TRANSLOCATION AND CELLULAR ENTERING OF METALLOTHIONEIN USING A CELL-PENETRATING PEPTIDE TO STUDY BASIC FUNCTIONS IN HUMAN SPERM

BERBERIAN MARIA VICTORIA; CRISTIAN A. POCOGNONI; LUIS S. MAYORGA

MENDOZA

Congreso; XXXIV Reunión Científica Anual organizada por la Sociedad de Biología de Cuyo; 2016

Sociedad de Biología de Cuyo

TRANSLOCATION AND CELLULAR ENTERING OF METALLOTHIONEIN USING A CELL-PENETRATING PEPTIDE TO STUDY BASIC FUNCTIONS IN HUMAN SPERMBerberian MV1,2, Pocognoni CA1, Mayorga LS1. 1Instituto de Histología y Embriología de Mendoza (IHEM-CONICET/UNCUYO) y 2Facultad de Ciencias Exactas y Naturales (FCEN-UNCUYO). E-mail: victoria.berberian@gmail.comThe cell membrane is an essential semi-permeable barrier effective for cell survival and function. However, it is also an important obstacle for intracellular delivery of molecules for diagnosis and treatment of pathological situations. Access to the cytosol is also crucial for basic studies on the functioning of the cell. Therefore, the development of tools that facilitate cellular uptake of large molecules is an important field of study nowadays. Cell Penetrating Peptides (CPPs) have been considered a promising delivery system because they have the benefit of being able to translocate into the intracellular compartment in a receptor-independent manner without causing any cell membrane damage. Most importantly, when conjugated to molecules such as a protein or nucleic acid or even nanoparticles, confers the property of crossing cell membranes. Therefore, the possibility of introducing a macromolecule directly to the cytosol opens new possibilities, especially for cells that do not possess the ability to transcribe and translate, such as spermatozoa. The work presented here characterizes the ability of a small metal-binding protein, metallothionein (MT) attached to a CPP to translocate the cell membrane of human sperm in order to study basic cellular functions. For these studies a construct encoding MT fused with a CPP was generated. The permeable CPP-MT was tested with and without gold nanoparticles (AuNP) conjugation. The labelling of CPP-MT with AuNP was conducted to visualize the cellular uptake by electron microscopy. Human sperm motility, membrane integrity and morphology were examined after co-incubation with CPP-MT or CPP-MT-AuNP. As a result, none of these parameters were affected by the presence of these conjugates. Electron microscopy and indirect immunofluorescence assays showed that CPP-MT and CPP-MT-AuNP, translocates inside the cell and were located in the head, middle and principal piece of the sperm. Furthermore, the role of CPP-MT was studied in human sperm and these tests showed that CPP-MT inhibits the acrosome reaction in a dose-dependent manner.Área Temática: BM Provincia de origen: Mendoza.