Despite numerous studies on mammalian fertilization, the mechanisms of fertilization—including the timing of acrosome reaction—remain largely unknown; more accurately described, the classical theory built upon years of layered experimental data is being challenged by recent conflicting evidence provided by gene-manipulated animals. Although in vitro fertilization remains our central research tool, the classical theory’s decline reminds us of the importance of in vivo observations. Here, I describe the essential roles of gene-manipulated animals in elucidating the mechanism of fertilization and the pitfalls of in vitro fertilization studies trapping many researchers.
Keywords: acrosome reaction, fertilization, gene-manipulated animals, live imaging, sperm-egg fusion
ntroduction
The role of spermatozoa is to fertilize eggs. However, mammalian spermatozoa produced in large numbers compared to eggs are incapable of fertilizing upon ejaculation. They must first undergo a physiological change called capacitation and a subsequent morphological change known as the acrosome reaction [59]. 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 has an intrinsic disadvantage of small cell numbers to be examined. Fertilization was originally studied for the purpose of establishing an in vitro fertilization system [60]. Initially, the system required the female factors, but soon a defined medium was reported in which spermatozoa could be capacitated, acrosome reacted and eggs fertilized. This in vitro fertilization system was utilized by many researchers to analyze the mechanism of fertilization, with numerous factors contributing to sperm-egg interaction identified and classical models for the mechanisms of fertilization formed
Although the in vitro fertilization (IVF) system produced findings and clinical applications (such as the Nobel-recognized human in vitro fertilization), it has an inherent scientific flaw. Table 1 lists our in-house in vitro fertilization results using frozen BALB/C spermatozoa. As indicated, the fertilization rate using the same spermatozoa fluctuates from 12 to 98%. In the in vitro fertilization system, the same spermatozoa show very different fertilizing abilities when different media are used. In other words, in vitro fertilization is susceptible to external influences and therefore cannot offer an ideal examination of factors impacting fertilization. The fertilizing ability of the same spermatozoa can be evaluated in many ways depending on the condition of the medium. Moreover, a larger number of spermatozoa are required for fertilization in vitro than in vivo. We must keep in mind that the in vitro fertilization system is very different from natural fertilization.