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(155) CHARACTERIZATION OF A NOVEL A-KINASE-ANCHORING PROTEIN (WAVE1) DURING MAMMALIAN SPERMATOGENESIS. Rawe, Vanesa1, Ramalho-Santos , Joćo 1,2, Payne, Christopher 1, Schatten, Gerald1, 1 Pittsburgh Development Center, MWRI, Pittsburgh, PA2 Center for Neuroscience and Cell Biology, Coimbra, Portugal ABSTRACT- Binding of the second messenger cyclic AMP (cAMP) to cyclic AMP-dependent kinase (PKA) results in protein phosphorylation and modulation of downstream activity. A-kinase-anchoring proteins (AKAPs), in turn, sequester PKA isoforms, which can shift signaling from the cytoplasm to other cellular structures. While studies have been carried out in somatic cells, little is known about the interaction of AKAPs and PKA in germ cells or gametes. Here we examine the distribution of three signaling molecules during mammalian spermatogenesis: AKAP WAVE1, a Wiskott-Aldrich syndrome (WASP) family protein, PKA regulatory subunit II (PKA RII), and small GTPase RAC1. For this purpouse, Immunofluorescence and Western blot analysis of mouse, bull, baboon and human spermatozoa and spermatogenic cells is performed. For immunofluorescence, samples are fixed with 2% formaldehyde and blocked before incubating with primary and secondaries antibodies. DNA is labeled using DAPI, and examined using a Nikon Eclipse E1000 epifluorescence microscope. For Western blotting, ejaculated sperm are lysed and proteins are separated by SDS-PAGE, transferred to membranes, blocked and incubated with antibodies. Bands are detected using chemiluminescence on autoradiographic film. WAVE1 localizes primarily to the Golgi apparatus in spermatocytes and round spermatids, and can also be found near the presumptive mitochondrial sheath in elongated spermatids and testicular sperm. In mature sperm, the distribution of WAVE1, PKA RII and RAC1 is consistent in all of the studied species. Both WAVE1 and PKA RII are enriched along the mitochondrial sheath, and PKA RII is also found on the principal piece. Small GTPase RAC1 distributes to the subequatorial region and the connecting piece. The identification of WAVE1, PKA RII and RAC1 in mammalian spermatozoa and spermatogenic cells suggests that their presence may be necessary for the normal development and function of sperm. In addition to possible functions in spermatogenesis, in mature sperm, the AKAP may be involved in the regulation of apoptosis by anchoring PKA to mitochondria and interacting with complexes with apoptotic function. Further investigation of these proteins should enhance our understanding of their contribution during mammalian fertilization. KEY WORDS: sperm, AKAPs, spermatogenesis, WAVE1 |
