The mechanism of sperm-egg interaction and the involvement of IZUMO1 in fusion

Abstract

An average human ejaculate contains over 100 million sperm, but only a few succeed in accomplishing the journey to an egg by migration through the female reproductive tract. Among these few sperm, only one participates in fertilization. There might be an ingenious molecular mechanism to ensure that the very best sperm fertilize an egg. However, recent gene disruption experiments in mice have revealed that many factors previously described as important for fertilization are largely dispensable. One could argue that the fertilization mechanism is made robust against gene disruptions. However, this is not likely, as there are already six different gene-disrupted mouse lines (Calmegin, Adam1a, Adam2, Adam3, Ace and Pgap1), all of which result in male sterility. The sperm from these animals are known to have defective zona-binding ability and at the same time lose oviduct-migrating ability. Concerning sperm-zona binding, the widely accepted involvement of sugar moiety on zona pellucida 3 (ZP3) is indicated to be dispensable by gene disruption experiments. Thus, the landscape of the mechanism of fertilization is revolving considerably. In the sperm-egg fusion process, CD9 on egg and IZUMO1 on sperm have emerged as essential factors. This review focuses on the mechanism of fertilization elucidated by gene-manipulated animals.

Figure 1

Mechanisms of sperm–egg interaction emerging from gene-manipulated animals. Some factors were found to be ‘essential' after gene disruption. After being judged by gene disruption, ADAM3, IZUMO1 and CD9 are concluded to be indispensable factors in zona-binding on sperm, gamete fusion on sperm and on egg, respectively. As a whole, the explanation of sperm–egg interaction requires significant modification from the gene manipulation point of view. ZP consists of ZP1, ZP2 and ZP3 in mouse. Recently, it has been proved that sperm–egg recognition depends on the cleavage status of ZP2 by gene-manipulated mice. ADAM, a disintegrin and metalloprotease; ZP, zona pellucida.

Figure 2

Gamete fusion-related factor IZUMO1. (a) IZUMO1 is an acrosomal membrane protein that is not exposed before the completion of an acrosome reaction (1). Acrosome-reacted sperm can be classified into three major groups by their IZUMO1-staining pattern: acrosomal cap (2), equatorial (3) and whole-head (4). (b) Accumulation of many sperm in the perivitelline space of the eggs recovered from the females mated with Izumo1^−/− males. Sperm in the perivitelline space were labeled with acrosome-reacted sperm-specific monoclonal antibody MN9.

Figure 3

N-linked glycan of IZUMO1 is not essential for fusion. (a) Comparison of the fusing ability of wild-type and N183Q-IZUMO1 sperm. The arrowheads indicate fused sperm. N183Q-IZUMO1 sperm are able to fuse with eggs, albeit in low yield (fusion index: 0.05 fused sperm/egg). (b) Fragmentation of N183Q-IZUMO1 protein in cauda epididymal sperm. N183Q-IZUMO1 is fragmented by protease in cauda epididymal sperm (filled arrowheads). Lane 1, Wild-type; Lane 2, Izumo1^−/−Izumo1-Tg; Lane 3, Izumo1^−/−Izumo1N183Q-Tg#1; Lane 4, Izumo1^−/−Izumo1N183Q-Tg#2.

Figure 4

Identification of IZUMO1-interacting proteins. (a) The purified IZUMO1 protein complex was separated by SDS–PAGE and then silver stained. Two specific 80- and 56-kDa bands appeared corresponding to ACE3 and IZUMO1, respectively. (b) Subcellular localization of ACE3 protein in mature sperm was examined in incubated cauda epididymal sperm. They were stained with anti-ACE3 (red) and anti-IZUMO1 (green) antibodies. The anti-ACE3 antibody stained acrosome-intact sperm head, but did not react to acrosome-reacted sperm (asterisk). Scale bar=20 µm. ACE3, angiotensin I-converting enzyme 3; PAGE, polyacrylamide gel electrophoresis.

Figure 5

SPESP1. (a) Many of the Spesp1-disrupted sperm showed abnormal spreading of IZUMO1 after acrosome reaction (indicated by asterisks in the figure). The localization pattern of MN9 antigen was also significantly affected by the disruption of Spesp1. Scale bars=10 µm. (b) Scanning electron microscopy of Spesp1-deficient and wild-type sperm. In wild-type sperm, the plasma membrane in the acrosome cap area disappeared down to the border of the equatorial segment after acrosome reaction. However, in Spesp1-deficient sperm, the plasma membrane disappeared from a wider area. As a result, the inner acrosomal membrane was exposed in most of the equatorial segment area (indicated by arrowheads). Scale bars=1 µm. SPESP1, sperm equatorial segment protein 1.