg zona pellucida (ZP)[1,2]. Upon binding to the sperm receptor on the mammalian ZP (ZPC protein in the mouse or ZPB-ZPC heterodimer in the pig), the fertilizing spermatozoon undergoes acrosomal membrane vesiculation and exocytosis of the acrosomal cap, referred to as the acrosome reaction or acrosomal exocytosis (AE)[3,4]. This event results in the formation of the acrosomal shroud, a sperm head-enveloping cluster of acrosomal membrane vesicles and matrices that exposes the acrosome-borne proteolytic enzymes. The second theory, introduced as early as 1958 by Austin and Bishop [10], proposes that the fertilizing spermatozoa release an enzyme, a putative zona ‘‘lysin’’, present in the sperm head acrosomal matrix [10,11]

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Conclusion