[Frontiers in Bioscience 1, d234-240, September 1, 1996]


Christopher De Jonge, Ph.D., HCLD

Department of Obstetrics and Gynecology, University of Nebraska Medical Center, Omaha, Nebraska, USA

Received 05/30/96; Accepted 07/14/96; On-line 09/01/96


3.1 Assay conditions

A procedure used for testing the effect(s) of stimulators and inhibitors on the acrosome reaction is the synchronous acrosome reaction assay (54). In this assay, spermatozoa are incubated for 3 hours at 37°C in 5% CO2 atmosphere to induce capacitation. An acrosome reaction stimulator is then added to one of two tubes and the incubation is continued for an additional 15 minutes. An aliquot of capacitation medium containing spermatozoa is removed from each tube and assessed for the percentage of motile cells ("motility") before stopping the reaction. Inhibitors of the acrosome reaction are added at the end of the incubation period, i.e., after spermatozoa had become capacitated, 5 minutes prior to the addition of activator and the assay is continued as described above.

3.2 cAMP and calcium

The first step in the acrosome reaction, after the spermatozoa are fully capacitated, is generally considered to be an influx of exogenous calcium ions across the sperm membranes. The addition of exogenous calcium alone to capacitated human spermatozoa does not stimulate the acrosome reaction (37). However, the use of a calcium transporting agent, such as, calcium ionophore A23187, to facilitate calcium entry results in a significant stimulation of the acrosome reaction (37, 54). However, calcium ionophores, such as A23187, have been shown to cause the liberation of calcium from internal stores, which contributes to the exocytotic process (e.g., 57). An argument against this concept is that A23187 is unable to induce an acrosome reaction when calcium is not included in the capacitation medium (37), suggesting that intracellular storage sites for calcium that can effect an acrosome reaction may not exist in human spermatozoa. By contrast, dbcAMP does induce the AR in the nominal absence of calcium, indicating that dbcAMP is able to bypass the calcium transporting requirement (37). These findings can be interpreted to mean that cAMP exerts its effect after the influx of calcium during the cascade of events which lead to acrosomal exocytosis. This has been shown to be the case for some somatic cell processes (e.g., 41). Therefore it is possible that in human spermatozoa an influx of calcium either directly or indirectly activates adenylate cyclase, resulting in an increase in cAMP levels.

Addition of xanthine and non-xanthine phosphodiesterase inhibitors to capacitated spermatozoa stimulates the AR to the same extent as the cAMP analogues (37). However, methylxanthines have also been shown to alter calcium homeostasis and transport. Yet based on the diversity of the phosphodiesterse inhibitors used, and their varying specificity's, it can be reasoned that the stimulatory effect on the AR was due to an increase in cAMP levels rather than a change in Ca2+ transport. Furthermore, in contrast to some other cells, cAMP does not function by increasing the Ca2+ transport across the cell membrane (58).

3.3 Adenylate cyclase

Forskolin is a diterpine isolated from the Indian plant Coleum forskoli. In isolated cell membranes and intact cells from a variety of mammalian tissues, forskolin has been shown to activate adenylate cyclase (e.g., 59). The same activating response would appear to be the case when capacitated spermatozoa are treated with forskolin. Forskolin stimulates the acrosome reaction of capacitated human spermatozoa, and to the same extent as the cAMP analogues (37). The ability of an adenylate cyclase activator to stimulate the AR strongly suggests that adenylate cyclase has a role in the AR of human spermatozoa.

Adenosine interacts with adenylate cyclase at two locations. One position, called the "R-site," has properties associated with extracellular hormone receptor-mediated responses of the membrane adenylate cyclase. The second position, which is not a cell surface receptor, is called the "P-site" and mediates the inhibition of adenylate cyclase (60). The addition of adenosine and two adenosine analogues, 2'-O-methyladenosine and 2',3'-dideoxyadenosine, to spermatozoa prevented stimulation of the acrosome reaction by forskolin and independent of whether they were added at the onset of incubation or after incubation to induce capacitation (37). The latter two inhibitors have been shown to act as agonists of the P-site (60). Since the two adenosine analogues inhibited the forskolin-induced AR, it is possible that the sperm cyclase is regulated via a P-site. Further evidence for the involvement of the adenylate cyclase/cAMP pathway in the human sperm AR comes from the ability of dbcAMP to overcome adenosine inhibition and stimulate the reaction. These results offer compelling support for the involvement of adenylate cyclase in the acrosome reaction.

3.4 cAMP-dependent kinase

Inhibitors of cAMP-dependent kinase (PKA) typically bind to the catalytic subunit causing the displacement of the regulatory subunit thereby inhibiting the phosphorylating activity of the kinase. Inhibitors with good specificity for their target enzyme, i.e., kinases, have recently been shown to prevent stimulation of the AR by compounds that act at that same target or a target upstream from the site of inhibition (37). KT5720, a competitive and reversible inhibitor of PKA, completely prevented stimulation of the AR by forskolin and by dbcAMP when used at a maximum test concentration (100nM). However, two lower concentrations of KT5720 (25nM and 50nM) caused a 58% inhibition of the forskolin-induced AR and a 50% and 67% inhibition of the dbcAMP-induced AR (37). The dose-dependent decreases in the AR achieved using KT5720 would appear to be reflective of an overall decrease in PKA activity. Furthermore, the concentrations used that resulted in the in vivo inhibition of PKA, i.e., referring to inhibition of the enzyme in the viable cell and not of the extracted enzyme, are commensurate with the reported inhibition constant for this compound (Ki=0.056 mM) as tested on somatic cell PKA in vitro (62).

3.5 Solubilized zona pellucida

The AR is initiated during the penetration of the spermatozoon through the follicle cell layer of the oocyte, either just prior to or after contact with the zona pellucida. At present, although the oocyte stimulus or stimuli of the AR remain to be definitively identified, it is likely that they include one or more of the zona glycoproteins.

It has been shown that the human sperm AR can be induced by solubilized human zonae (sZP)(46). Further, when several PKA inhibitors were tested to determine if the signal transduction pathway stimulated in the spermatozoon by sZP might involve the cAMP-dependent kinase pathway, a significant reduction in the AR was detected. For example, the addition of KT5720 to medium containing sperm, and just prior to addition of sZP, caused a reduction in the AR but not to the same level as the control. However, when a combination of inhibitors of different kinases was tested, the sZP-induced AR was reduced to levels approaching that of the control. These results suggest that human sZP induces the AR via stimulation of kinases from three different signaling pathways, and one of which appears to be the cAMP-dependent kinase pathway.

3.6 Human follicular fluid

Previous data have shown that human follicular fluid stimulates an acrosome reaction in capacitated human spermatozoa (32-35). When an inhibitor of protein kinase A (KT5720) was added to spermatozoa at the end of the capacitation period and 5 min prior to the addition of inducer complete inhibition of the AR occurred (32). These data suggest that periovulatory hFF stimulates the human sperm AR and by activating the cAMP-dependent kinase pathway. Finally, it is clear that additional work is required to delineate the precise sequence in which the PKA pathway becomes involved in the exocytotic process following exposure to either hFF or ZP3.

[Table of Contents ] [Next Section] [Previous Section]