Signaling Pathways for Modulation of Mouse Sperm Motility by Adenosine and Catecholamine Agonists1

Abstract

Capacitation of mammalian sperm, including alterations in flagellar motility, is presumably modulated by chemical signals encountered in the female reproductive tract. This work investigates signaling pathways for adenosine and catecholamine agonists that stimulate sperm kinetic activity. We show that 2-chloro-2'-deoxyadenosine and isoproterenol robustly accelerate flagellar beat frequency with EC50s near 10 and 0.05 microM, respectively. The several-fold acceleration is maximal by 60 sec. Although extracellular Ca2+ is required for agonist action on the flagellar beat, agonist treatment does not elevate sperm cytosolic [Ca2+] but does increase cAMP content. Acceleration does not require the conventional transmembrane adenylyl cyclase ADCY3, since it persists in sperm of ADCY3 knockout mice and in wild-type sperm in the presence of the inhibitors of conventional adenylyl cyclases SQ-22536, MDL-12330A, or 2', 5'-dideoxyadenosine. In contrast, the acceleration by these agents is absent in sperm that lack the predominant atypical adenylyl cyclase, SACY. Responses to these agonists are also absent in sperm from mice lacking the sperm-specific Calpha2 catalytic subunit of protein kinase A (PRKACA). Agonist responses also are strongly suppressed in wild-type sperm by the protein kinase inhibitor H-89. These results show that adenosine and catecholamine analogs activate sperm motility by mechanisms that require extracellular Ca2+, the atypical sperm adenylyl cyclase, cAMP, and protein kinase A.

Testicular Expression of Adora3i2 in Adora3 Knockout Mice Reveals a Role of Mouse A3Ri2 and Human A3Ri3 Adenosine Receptors in Sperm*

“…Adenosine Transporters in Acceleration of the Sperm Flagellar Beat-Our past work suggested that adenosine and related analogs may enter sperm via equilibrative and concentrative nucleoside transporters (ENTs and CNTs) to engage a cAMPmediated signaling pathway that increases sperm flagellar beat frequency (10,11). For the mouse, the Slc28a1-3 genes encode CNT1-3 (25) and Slc29a1-4 genes encode ENT1-4 (26).…”

“…We have combined biophysical methods with a genetic approach, using sperm from null-mutant (knock-out) mice that carry targeted disruptions of the genes for several of the components of the signaling pathways that operate in the sperm flagellum. This approach has provided definitive evidence for required roles of the unique CatSper ion channel proteins (1)(2)(3)(4)(5)(6)(7), for the atypical sperm adenylyl cyclase SACY 2 (3,8), and for the sperm-specific C␣2 catalytic subunit of PKA (9,10). It also produced the unexpected finding that adenosine analogs engage cAMP-mediated signaling to increase flagellar beat frequency (10,11).…”

“…However, in sperm the predominant (perhaps the only) adenylyl cyclase is the atypical SACY (ADCY10) (8), which lacks transmembrane domains and is unaffected by G-proteins (14,15). The apparent absence of conventional adenylyl cyclases led us to propose that adenosine analogs may enter sperm via cell surface nucleoside transporters to engage the SACY/PKA signaling pathway (10,11). We now reconsider this proposal, evaluating the expression of Slc28a and Slc29a nucleoside transporters in adult mouse testis, and examining the phenotype of sperm from the Slc29a1 knock-out mouse (16).…”

Acetylproteomic Analysis Reveals Functional Implications of Lysine Acetylation in Human Spermatozoa (sperm)

“…Of the proteins tagged with sperm capacitation, PRKACA, a catalytic subunit of cAMP-dependent protein kinase, was the only one that was not identified in the present study (Table II). It is known that mouse sperm lacking the sperm-specific PRKACA fail to respond to adenosine and catecholamine agonists that stimulate sperm flagellar beat frequency (40). Therefore, identification of PRKACA acetylation in capacitated sperm (31) rather than uncapacitated sperm implied that PRKACA acetylation might be correlated with stimulated sperm kinetic activity in capacitation.…”

Role(s) of the Serine/Threonine Protein Phosphatase 1 on Mammalian Sperm Motility

“…Both the cAMP and Ca 2þ signal transduction pathways are involved in activation of motility in immotile spermatozoa from the cauda epididymis in rat and mouse [Wade et al 2003;Schulh et al 2006]. A Ca 2þ dependent increase in cAMP initiates a signal transduction cascade for motility activation, which is independent of protein kinase A-mediated phosphorylation of flagellar proteins in immotile rat spermatozoa [Wade et al 2003].…”