Molecular and Cellular Mechanisms Involved in the Regulation of Equine Sperm Motility

     Sperm motility is of central importance to male fertility.  Thus, significant research has been devoted to understanding the basic mechanisms that regulate flagellar function.  Among the most important sperm motility regulatory mechanisms are calcium signaling and the cAMP/Protein Kinase-A (PK-A) pathways (1-7).  In sperm, the majority of cAMP is produced by an atypical, “soluble” adenylyl cyclase (sAC) that is genetically and biochemically distinct from the classical transmembrane cyclase(8-15).  sAC, and the cAMP pathway, are required for normal sperm motility and male fertility (16).  One goal of this laboratory is to improve fertility and reproductive efficiency of male domestic animals, in particular stallions.  However, there are few studies designed to elucidate the mechanism(s) for regulation and modulation of equine sperm motility.

     Most studies on the molecular biology of sperm motility have been performed either in the mouse model system or in the human.  Fortunately, flagellar anatomy and function appear to be highly conserved among mammals and it is likely that much of the existing data also will apply to domestic species including the horse.  Since sperm motility is a central, necessary component of the natural fertilization process, a better understanding of the signaling pathways responsible for flagellar motility will benefit the veterinary clinician’s ability to manipulate and potentially enhance equine sperm motility and therefore fertility.

     This study is composed of three parts.  The first part involves adding N-(6-Aminohexyl)-5-chloro-1-napthalenesulfonamide hydrochloride (W-7), a calmodulin inhibitor, in varying concentrations to equivalent samples of stallion semen in order to test the hypothesis that part of calcium’s  regulation of sperm motility is achieved through calmodulin.  Once this is accomplished, part two will consist of using consistent concentrations of W-7 to inhibit sperm motility without affecting cell viability.  Then attempts will be made to restore motion through the addition of the following components: Thimerasol (a compound that causes release of calcium from intracellular stores), dibutyrl cyclic adenosine monophosphate (db cAMP - a second messenger that is responsible for activating PK-A and so is important for the regulation of sperm motility), and 5-isobutryl-1-methylxanthine (IBMX - a phosphodiesterase inhibitor that prolongs the activity of PK-A.  Finally, the last part of the study will involve the addition of these same compounds to samples of extended, cooled, stored semen once sperm motility has dropped below a set value of 60%.  It is hypothesized that the addition of these compounds to viable, yet nonmotile cells will cause an increase in total and progressive motility and possibly prolongs the useful lifespan of cooled, stored horse sperm.

     Thus far, results have shown that W-7 inhibits sperm motility in a concentration dependent manner without affecting sperm viability.  This indicates that calmodulin is important for the normal signaling pathways that regulate sperm motility.  Results for the remaining two portions of the study are pending.  

References 

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2.                Suarez SS, Vincenti L, Ceglia MW. Hyperactivated motility induced in mouse sperm by calcium ionophore A23187 is reversible. J Exp Zool. 1987;244(2):331-336.

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4.                White DR, Aitken RJ. Relationship between calcium, cyclic AMP, ATP, and intracellular pH and the capacity of hamster spermatozoa to express hyperactivated motility. Gamete Res. 1989;22(2):163-177.

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7.                Ho HC, Granish KA, Suarez SS. Hyperactivated motility of bull sperm is triggered at the axoneme by Ca2+ and not cAMP. Dev Biol. 2002;250(1):208-217.

8.                Braun T. The effect of divalent cations on bovine spermatozoal adenylate cyclase activity. J Cyclic Nucleotide Res. 1975;1(6):271-281.

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12.            Pastor-Soler N, Beaulieu V, Litvin TN, et al. Bicarbonate-regulated adenylyl cyclase (sAC) is a sensor that regulates pH-dependent V-ATPase recycling. J Biol Chem. Dec 5 2003;278(49):49523-49529.

13.            Litvin TN, Kamenetsky M, Zarifyan A, Buck J, Levin LR. Kinetic properties of \"soluble\" adenylyl cyclase. Synergism between calcium and bicarbonate. J Biol Chem. May 2 2003;278(18):15922-15926.

14.            Zippin JH, Levin LR, Buck J. CO(2)/HCO(3)(-)-responsive soluble adenylyl cyclase as a putative metabolic sensor. Trends Endocrinol Metab. Oct 2001;12(8):366-370.

15.            Jaiswal BS, Conti M. Calcium regulation of the soluble adenylyl cyclase expressed in mammalian spermatozoa. Proc Natl Acad Sci U S A. Sep 16 2003;100(19):10676-10681.

16.            Esposito G, Jaiswal BS, Xie F, et al. Mice deficient for soluble adenylyl cyclase are infertile because of a severe sperm-motility defect. Proc Natl Acad Sci U S A. Feb 19 2004.