Background

Unique to the male gamete, the acrosome is a lysosome-like membranous organelle that adorns the anterior region of the sperm head and is delineated by both inner and outer acrosomal membranes (Hermo et al., 2010a and 2010b). The acrosomal vesicle, so formed, encapsulates a myriad of structural and enzymatic components that are compartmentalized into either soluble or insoluble fractions (Guyonnet et al., 2012; Guyonnet et al., 2014). The latter of these, termed the acrosomal matrix, functions as a stable scaffold that allows for the controlled release of matrix associated proteins necessary for fertilization during the acrosome reaction (Guyonnet et al., 2014). This reaction is a distinctive exocytotic event that is initiated by the formation of multiple sites of fusion between the outer acrosomal membrane and the overlying plasma membrane. As this vesiculation process propagates across the anterior region of the sperm head, it destabilizes the acrosomal vesicle leading to the formation of hybrid membrane vesicles and the concomitant release of acrosomal contents (Barros et al., 1967). This, in turn, renders the fertilizing spermatozoon competent to penetrate the formidable outer vestments of the oocyte and access the underlying oocyte plasma membrane (Buffone et al., 2008).

Recent work suggests that the acrosome reaction proceeds with relatively slow kinetics through several intermediary steps (Buffone et al., 2012; Stival et al., 2016). These distinctive features underscore the highly specialized nature of this secretory process and the likelihood that it is governed by complex molecular mechanisms. Accordingly, the ability to complete a physiological acrosome reaction necessitates that spermatozoa must have first undergone a functional maturation process known as capacitation (Aitken and Nixon, 2013). Capacitation occurs during sperm ascent of the female reproductive tract and incorporates numerous biochemical and biophysical changes, including the modulation of intracellular ion concentrations, hyperpolarization of the sperm plasma membrane, and the induction of signaling cascades, which culminate in increased protein phosphorylation (Nixon and Bromfield, 2018). While these collective processes prime the sperm cell for completion of an acrosome reaction, the nature of the physiological stimulus responsible for initiating this exocytotic event remains a matter of some controversy (Buffone et al., 2014); with candidate agonists including zona pellucida ligands, follicular fluid and/or progesterone (a steroidal hormone that sperm encounter within the oviduct prior to zona pellucida adhesion) (Tesarik, 1985). Alternatively, acrosomal exocytosis can be recapitulated in vitro using non-physiological pharmacological reagents, such as the divalent cation ionophore, A23187 (Jamil and White, 1981). Indeed, by directly facilitating Ca²⁺ influx, the A23187 ionophore is relatively forgiving of the need for intracellular signaling mechanisms and effector pathways that otherwise exert strict control over the progression of the acrosome reaction (Reid et al., 2012; Zhou et al., 2017).

Notably, the characteristics of acrosomal exocytosis driven by calcium ionophore differ from those elicited in response to physiological stimuli, such as solubilized zonae pellucidae (Liu and Baker, 1996). Specifically, ionophore induction leads to random vesiculation across the sperm head. By contrast, solubilized zonae pellucidae stimulates a more orderly loss of acrosomal components that begins at the posterior acrosome before proceeding in an anterograde direction (Buffone et al., 2009). Whilst these data lend support to the concept of receptor-mediated events controlling membrane fusion and release of acrosomal components, the limited availability of human zonae pellucidae prevents this resource being widely used as a physiological stimulus in assisted reproductive settings (Liu et al., 2003). Thus, despite its limitations, monitoring of the ability of spermatozoa to undergo an ionophore-induced acrosome reaction has become an important diagnostic criterion of human sperm function testing in clinical laboratories (Nixon and Bromfield, 2020).

Within the clinical setting, the impetus to study acrosomal exocytosis stems from evidence that a failure to complete this event represents a relatively common etiology associated with the defective spermatozoa of male infertility patients; potentially accounting for up to one-third of all cases of failed in vitro fertilization in couples taking recourse to assisted reproductive programs due to male factor infertility (Liu et al., 2001; Liu and Baker, 2003). However, in addition to prospective evaluation of semen quality, assaying the ability spermatozoa to complete an acrosome reaction has utility in the context of andrological research for development of male contraceptives and/or detection of the cytotoxic effects of various environmental insults (Katen et al., 2016; Houston et al., 2018 and 2019). Accordingly, here we describe optimized protocols for the induction and monitoring of human sperm acrosomal exocytosis in an in vitro setting.