PARENT SESSION

(M277) SIGNALING PATHWAYS FOR MODULATION OF MAMMALIAN SPERM MOTILITY BY GPCR AGONISTS.

Schuh, Sonya¹, Carlson, Anne¹, Xie, Fang², Conti, Marco², Hille, Bertil¹, Babcock, Donner¹, ¹ University of Washington, Seattle, WA² Stanford University School of Medicine, Stanford, CA

ABSTRACT- Although the sperm's role in fertilization is essential for survival of mammalian species, we understand relatively little about the processes, known collectively as capacitation, that prepare sperm to meet the egg. During capacitation in vivo, sperm motility may be modulated by signals encountered in the female reproductive tract. Adenosine (Ado) and norepinephrine (NE) present in reproductive fluids may have such modulatory roles. In vitro, these agonists have dose-dependent actions on sperm. Ado analogues and the catecholamines NE, epinephrine (E), and isoproterenol (ISO) rapidly and reversibly accelerate the flagellar beat of mouse epididymal sperm. In somatic cells these agonists operate though G-protein-coupled receptors (GPCR) that link to stimulation of a conventional adenylyl cylclase. In sperm, enzyme immunoassays confirmed that 45 s treatment with the Ado analogue, 2-chloro-2′-deoxyadenosine (Cl-dAdo), or ISO increased cAMP content from 2.6 +/- 0.1 to 4.1 +/- 0.2 and 3.8 +/- 0.3 pmol /10^7 cells, respectively. To determine whether these agonists couple to the atypical adenylyl cyclase of sperm (sAC), we examined sperm from sAC-null mice. Before exposure to the agonists, resting beat frequencies of WT and sAC-null sperm were similar. However, the accelerating action of Cl-dAdo on WT sperm was completely absent from sAC-null sperm (7.6 +/- 0.3 vs 2.7 +/- 0.2 Hz). ISO was similarly effective in WT and ineffective in sAC-null sperm (7.4 +/- 0.4 vs 2.8 +/- 0.1 Hz). In contrast, the beat of sAC-null sperm still accelerated from 2.6 +/- 0.1 Hz before to 6.3 +/- 0.4 Hz after cAMP content was elevated by incubation with 60 M of the cell-permeant cAMP-AM ester. Past work showed that cAMP-mediated actions of bicarbonate require the sperm-specific catalytic subunit of PKA (PKA C2). We now find that sperm of C2 null mice (from G. Stanley McKnight) also lack responses to either catecholamine or Ado agonists, and that the PKA inhibitor H-89 blocks stimulatory responses to these agonists. Together, these results indicate that the classical GPCR agonists, adenosine and catecholamines, modulate sperm motility in a pathway that uses sAC, cAMP, and PKA. Support: U54-HD12629 of the SCCPR program of NICHD; NIH R01-31544 (to M.C.); S.M.S. and A.E.C. supported in part by NIH T32 grants HD07183 and GM07270, respectively.

KEY WORDS: g-protein-coupled receptor, adenosine, catecholamines, sperm adenylyl cyclase (sAC)