The role of spermatozoa is to fertilize eggs.  However, mammalian spermatozoa produced in large numbers compared to eggs are incapable of fertilization upon ejaculation. They must first undergo a physiological change called capacitation and a subsequent morphological change known as the acrosome reaction. Both of these events occur in the female reproductive tract, with the sperm number ultimately decreasing to one, as a single spermatozoon fertilizes one egg. Thus, the study of fertilization is intrinsically disadvantaged be the small number of cells to be examined in vivo. The in vitro fertilization system was introduced to circumvent this problem. Initially, it required female factors in the fertilization medium, but soon, a defined medium was reported in which spermatozoa could be capacitated, acrosome reacted and eggs fertilized.  The success of in vitro fertilization allowed many researchers to investigate the mechanism of fertilization under a range of controlled conditions and many factors contributing to sperm-egg interaction were identified.

The clinical applications of in vitro fertilization (IVF) were very successful and resulted in the 2010 Nobel Prize won by Dr. Edwards.  Despite publication of many contributing factors, however, the precise mechanisms of fertilization remained unspecified. This might have been due to inherent scientific weaknesses of IVF.  First, IVF results are easily influenced by various conditions.  Thus, the fertilizing ability of the same spermatozoa can be evaluated differently depending on the medium used. Second, a relatively larger number of spermatozoa are required for fertilization in vitro than in vivo.  Therefore, the mechanism of fertilization in vitro might not faithfully reflect fertilization in vivo.

In the 1990s, usage of gene-manipulated animals such as acrosin-disrupted mice was incorporated as an alternative means to elucidate the mechanism of fertilization. Initially, genes thought to be important for fertilization were disrupted, but many demonstrated no severe phenotype in fertilization. However, many genes essential for fertilization have emerged from gene disruption experiments. To our surprise, among these genes, more than 10 gene-disrupted mouse lines shared common phenotypes, with i) no migration into the oviduct in vivo and ii) aberrant zona binding ability in vitro. Investigation of these many gene-disrupted mouse lines would help elucidate the mechanism of sperm-egg interaction in vivo.

The mechanism of sperm-egg fusion is also beginning to be clarified by using gene-manipulated animals such as disruption of Izumo1 on spermatozoa and CD9 and Juno on eggs. Recently, a direct binding was reported in IZUMO1 and JUNO. However, no fusogenic domain was found in either of these factors. An additional factor or factors, or unknown modifications of these factors, must exist or occur before fusion.

Gene-manipulation now allows us to view live sperm inside uterus or in oviduct (green fluorescent acrosome, green or red fluorescent mitochondria spermatozoa, etc.). The visualization of important factors on spermatozoa and eggs is also possible (mCherry-IZUMO1, GFP-CD9 etc.)   Use of gene-manipulated animals seems to be ideally suited to the field of fertilization research, as the mysterious behavior of gametes is challenging to reproduce in vitro.