ABSTRACT
To interact with the egg, the spermatozoon must undergo several biochemical and motility modifications in the female reproductive tract, collectively called capacitation. Only capacitated sperm can undergo acrosomal exocytosis, near or on the egg, a process that allows the sperm to penetrate and fertilize the egg. In the present study, we investigated the involvement of cyclic adenosine monophosphate (cAMP)-dependent processes on acrosomal exocytosis. Inhibition of protein kinase A (PKA) at the end of capacitation induced acrosomal exocytosis. This process is cAMP-dependent; however, the addition of relatively high concentration of the membrane-permeable 8-bromo-cAMP (8Br-cAMP, 0.1 mmol l-1) analog induced significant inhibition of the acrosomal exocytosis. The induction of acrosomal exocytosis by PKA inhibition was significantly inhibited by an exchange protein directly activated by cAMP (EPAC) ESI09 inhibitor. The EPAC selective substrate activated AE at relatively low concentrations (0.02-0.1 μmol l-1), whereas higher concentrations (>5 μmol l-1) were inhibitory to the AE induced by PKA inhibition. Inhibition of PKA revealed about 50% increase in intracellular cAMP levels, conditions under which EPAC can be activated to induce the AE. Induction of AE by activating the actin severing-protein, gelsolin, which causes F-actin dispersion, was inhibited by the EPAC inhibitor. The AE induced by PKA inhibition was mediated by phospholipase C activity but not by the Ca2+-channel, CatSper. Thus, inhibition of PKA at the end of the capacitation process induced EPAC/phospholipase C-dependent acrosomal exocytosis. EPAC mediates F-actin depolymerization and/or activation of effectors downstream to F-actin breakdown that lead to acrosomal exocytosis.
ABSTRACT
To interact with the egg, the spermatozoon must undergo several biochemical and motility modifications in the female reproductive tract, collectively called capacitation. Only capacitated sperm can undergo acrosomal exocytosis, near or on the egg, a process that allows the sperm to penetrate and fertilize the egg. In the present study, we investigated the involvement of cyclic adenosine monophosphate (cAMP)-dependent processes on acrosomal exocytosis. Inhibition of protein kinase A (PKA) at the end of capacitation induced acrosomal exocytosis. This process is cAMP-dependent; however, the addition of relatively high concentration of the membrane-permeable 8-bromo-cAMP (8Br-cAMP, 0.1 mmol l-1) analog induced significant inhibition of the acrosomal exocytosis. The induction of acrosomal exocytosis by PKA inhibition was significantly inhibited by an exchange protein directly activated by cAMP (EPAC) ESI09 inhibitor. The EPAC selective substrate activated AE at relatively low concentrations (0.02-0.1 μmol l-1), whereas higher concentrations (>5 μmol l-1) were inhibitory to the AE induced by PKA inhibition. Inhibition of PKA revealed about 50% increase in intracellular cAMP levels, conditions under which EPAC can be activated to induce the AE. Induction of AE by activating the actin severing-protein, gelsolin, which causes F-actin dispersion, was inhibited by the EPAC inhibitor. The AE induced by PKA inhibition was mediated by phospholipase C activity but not by the Ca2+-channel, CatSper. Thus, inhibition of PKA at the end of the capacitation process induced EPAC/phospholipase C-dependent acrosomal exocytosis. EPAC mediates F-actin depolymerization and/or activation of effectors downstream to F-actin breakdown that lead to acrosomal exocytosis.
Subject(s)
Humans , Male , 8-Bromo Cyclic Adenosine Monophosphate/pharmacology , Acrosome/metabolism , Acrosome Reaction/drug effects , Calcimycin/pharmacology , Cyclic AMP/metabolism , Cyclic AMP-Dependent Protein Kinases/antagonists & inhibitors , Exocytosis/drug effects , Guanine Nucleotide Exchange Factors/metabolism , Protein Kinase Inhibitors/pharmacology , Signal Transduction/drug effects , Spermatozoa/metabolism , Thapsigargin/pharmacologyABSTRACT
Objective To investigate effect of exchange protein directly activated by cyclic adenosine monophosphate 1 on mechanical pain in diabetic rats.Methods Male SD rats were randomly divided into CON group and STZ group. CON group were further randomly divided into CONshRNA group,and CONEpac1shRNA group. STZ group induced diabetic mechanical pain (DMA) model were randomly divided into DMA shRNA group, DMA Epac1shRNA group,DMANS group,and DMA 8-pCPT group. The Epac1shRNA lentivirus vector is used to inhibit the expression of Epac1, and the shRNA lentivirus vector is negative control, and 8-pCPT is the activator of Epac1. Group CONshRNA and group DMAshRNA were given intrathecal injection of control shRNA lentivirus vector. Group CONEpac1shRNA and group DMAEpac1shRNA were injected with Epac1shRNA lentivirus carrier, DMANS group was injected into the plantar saline,and 8-pCPT in group DMA8-pCPT was injected into the foot. The changes of hind paw retraction threshold(PWT) were observed and the expression of guanine nucleotide transforming factor 1(Epac1) mRNA was detected by Real Time-PCR and Western blot in rat dorsal root ganglion(DRG) And protein expression changes.Results Compared with CON rats,the mechanical pain threshold of STZ rats decreased (P=0.035). Compared with saline group,the pain of injection Epac1 activator 8-pCPT group was prolonged(2h, P=0.012;4h,P=0.020). The expression of Epac1 mRNA and protein was significantly higher in the DMA group than in the CON group(both P<0.01). Intrathecal injection of shRNA reduced the expression of Epac1 mRNA and protein(P<0.01,P=0.020),and the PWT of the DMA group was significantly lower than that of the CON group (P=0.006).Conclusion Epac1 expression in diabetic rats with increased pain,and down-regulation of Epac1 may relieve pain.