Roles of cAMP in regulating microtubule sliding and flagellar bending in demembranated hamster spermatozoa

Abstract

To understand the mechanism regulating spermatozoa motility, it is important to investigate the mechanism regulating the conversion of microtubule sliding into flagellar bending. Therefore, we analyzed microtubule sliding and its conversion into flagellar bending using a demembranated spermatozoa model in which microtubule sliding and flagellar bending could be analyzed separately by treating the demembranated spermatozoa with and without dithiothreitol, respectively. Using this model, we examined the roles of cAMP and its target molecules in regulating flagellar bending and microtubule sliding. Although flagellar bending did not occur in the absence of cAMP, microtubule extrusion occurred without it, suggesting that cAMP is necessary for the conversion of microtubule sliding into flagellar bending, but not for microtubule sliding itself. The target of cAMP for regulating flagellar bending was not cAMP-dependent protein kinase (PKA), since flagellar bending was still observed in the spermatozoa treated with a PKA-specific inhibitor. Alternatively, the Epac/Rap pathway may be the target. Epac2 and Rap2 were detected in hamster spermatozoa using immunoblotting. Since Rap2 is a GTPase, we investigated the flagellar bending of demembranated spermatozoa treated with GTPgammaS. The treatment markedly increased the beat frequency and bending rate. These results suggest that cAMP activates the Epac/Rap pathway to regulate the conversion of microtubule sliding into flagellar bending.

Evidence for Existence of cAMP-Epac Signaling in the Heads of Mouse Epididymal Spermatozoa

“…The other isoform, Epac2, is prominently expressed in limited organs including the brain and adrenal gland. However, only limited data is available regarding the existence of Epac in the mammalian testes and spermatozoa (human [11]; hamster [12]). In this study, we investigated whether these two types of Epac mRNAs are expressed in the germ cells of the mouse testis using Northern blot analysis with DIG-labeled antisense cRNA probes.…”

“…In other words, PKA has been considered the main target of in t r a c e ll u l a r c A M P i n s p e r m a t o z o a [ 6 -9 ] . However, recent articles suggest that other cAMP signaling cascades [cAMP-cyclic nucleotide-gated cation channel (cNGC) (mouse [10]) and cAMPEpac signaling cascades (human [11], hamster [12])] are also involved in the regulation of sperm motility and acrosome reaction. However, the universality of sperm PKA-independent cAMP signaling among different animal species remains to be demonstrated.…”

Evidence of the Existence of Adenylyl Cyclase 10 (ADCY10) Ortholog Proteins in the Heads and Connecting Pieces of Boar Spermatozoa

“…The intracellular acceptor for bicarbonate in spermatozoa is an adenylyl cyclase 10 (ADCY10, formerly called soluble adenylyl cyclase) that is distinguished from the other isoforms by G protein-independent activation and lack of a membrane-binding domain. Binding of bicarbonate to ADCY10 obviously stimulates catalysis of adenosine 5'-triphosphates (ATP) to cyclic adenosine 3',5'-monophosphate (cAMP) [22,23], which is an important second messenger that can activate the protein kinase A (PKA)-mediated signaling cascades [11,24,25] and exchange protein directly activated by cAMP (Epac)-mediated signaling cascades, leading to the expression of sperm fertilizing ability [26][27][28][29]. Moreover, the PKA-mediated signaling cascades are also modulated by the protein phosphatase 1 [30,31].…”

Cryopreservation of Rat Sperm

“…Exogenous ATP in the freezing medium may be responsible for the generation of multiple metabolic signals that appear to be related to the sperm motility through a rise in calcium levels (Gibbons.1963, Kinukawa et al, 2006, Litvin et al, 2003, Luria et al, 2002, Ren et al, 2001, Rodriguez-Miranda et al, 2007; this reaction increases de novo ATP synthesis before freezing and may contribute to the remobilization of sperm after freezing-thawing. The motility of ram sperm was restored by exogenous ATP that crossed plasma membrane when the membrane was damaged by cryopreservation (Holt et al, 1992).…”

“…2001, Ren et al, 2001. cAMP induces protein phosphorylation by activating protein kinase A (Fujinoki et al, 2004, Kinukawa et al, 2006 and mediates calcium influx into sperm via the CatSper calcium ion channels (Cook and Babcock, 1983, Garbers. 2001, Ren et al, 2001.…”