ISOLATION AND CHARACTERIZATION OF BOAR SPERM PROTEINS WITH AFFINITY TO Concanavalia ensiformis AGGLUTININ

Abstract

Capacitation is the process by which the spermatozoa from mammals acquire the capacity to fertilize the oocyte. It is characterized by changes in the composition and distribution of the components in the surface of the spermatozoa. Among these changes, the plasma membrane glycoproteins are lost or redistributed. The objective of this study was to isolate and characterize the proteins from capacitated boar spermatozoa, which have affinity for Concanavalia ensiformis Agglutinin (Con-A). Samples from capacitated and non-capacitated boar spermatozoa were analyzed. Sperm glycoproteins were isolated by affinity chromatography. Protein extract was added to a sepharose column conjugated to Con-A. The retained fraction by the affinity column was analyzed by PAGE-SDS and 2D electrophoresis. Protein band was excised and subjected to limited Edman degradation sequencing. The amount of protein extracted from capacitated sperm was lower, compared to that of non-capacitated sperm. It was found that after capacitation there is a reduction in the proportion of the proteins with less than 32 kDa. By affinity chromatography it was found that the plasma membrane from boar spermatozoa has a large number of proteins linked to Con-A. Total proteins from capacitated spermatozoa showed molecular masses between 14 and 102 kDa. Bands from different molecular masses were found, corresponding to arylsulfatase (AS-A), hialuronidase and lactadherine, respectively. The pattern of retained proteins by Con-A (RP) showed bands of 14 to 98 kDa. After sequencing and BLAST analysis, it was found that 62 kDa band corresponded to ASA. This study showed that the amount of proteins obtained after capacitation decreases considerably in relation to the amount obtained from non-capacitated spermatozoa. It was possible to isolate sperm proteins by affinity chromatography with Con-A, this indicates that the carbohydrates with affinity to Con-A are abundant in the plasma membrane of capacitated spermatozoa as glycoproteins. The presence of lactadherine and hialuronidase, reported as proteins participating in the fertilization process, was confirmed in capacitated boar spermatozoa. It was confirmed the presence of AS-A in boar spermatozoa and it was found that this protein is still present in capacitated spermatozoa and that it contains mannose residues as part of its glycosilation. This study was partially supported by CONACYT (Mexico). (poster)