Extraction of proteins and location of the L-ype Voltage-Dependent Ca2+ channel (CaV1.2 α1c) in pig sperm extracted from fresh, frozen and permeated semen

Abstract

Ion channels are protein structures located in the cell plasma membrane. Sperm cells require calcium ions (Ca2+) to carry out fundamental physiological processes, such as hypermotility, capacitation and acrosomal reaction. Protein extraction is the first, and most critical, stage of molecular tests (Western Blot, proteomic tests, etc). The implementation of these techniques in the study of sperm cells will allow us to understand their physiology (biomarkers) and ways of manipulation (refrigeration, cryopreservation, permeation using streptolysin O (SLO), etc.). The goal of this project was to determine whether if changes in the plasma membrane, brought on by cryopreservation or permeation with SLO, modify the Ca2+ CaV1.2 α1C channel in pig sperm cells. 2 ½ pig ejaculates were taken as working simples, and they were treated as follows: -Control (C): refrigerated semen (SR) 16°/24h; Treatment 1 (T1): SR with SLO T2: cryopreserved semen (SC); T3: SC, previously treated with SLO. It was determined that a better quantitation of proteins from the samples (p<0.001) is obtained with a (total) protein extraction buffer composed of SDS 2% + β-Mercaptoethanol 3%. The detection of the CaV 1.2 α1C protein was done through Western Bloting, using the Rabbit anti-CaV 1.2 α1C (USA, Sigma-AldrichTM) antigen. Better visibility of the protein band of the Ca2+ channel was achieved in the C and T1 experimental groups; whereas its’ presence was not observed in T2 and T3. It was determined that the Ca2+ CaV 1.2 α1C was present in the sperm cells, and that it deteriorates once the sperm cell is frozen and permeated with SLO, which is linked to the decrease of cell viability percentages. alibri font, size 10, and single line spacing.