Incorporation of membrane- permeable cAMP sponge protein in human sperm.

LUCCHESI O; RUETE MC; BUSTOS MA; QUEVEDO MF; TOMES CN

Mendoza

Congreso; XLVIII Reunión Anual Sociedad Argentina de Investigaciones en Bioquímica y Biología Molecular (SAIB); 2012

Sociedad Argentina de Investigaciones en Bioquímica y Biología Molecular (SAIB)

Acrosomal reaction (RA) is a type of regulated exocytosis that leads to the release of the acrosomal granule content, a key event in fertilization. Sperm are transcriptionally and translationally inactive; hence, overexpression or silencing experiments cannot be performed. An alternative method to overcome this limitation is the use of membrane-permeant proteins. We have designed a cAMP sponge that possesses the sequence of the C-terminal domain of human PKA-RIβ (AA 133-380) coupled to a TAT peptide that confers membrane permeability and a His6-tag for purification and detection. We analyzed incorporation into human sperm by indirect immunofluorescence and found a high number of cells stained. cAMP-induced tyrosine phosphorylation is a hallmark of sperm capacitation. When we capacitated human sperm in the presence of the sponge, we observed a decrease in tyrosine phosphorylation. To evaluate the effect of cAMP depletion in the AR, we performed functional assays on intact and streptolysin-O permeabilized sperm. The sponge inhibited the AR elicited by progesterone, A23187 and calcium. These results indicate that the protein can transduce into cells and cause a decrease in intracellular cAMP, reflected in tyrosine phosphorylation diminution and AR inhibition. The development of permeant versions of proteins brings new perspectives to the study of sperm physiology.