© 2013. Published by The Company of Biologists Ltd | Development (2013) 140, 4471-4479 doi:10.1242/dev.090613 4471 ABSTRACT Fertilization is the process by which eggs and spermatozoa interact, achieve mutual recognition, and fuse to create a zygote, which then develops to form a new individual, thus allowing for the continuity of a species. Despite numerous studies on mammalian fertilization, the molecular mechanisms underpinning the fertilization event remain largely unknown. However, as I summarize here, recent work using both gene-manipulated animals and in vitro studies has begun to elucidate essential sperm and egg molecules and to establish predictive models of successful fertilization. KEY WORDS: Fertilization, Izumo1, Acrosome reaction, Eggs, Hyperactivation, Spermatozoa Introduction Our individual body has a limited lifetime. However, through fertilization we are able to continue life as a species. The role of spermatozoa is to fertilize eggs. However, mammalian spermatozoa cannot accomplish this task when ejaculated. They must first undergo a physiological change called capacitation and a subsequent morphological change known as the acrosome reaction in the female reproductive tract. Spermatozoa also harbor the ability to migrate into the oviduct, where they interact with and subsequently fuse with the egg. A number of factors that contribute to sperm-egg interactions have been identified, based on observations using enzyme inhibitors and antibodies in in vitro fertilization systems (Box 1). This research led to the conclusion that various sperm enzymes within the acrosome dissolved the egg components and that various membrane proteins were used for binding with eggs. However, recent experiments using gene disruption of these factors did not result in an infertile phenotype, suggesting that they are not essential for fertilization, although they may indeed play a role during the fertilization event. By contrast, using in vivo gene- targeting experiments, a number of proteins have unexpectedly emerged as being essential factors for fertilization. In this Primer, I discuss the factors that have been implicated in the various stages of fertilization, ranging from sperm capacitation and migration to sperm-egg fusion. Newly arising views of mammalian fertilization are reviewed and compared with previously postulated models. The nature of eggs Ovaries are endowed at birth with a fixed number of oocytes enclosed in primordial follicles. This number declines as a result of ovulation and atresia during the reproductive life of the female (Faddy, 2000). Oocytes are arrested in the dictyate stage of first meiotic division. Over time, cohorts of oocytes enter into a growth phase and become among the largest cells in the body, their diameters reaching about 80 μm and 100 μm in mouse and human, PRIMER Center for Genetic Analysis for Biological Responses Research Institute for Microbial Diseases Osaka University, Yamadaoka 3-1, Suita, Osaka 565-0871, Japan. *Author for correspondence (okabe@biken.osaka-u.ac.jp) respectively. During growth, eggs form an extracellular matrix called the zona pellucida (ZP) by secreting glycoproteins (Fig. 1A). The ZP of human eggs consists of four ZP glycoproteins (ZP1 to ZP4), whereas that of the mouse egg consists of three ZP proteins (ZP1 to ZP3; mouse Zp4 is a pseudogene). Full oocyte growth must be supported by surrounding granulosa cells, which proliferate and form multiple layers of cumulus cells that surround the ovulated egg (Fig. 1A). Cumulus cells support fertilization, and in vitro fertilization can be achieved more efficiently with them than without them (Jin et al., 2011; Tokuhiro et al., 2012). The molecular basis of this observation remains obscure; however, a few studies have addressed the function of cumulus cells (Oren-Benaroya et al., 2008; Shimada et al., 2008). Following meiotic maturation, eggs are ovulated in preparation for fertilization. After ovulation, the eggs are picked up by adhesion between the extracellular matrix of the cumulus cells and oviductal cells of the oviductal fimbriae and are subsequently transferred into the oviduct (Talbot et al., 2003). The eggs are then transported to the ampulla portion of the oviduct, where they await spermatozoa for fertilization. However, there is only a short ‘fertile window’, which is less than a day after ovulation in humans (Wilcox et al., 1995) and a few hours in the mouse, during which fertilization can successfully occur. The nature of spermatozoa In human testes, ~1000 spermatozoa are produced per second (Amann and Howards, 1980), although the reason why mammalian males produce so many spermatozoa to fertilize so few eggs is not understood. Spermatozoa produced in the testes are transferred to epididymides, where they receive various proteins (Busso et al., 2007), probably in part through a structure called the epididymosome (Frenette et al., 2010). The spermatozoa are Box 1. Studying fertilization in vitro In vitro fertilization (IVF) requires different elements for different species. In mice, eggs are usually collected from the oviduct after treatment with hormones that induce super ovulation. The eggs are introduced into a tiny drop of IVF medium on a dish covered by paraffin oil and cultivated under 5% CO 2 in air. Spermatozoa from mice are normally prepared by squeezing them out from an opening made in the epididymis, followed by suspension in IVF medium; alternatively, ejaculated spermatozoa are utilized in larger animals after washing with medium. The spermatozoa are introduced into the egg culture drop at a final concentration of ~1×10 5 spermatozoa/ml (note that IVF requires a large number of spermatozoa compared with fertilization in vivo, in which only a few spermatozoa are required per egg). The success of fertilization can be assessed by observing: (1) spermatozoa inside the ZP; (2) the formation of pronuclei; or (3) the formation of two-cell embryos. The eggs fertilized by IVF can also be transferred into oviducts of pseudo-pregnant females to assess the developmental outcome of these embryos. When antibodies or inhibitors added to the IVF medium successfully inhibit fertilization, the factors that these antibodies and inhibitors target have traditionally have been viewed as fertilization-related factors. The cell biology of mammalian fertilization Masaru Okabe* Development