|[Frontiers in Bioscience 1, d234-240, September 1, 1996]|
THE cAMP-DEPENDENT KINASE PATHWAY AND HUMAN SPERM ACROSOMAL
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
From all cells, only the human spermatozoa in seminal plasma have the potential to fertilize the ovum. It is not until they are removed from seminal plasma, either by passage through cervical mucus or in vitro washing techniques, that spermatozoa become competent to fertilize a fully developed human oocyte. It is thought that proteins present in seminal plasma stabilize the sperm plasma membrane. These proteins also may serve to occupy receptors integral to the exocytotic process until such a time when it is beneficial for them to be shed, e.g., after sperm passage through the cervix. A number of different terms, such as, decapacitation factor and acrosome stabilizing factor, have been applied to these proteins. It is at the time when the aforementioned proteins have been removed that changes begin to occur in the spermatozoon that prepare it for fertilization. These processes, collectively termed capacitation, were first described by Chang and Austin (1,2). Since these initial reports, a large number of review articles have been published addressing the physicochemical aspects of capacitation (3-23). While it is acknowledged that capacitation is a requisite preparatory process for fertilization, no clear recognizable marker for capacitation can be identified. At present, the only available marker to indicate the completion of capacitation is the occurrence of the acrosome reaction (see below). However, several characteristics have been associated with capacitation, and they are: 1) an increase in membrane fluidity, perhaps facilitated by the removal of seminal plasma proteins; 2) a decrease in the plasma membrane cholesterol to phospholipid ratio; 3) a decrease in net surface charge; 4) an increase in oxidative processes and production of cAMP; and 5) changes in swimming patterns of sperm. When sufficient time is allowed for capacitation to occur (the duration of this period is dependent upon the incubation conditions) sperm become susceptible to stimuli that will induce a process called the acrosome reaction.
The acrosome reaction is an exocytotic process and is a necessary step for successful penetration of the oocyte vestments and fertilization. On the basis of a number of investigations, it has long been held that mammalian spermatozoa must undergo a capacitative process, otherwise the acrosome reaction would not take place. However, this idea has recently been challenged by several investigators who have demonstrated that the human sperm acrosome reaction can be biochemically stimulated in a non-capacitated spermatozoa (24,25). Potentially, this occurs by bypassing membrane-mediated events. The conclusion from these and other findings is that capacitation and the acrosome reaction are distinct and distinguishable processes.
Morphologically, the acrosome reaction is characterized by point fusions between the plasma membrane and outer acrosomal membrane. These fusions are followed by the formation of fenestrations and hybrid vesicles. The vesicles consist of both plasma and outer acrosomal membranes. The interior of the acrosome, termed the acrosomal matrix, becomes exposed and solubilized by processes that are not completely understood. Upon or coincident with solubilization, an acrosomal serine glycoproteinase is converted from the zymogen form of the enzyme, proacrosin, to its active form, acrosin. Acrosin is thought to be a key enzyme for facilitating sperm binding and penetration through the oocyte vestments (10,12-14,19-22,26-31).
Surrounding the oocyte is an acellular glycoprotein matrix called the zona pellucida. The zona pellucida serves as a species specific barrier for fertilization. In addition, it is commonly held that a zona pellucida glycoprotein, termed ZP3, is primarily responsible for acrosome reaction induction (21,27,28). It is critical that the sperm membrane receptor or receptors for ZP3 be properly situated and primed to allow for the appropriate signaling and subsequent cascade of events that will culminate in the acrosome reaction, and lead to the localized activation and release of the acrosomal enzymes (19-22,27-31). A breakdown in this sequence of events can influence the outcome of fertilization. On the other hand, follicular and oviductal fluids, and cumulus oophorus can induce the acrosome reaction (30-35) in certain spermatozoa that are not yet temporally or spatially competent to fertilize an ovum. Furthermore, dysfunction in the intracellular signaling processes of the acrosome reaction (22,36-38) is likely to lead to reduced fertilization potential of the spermatozoon.
The acrosome reaction is analogous to exocytotic processes that occur in various somatic cells, such as, nerve terminals and mast cells. The exocytotic process in human spermatozoa appears to involve the following sequence of events (21,22,26,31,32,36-38,42-53). The interaction of an extracellular ligand, e.g., ZP3, with a sperm membrane-bound receptor causes a conformational change in a receptor-linked guanine nucleotide binding protein, G-protein. The G-protein influences one or more targets, including: ion channels, other G-proteins or membrane-associated amplifying enzymes (39,40). If the G-protein interacts with an amplifying enzyme, e.g., adenylyl cyclase, then the result is the hydrolysis of a precursor molecule, e.g., adenosine 5'-triphosphate (ATP), into a second messenger molecule, e.g., adenosine 3':5'-cyclic monophosphate (cyclic AMP). The second messenger activates a protein kinase, e.g., cAMP-dependent kinase, and thus phosphorylation of a protein integral to the exocytotic process (41). It is puzzling that no clear link between G-protein and target amplifying enzyme, second messenger or protein kinase has yet been demonstrated. Alternatively, several second messenger pathways have been shown to participate in the acrosome reaction (21,22,26,31,32,36-38,42-53).
This review will describe results from several reports in which the addition of cAMP analogues (dibutyryl cyclic AMP, 8-bromo cAMP), a stimulator of cAMP production (forskolin) or phosphodiesterase inhibitors (isobutylmethy lxanthine, papaverine, SQ 20009) to capacitated human spermatozoa culminates in the acrosome reaction. In addition, the use of inhibitors (adenosine, 2'-O-methyladenosine, 2',3'-dideoxyadenosine, KT5720, H-8, Walsh cAMP-dependent protein kinase inhibitor) that target specific pivotal enzymes in the cAMP-dependent kinase (PKA) pathway prevent induction of the AR when upstream components of the pathway are stimulated. These findings give credence to the possible role of the cAMP-dependent kinase signal transduction pathway in human sperm acrosomal exocytosis.