Membrane-permeant Rab3A triggers acrosomal exocytosis in living human sperm.
The acrosome reaction is a regulated Ca2+-dependent secretion event required for sperm-egg interaction. Previous studies indicate that the process requires Rab3-dependent tethering of membranes, SNARE complex assembly, and Ca2+-mediated activation of synaptotagmin. Sperm are transcriptionally and translationally inactive; hence, most studies of the exocytosis mechanism are limited to membrane-permeant reagents. The effect of proteins involved in exocytosis has been assessed only in permeabilized cells. Polyarginine peptides are a powerful tool for delivering macromolecules to cells. Most reports indicate that membrane translocation of arginine-containing proteins requires endocytosis; therefore, this strategy might not be useful in sperm. However, our results indicate that GST and Rab3A, when fused with an arginine-rich peptide, were able to translocate into sperm. Moreover, membrane-permeant Rab3A initiated exocytosis when prenylated and activated with GTP. We show here that a key event after the cytoplasmic Ca2+ increase caused by progesterone is the activation of Rab3A. When active Rab3A is introduced into sperm, Ca2+ in the extracellular medium and in the cytoplasm is dispensable. However, a Ca2+ efflux from inside the acrosome is still required to achieve exocytosis. In conclusion, arginine-containing proteins can penetrate the sperm plasma membrane and thus are valuable tools to study sperm physiology in intact cells.
Lopez
Cecilia I
CI
Laboratorio de Biología Celular y Molecular, Instituto de Histología y Embriología (IHEM-CONICET), Facultad de Ciencias Médicas, Universidad Nacional de Cuyo, Mendoza, Argentina.
Belmonte
Silvia A
SA
De Blas
Gerardo A
GA
Mayorga
Luis S
LS
D000177:Acrosome / Q000201:enzymology / Q000378:metabolism*; D000818:Animals; D016466:CHO Cells; D002463:Cell Membrane Permeability / Q000502:physiology*; D006224:Cricetinae; D003412:Cricetulus; D004789:Enzyme Activation / Q000502:physiology; D005089:Exocytosis / Q000502:physiology*; D005260:Female; D006160:Guanosine Triphosphate / Q000502:physiology; D006801:Humans; D008297:Male; D017368:Protein Prenylation; D013094:Spermatozoa / Q000201:enzymology / Q000378:metabolism*; D020826:rab3A GTP-Binding Protein / Q000502:physiology*
D006160:Guanosine Triphosphate; D020826:rab3A GTP-Binding Protein