Acrosomal exocytosis of mouse sperm progresses in a consistent direction in response to zona pellucida

Abstract

Sperm acrosomal exocytosis is essential for successful fertilization, and the zona pellucida (ZP) has been classically considered as the primary initiator in vivo. At present, following what is referred to as primary binding of the sperm to the ZP, the acrosome reaction paradigm posits that the outer acrosomal membrane and plasma membrane fuse at random points, releasing the contents of the acrosome. It is then assumed that the inner acrosomal membrane mediates secondary binding of the sperm to the ZP. In the present work we used a live fluorescence imaging system and mouse sperm containing enhanced green fluorescent protein (EGFP) in their acrosomes. We compared the processes of acrosomal exocytosis stimulated by the calcium ionophore ionomycin or by solubilized ZP. As monitored by the loss of EGFP from the sperm, acrosomal exocytosis driven by these two agents occurred differently. When ionomycin was used, exocytosis started randomly (no preference for the anterior, middle or posterior acrosomal regions). In contrast, following treatment with solubilized ZP, the loss of acrosomal components always started at the posterior zone of the acrosome and progressed in an anterograde direction. The exocytosis was slower when stimulated with ZP and on the order of 10 sec, which is in accordance with other reports. These results demonstrate that ZP stimulates acrosomal exocytosis in an orderly manner and suggest that a receptor-mediated event controls this process of membrane fusion and release of acrosomal components. These findings are incorporated into a model.

A model of the acrosome reaction progression via the acrosomal membrane-anchored protein equatorin

“…Another report by Buffone et al (2009b) using acrosin-GFP transgenic mice showed the differences of the rate and region of intra-acrosomal GFP signals loss during the acrosome reaction after comparison with those induced by solubilized zona pellucida proteins …”

“…under spontaneous acrosome reaction) and since the progression speed of the acrosome reaction was slower than that of artificial acrosome reaction. In addition, Buffone et al (2009b) proposed that the acrosome reaction orderly occurs with membrane fusion between plasma membrane and OAM under the controlled receptor-mediated manner, forming hybrid vesicles and releasing acrosomal components. In this context, the present model of the acrosome reaction progression based on the equatorin behavior will be useful to further analyze the molecular mechanism during the acrosome reaction.…”

“…zonadhesin (Hardy et al 1991, Gao & Garbers 1998, Olson et al 2004, ACRV1/SP-10 (Foster et al 1994), AM67/ZP3R/sp56 (Cheng et al 1994, Foster et al 1997, Kim et al 2001a, ZPBP1/sp38/IAM38 (Mori et al 1995, Yu et al 2006, AM50 (Westbrook-Case et al 1994, Kim et al 2001b, MC41 (Tanii et al 2002), SAMP32 (Hao et al 2002), ESP , SAMP14 , and acrosomal enzymes (reviewed by Moreno & Alvarado (2006)). In previous reports relating acrosomal molecules as described above, the status of the acrosome and the acrosome reaction progression were often measured by acrosome-related lectin stains such as Arachis hypogaea agglutinin (PNA), concanavalin A, and Pisum sativum agglutinin (Kallajoki et al 1986, Mortimer et al 1987, Holden et al 1990, Holden & Trounson 1991, Kinger & Rajalakshmi 1995, De Blas et al 2005, Harper et al 2008, Mugnier et al 2009, and in transgenic mice such as acrosin-green fluorescent protein (GFP) transgenic mice (Nakanishi et al 1999, Kim & Gerton 2003, Buffone et al 2009b. In human sperm, the acrosome reaction progression was identified as having six stages, with the intermediate ones based on transmission electron microscopic observations, in which acrosomal matrix material was lost while outer membranes appeared to retain their integrity (Stock & Fraser 1987, Yudin et al 1988.…”

Membrane Fusions During Mammalian Fertilization

“…The now exposed intra-acrosomal layer of proteins consists of an array of proteins that interact with the zona pellucida (for review see [7]). This so-called secondary zona binding was thought to be specific to ZP2 [66][67][68] but recently it has been shown that the intra-acrosomal protein sp56 binds to ZP3 [4,12].…”

“…The membrane fusions are induced when the sperm cell binds to specific zona binding proteins at the sperm surface [3][4][5][6][7]. The acrosome exocytosis is a multipoint membrane fusion event between the sperm plasma membrane and the outer acrosomal membrane (see Fig.…”