Involvement of cytoplasmic free calcium in boar sperm: head-to-head agglutination induced by a cell-permeable cyclic adenosine monophosphate analog.

摘要: When boar spermatozoa are incubated in a medium designed for vitro fertilization, many of them become agglutinated at the acrosomes. We previously reported that bicarbonate and cyclic adenosine 3',5'-monophosphate (cAMP) promote agglutination. The aim present study is to examine role cytoplasmic free Ca(2+) sperm agglutination induced by cell-permeable cAMP analogue. Spermatozoa were collected from five mature boars, washed, resuspended modified Krebs-Ringer-Hepes solution lacking calcium chloride. suspensions water bath (38.5 degrees C) 60 minutes then used determine percentages head-to-head spermatozoa. Percentages samples rose significantly after incubation, 28% 61%-62%, adding cell-permeable, phosphodiesterase-resistant analogue (cBiMPS, 10 microM) or an adenylyl cyclase stimulator (sodium bicarbonate, 5 mM) plus phosphodiesterase inhibitor (IBMX, 25 microM). However, promoting effects these reagents blocked when pretreated with chelator (BAPTA-AM, microM), whereas same pretreatment cell-impermeable (BAPTA, had almost no influence on Adding thapsigargin, potential Ca(2+)-ATPase inhibitor, raised concentration-dependent manner concentrations up 4 microM. microM thapsigargin cBiMPS examined their levels heads using indicator (fluo-3/AM), both either contained cells exhibited intense fluorescence heads. In control without contrast, most (unagglutinated) characterized only slight Moreover, morphological observation Giemsa-stained preparations revealed possessed darkly stained acrosomes, which distinguished acrosomereacted This indicated was not result acrosome reaction. Furthermore, indirect immunofluorescence Ca(2+)-ATPases, mouse monoclonal antibody this enzyme demonstrated high affinity acrosomes permeabilized Based results, we conclude involved