Article ID | Journal | Published Year | Pages | File Type |
---|---|---|---|---|
10801766 | Biochimica et Biophysica Acta (BBA) - Molecular Cell Research | 2016 | 59 Pages |
Abstract
Exocytosis of the sperm's single secretory granule, or acrosome, is a regulated exocytosis triggered by components of the egg's investments. In addition to external calcium, sperm exocytosis (termed the acrosome reaction) requires cAMP synthesized endogenously and calcium mobilized from the acrosome through IP3-sensitive channels. The relevant cAMP target is Epac. In the first part of this paper, we present a novel tool (the TAT-cAMP sponge) to investigate cAMP-related signaling pathways in response to progesterone as acrosome reaction trigger. The TAT-cAMP sponge consists of the cAMP-binding sites of protein kinase A regulatory subunit RIβ fused to the protein transduction domain TAT of the human immunodeficiency virus-1. The sponge permeated into sperm, sequestered endogenous cAMP, and blocked exocytosis. Progesterone increased the population of sperm with Rap1-GTP, Rab3-GTP, and Rab27-GTP in the acrosomal region; pretreatment with the TAT-cAMP sponge prevented the activation of all three GTPases. In the second part of this manuscript, we show that phospholipase Cε (PLCε) is required for the acrosome reaction downstream of Rap1 and upstream of intra-acrosomal calcium mobilization. Last, we present direct evidence that cAMP, Epac, Rap1, and PLCε are necessary for calcium mobilization from sperm's secretory granule. In summary, we describe here a pathway that connects cAMP to calcium mobilization from the acrosome during sperm exocytosis. Never before had direct evidence for each step of the cascade been put together in the same study.
Keywords
PLC8-pCPT-2′-O-Me-cAMP2-aminoethoxydiphenylborateGEFEpac2-APBIP3pKaGSTSACPBSPVPHTFIPTG1,4,5-trisphosphatecAMPSDS–PAGECyclic adenosine monophosphateStreptolysin Oslosodium dodecyl sulfate–polyacrylamide gel electrophoresisProtein transductionExocytosisisopropyl-β-d-thio-galactosideguanine nucleotide exchange factorphospholipase CPhosphate-buffered salineacrosome reactioncAMP-dependent protein kinasepolyvinylpyrrolidoneCalciumglutathione-S-transferase
Related Topics
Life Sciences
Biochemistry, Genetics and Molecular Biology
Biochemistry
Authors
Ornella Lucchesi, MarÃa C. Ruete, MatÃas A. Bustos, MarÃa F. Quevedo, Claudia N. Tomes,