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Master of Science (M.S.)
Year Degree Awarded
Month Degree Awarded
Sperm, Acrosome Reaction
REGULATION OF THE ACROSOME REACTION BY THE
TRANSMEMBRANE ADENYLYL CYCLASE
DOUGLAS HADDAD, B.S., UNIVERSITY OF MASSACHUSETTS AMHERST
M.Sc., UNIVERSITY OF MASSACHUSETTS AMHERST
Directed by: Pablo Visconti
Capacitation prepares mammalian sperm to undergo a process known as the acrosome reaction, which enables them to penetrate the zona pellucida. The standard method of measuring the acrosome reaction has been over the years the staining of the acrosome and visual counting using light or fluorescence microscopy. In this study we explored the intracellular signaling that results in the acrosome reaction using a novel method. This method employs the use of transgenic mice that contain green fluorescent protein, GFP, in the sperm acrosome. The quantity of sperm either containing or lacking GFP is precisely and rapidly quantified with flowcytometry. Currently little is known about the signaling processes that lead to the acrosome reaction. It has been proposed that this reaction is regulated by the cAMP activated guanine nucleotide exchange factor, Epac. In human sperm, stimulation of this pathway leads to an increase in acrosomal exocytosis. Furthermore, previous studies from our laboratory indicated that Gαs is present in the mouse sperm anterior head. These results suggest that in mouse sperm, the cAMP pathway leading to the acrosome reaction could be stimulated by Gαs associated with a transmembrane adenylyl cyclase. In this study we first validated the ability of known reaction inducers to increase the rate of the acrosome exocytosis in mouse sperm. Progesterone, solubilized zona pellucida and calcium ionophore A23187 all showed to be very effective at inducing the acrosome reaction in mouse sperm. We then investigated the role of the cAMP pathway using a battery of cAMP agonists and antagonists. We observed that stimulation of the cAMP dependent pathway through the transmembrane adenylyl cyclases, using forskolin, inhibit the increase in acrosome reaction induced by soluble zona pellucida, progesterone and the calcium ionophore A23187. This inhibitory effect was observed only when forskolin was used before the start of capacitation. Consistent with this observation, addition of cAMP analogues including an Epac specific cAMP analogue 8-pCPT-2’-O-Me-cAMP can also inhibit the increase in acrosome reaction by progesterone. This inhibition was seen only when the pathway was stimulated from the beginning of capacitation. Altogether, these data suggest that transmembrane cyclases are involved in the regulation of mouse sperm acrosome reaction.
Pablo E Visconti