Abstract
Cyclic AMP stimulates sperm motility in a variety of mammalian species, but the molecular details of the intracellular signaling pathway responsible for this effect are unclear. The type IIalpha isoform of protein kinase A (PKA) is induced late in spermatogenesis and is thought to localize PKA to the flagellar apparatus where it binds cAMP and stimulates motility. A targeted disruption of the type IIalpha regulatory subunit (RIIalpha) gene allowed us to examine the role of PKA localization in sperm motility and fertility. In wild type sperm, PKA is found primarily in the detergent-resistant particulate fraction and localizes to the mitochondrial-containing midpiece and the principal piece. In mutant sperm, there is a compensatory increase in RIalpha protein and a dramatic relocalization of PKA such that the majority of the holoenzyme now appears in the soluble fraction and colocalizes with the cytoplasmic droplet. Unexpectedly the RIIalpha mutant mice are fertile and have no significant changes in sperm motility. Our results demonstrate that the highly localized pattern of PKA seen in mature sperm is not essential for motility or fertilization.
Highlights
Cyclic AMP analogues and phosphodiesterase inhibitors increase the motility of mature spermatozoa [1,2,3,4], and the phosphodiesterase inhibitor, pentoxyfylline, is routinely used to enhance the motility and acrosome reaction in sperm from infertile men [5]
It has been proposed that elevated cAMP stimulates the phosphorylation of sperm proteins by protein kinase A (PKA)1 leading to increased motility [2, 3, 6, 7]
These results suggest that type II PKA is the primary isoform of PKA in mature sperm and that it is tightly anchored to the particulate fraction of the sperm flagellum
Summary
Animals—Wild type and RII␣ mutant males were generated as described previously [32]. Animals used in this study were 75, 87.5, or 97% C57BL/6 with the remaining genetic background as 129 Sv/J. Volumes were adjusted to provide equal concentrations (determined with a hemacytometer) of sperm from wild type and mutant mice. The pellet was washed once by dispersal and sedimentation as above and resuspended in the same volume of fortified buffer A as used for the supernatant fraction (above) Equal volumes of these fractions from wild type and mutant sperm were loaded on 10% polyacrylamide gels, and PKA subunits were visualized as described above by Western blot. After a 5-h incubation and a further 30 min in the presence or absence of either 1 mM Sp-cAMP or 1 mM Rp-cAMP (Research Biochemicals Inc.), sperm motility was examined For both protocols, quantitative sperm motility analysis on videotaped sperm samples was done by using an automated Hamilton-Thorn Motility analyzer as described [36]. Statistical Analysis—Unpaired t tests were performed when comparing between wild type and mutant groups
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