Investigating the Role of Soluble Adenylyl Cyclase and Protein Kinase A in Acrosomal Exocytosis in Equine Spermatozoa
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Capacitation involves the molecular changes that ejaculated sperm undergo within the female oviduct to become fertilization-competent, and can be achieved in vitro by incubating sperm in a defined medium. During this process, sperm gain the ability to undergo acrosomal exocytosis whereby the release of proteolytic enzymes from the acrosome facilitates penetration of the oocyte vestments at fertilization; this is often used as a marker of capacitation. This study aims to investigate the signal transduction pathway leading to acrosomal exocytosis in stallion spermatozoa, particularly the role of soluble adenylyl cyclase (SACY) and protein kinase A (PKA). In order to clarify their function in supporting this event in stallion sperm, we use the SACY-specific inhibitor KH7, and the PKA-specific activator 3´-5´ cyclic adenosine monophosphate (cAMP) analog SP6. We add these agents to sperm incubated in capacitating conditions and compare rates of acrosomal exocytosis with those achieved after the addition of the agonists calcium ionophore (CaI) or progesterone, or control dimethyl sulfoxide (DMSO). First, it was determined that inhibiting SACY has no effect on rates of acrosomal exocytosis when the inducer is CaI, but partially reduces rates when the inducer is progesterone, albeit not to control levels. The results suggest that some cAMP production by SACY is required for acrosomal exocytosis under physiological conditions, but there may be multiple pathways involved. Next, it was found that stimulation of PKA by a cAMP analog resulted in rates of acrosomal exocytosis sperm higher than rates achieved with DMSO, suggesting that it is a PKA-dependent process.
Protein kinase A; soluble adenylyl cyclase; acrosome reaction; stallion; spermatozoa
dissertation or thesis