Viability and protein phosphorylation patterns of boar spermatozoa agglutinated by treatment with a cell-permeable cyclic adenosine 3',5'-monophosphate analog.

摘要: Boar spermatozoa become agglutinated with one another at the head when their intracellular cyclic adenosine 3',5'-monophosphate (cAMP)-signaling cascades are activated in head. The aim of present study is to examine viability and protein phosphorylation patterns cAMP-dependently boar spermatozoa. Ejaculated were washed then incubated a modified Krebs-Ringer HEPES medium containing polyvinyl alcohol (mKRH-PVA) plus 0.1 mM Sp-5,6-dichloro-1-beta-D-ribofuranosyl-benzimidazole-3',5'-monophosphorothioate (cBiMPS, cell-permeable cAMP analog) 38.5 degrees C up 180 minutes. Aliquots sperm suspensions recovered after various incubation periods used state agglutination, by SYBR14-PI staining motility assay, Western blotting indirect immunofluorescence. In control samples without cBiMPS for minutes, less than 30% total heads, more 70% propidium iodide (PI)-positive (dead). However, rapidly increased percentages head-to-head approximately 60% within 30 but did not significantly change them thereafter. moreover, PI-positive cells (approximately 30%) lower those obtained (more 70%). This result was supported observation that motile much higher minutes cBiMPS. As revealed immunofluorescence, cBiMPS-induced serine/threonine proteins (eg, >220 kd, 220 84 54 kd) appeared mainly connecting principal pieces both free additional occurred middle piece later Moreover, tyrosine 190 93 59 32 induced intensely moderately almost half rarely These findings consistent following suggestions: activation cAMP-signaling leads rapid (within minutes) agglutination live spermatozoa; subsequent (later them; slow connecting, middle, Based on these suggestions, we conclude many which leading whole flagellum.