Most fertilizing mouse spermatozoa begin their acrosome reaction before contact with the zona pellucida during in vitro fertilization

Abstract

To fuse with oocytes, spermatozoa of eutherian mammals must pass through extracellular coats, the cumulus cell layer, and the zona pellucida (ZP). It is generally believed that the acrosome reaction (AR) of spermatozoa, essential for zona penetration and fusion with oocytes, is triggered by sperm contact with the zona pellucida. Therefore, in most previous studies of sperm-oocyte interactions in the mouse, the cumulus has been removed before insemination to facilitate the examination of sperm-zona interactions. We used transgenic mouse spermatozoa, which enabled us to detect the onset of the acrosome reaction using fluorescence microscopy. We found that the spermatozoa that began the acrosome reaction before reaching the zona were able to penetrate the zona and fused with the oocyte's plasma membrane. In fact, most fertilizing spermatozoa underwent the acrosome reaction before reaching the zona pellucida of cumulus-enclosed oocytes, at least under the experimental conditions we used. The incidence of in vitro fertilization of cumulus-free oocytes was increased by coincubating oocytes with cumulus cells, suggesting an important role for cumulus cells and their matrix in natural fertilization.sperm-egg interaction | acrosomal exocytosis | real-time imaging | video microscopy F ertilization is a critical moment in animal sexual reproduction and comprises sequential steps in the interaction of sperm with an egg. In eutherian mammals, natural fertilization is accomplished by sperm ascent to the oviduct ampulla, passage through the cumulus oophorus surrounding oocytes, loose binding to the oocyte's outer envelope, the zona pellucida (ZP), followed by tight sperm binding to the ZP and penetration through the ZP, culminating in sperm-oocyte membrane fusion (1). Before penetrating into the ZP, the fertilizing spermatozoon must undergo a morphological change involving disruption of the acrosome called the acrosome reaction (AR). In most studies of mouse sperm-oocyte interactions in vitro, the cumulus oophorus is removed by hyaluronidase treatment before insemination, allowing direct sperm contact with the ZP upon insemination. However, under natural in vivo conditions, spermatozoa first encounter the cumulus before reaching the ZP surface. The question this paper addresses is: Where does the fertilizing mouse spermatozoon begin the AR-in the cumulus or at the ZP?In the mouse, it has been reported that a ZP glycoprotein, ZP3, has both sperm-binding (sperm receptor) and AR-inducing activities and is essential for formation of the zona pellucida and therefore fertility (2, 3). The specificity of sperm binding to homologous zona might be conferred by the supramolecular structure (reviewed in ref. 4) or cleavage status of ZP glycoproteins (5). It is generally assumed that-at least in the mouse-the AR occurs after sperm binding to the ZP (6, 7). Even though spermatozoa of several species other than the mouse are known to be able to undergo the AR on the ZP surface (8-10), the presence of motile spermatozoa within the cumulus at...

Acrosome-reacted mouse spermatozoa recovered from the perivitelline space can fertilize other eggs

“…This was questioned by Jin et al (17), who inseminated mouse eggs in vitro, videorecorded them continuously, and analyzed all images retroactively to see which spermatozoa actually fertilized the eggs. They found that most fertilizing spermatozoa moving in the cumulus had initiated the AR (as judged by loss of a fluorescent marker for the acrosomal matrix) and then penetrated the ZP without spending much time on its surface.…”

Atg7 is required for acrosome biogenesis during spermatogenesis in mice

“…It plays an important role in the dispersion of cumulus cells and/or sperm penetration of the zona pellucida of the oocyte during fertilization [1,2]. Acrosome biogenesis begins with the initial phase of spermatid development and has been described as follows: in the Golgi phase of spermatogenesis, numerous proacrosomic granules are formed from trans-Golgi stacks, and they are transported to and accumulate in the concave region of the nuclear surface; they then fuse with each other to form a single large acrosomic granule that attaches to the nucleus.…”

Cl− Channels and Transporters in Sperm Physiology

“…Recent fi ndings have challenged this paradigm, and where, when, and what induces the AR are open questions again (Inoue et al 2011 ;Jin et al 2011 ;Kunzelmann et al 2011 ).…”

“…The fusion machinery involved in this reaction is regulated by Ca 2+ and similar to that found in many neuroendocrinal secretory cells (Bello et al 2012 ;Castillo Bennett et al 2010 ). Notably, where and what triggers the physiologically relevant AR is being reevaluated (Inoue et al 2011 ;Jin et al 2011 ;Visconti and Florman 2010 ;Yanagimachi 1998 ).…”