Fertilization with acrosome-reacted mouse sperm: implications for the site of exocytosis.

F or successful mammalian fertilization, sperm penetrate the outer investments of female gametes in the ampulla of the oviduct and fuse with the egg plasma membrane. The “fertilizing” sperm traverse the cumulus mass surrounding ovulated eggs, bind temporarily, and then penetrate through the zona pellucida, a structured extracellular glycomatrix, to enter into the perivitelline space (Fig. 1A). Underlying the anterior plasma membrane of the mouse sperm head is the acrosome, a Golgi-derived subcellular organelle. Sperm that approach ovulated eggs in the female reproductive tract have an intact acrosome. Before fertilization, the outer acrosomal membrane fuses with the sperm plasma membrane resulting in exocytosis of acrosomal contents (Fig. 1B). Only acrosome-reacted sperm are observed in the perivitelline space between the egg plasma membrane and the inner aspect of the zona pellucida and only acrosomereacted sperm fuse with the egg. Thus, at some point during sperm penetration through the investments of ovulated eggs, acrosome exocytosis must occur. But where (Fig. 1A)? This is the issue that concerns Inoue et al. (1) in their study in PNAS. Ovulated eggs in the ampulla of the oviduct are enmeshed in the cumulus oophorus composed of glycosaminoglycans interspersed with a gradient of residual cumulus cells from the ovarian follicle. Until recently, the prevailing model for mice was that acrosome-intact sperm pass through the cumulus oophorus and exocytosis is triggered by binding to the zona pellucida. Only acrosome-intact sperm are observed by EM on the surface of the zona pellucida and only acrosomereacted sperm are observed in the perivitelline space (2). The presence of vesiculated acrosomal shrouds on the zona surface (3–5) and the reported ability of solubilized zonae pellucidae or isolated mouse ZP3 mouse to induce acrosome exocytosis (6) are consistent with this model. However, the persistence of acrosome-intact Acr-EGFP sperm on the surface of the mouse zona pellucida for several hours is seemingly at odds with gamete binding transducing a signal that triggers acrosome exocytosis, and raises the possibility that initial penetration into the zona matrix plays the more important role (7). Alternatively, a recent investigation that used live imaging of Acr-EGFP sperm to fertilize eggs in cumulus reports that, with few exceptions, the fertilizing sperm is already acrosome-reacted when it encounters the zona pellucida (8). Earlier reports differ remarkably on the acrosome status of sperm within the cumulus mass, with some investigators observing acrosome-intact (9, 10) and others acrosome-reacted (8, 11) sperm. Further clouding the issue is the observation that some noneutherian mammals do not form a cumulus mass, and the efficacy of fertilizing cumulus-free compared with cumulus-intact eutherian eggs is variously reported as decreased or unchanged (12– 14). With these disparate reports, the site for induction of acrosome exocytosis continues to intrigue and remains a worthy focus of investigation. Inoue et al. (1) now provide evidence that acrosome-reacted mouse sperm can penetrate the zona pellucida and fertilize mouse eggs that develop to birth. These elegant studies take advantage of two genetically modified mouse lines. The first (Izumo I) lacks a sperm surface protein and is unable to fuse with normal eggs (15); the second (Cd9) lacks an egg plasma membrane protein and is unable to fuse with normal sperm (16). In each case, sperm accumulate in the perivitelline space and are acrosome-reacted. Using these sperm, the investigators report that acrosome-reacted sperm are able to fertilize normal eggs in cumulus. In a first experiment, normal female mice were mated to Izumo I male mice. Eight hours after coitus, eggs were collected from the oviduct and sperm were mechanically released from the perivitelline space. The Izumo-null sperm were incubated with normal eggs in cumulus, and sperm were observed in the perivitelline space of five of 74 normal eggs (7%) 12 h after reinsemination. These observations corroborate those from earlier studies in the rabbit and guinea pig. Normally, rabbit eggs accumulate multiple sperm in their perivitelline space because of a porous postfertilization block to zona penetration. Presumed to be acrosome-reacted, these sperm fertilize after reinsemination, albeit A