Identification of CRISP proteins in the dog for their potential use in immunocontraception

LA ROSA I; BRUKMAN N; DE SALVO MN; CICUTTIN GL; DA ROS VG; LENCINAS OE; CUASNICU PS

San Juan

Congreso; 48th Annual Meeting of the Society for the Study of Reproduction; 2015

Society for the Study of Reproduction

There is a growing need to reduce the dog population levels as a means to control diseases transmitted to human (zoonosis). Based on previous results from our group supporting epididymal CRISP1 (Cysteine-Rich Secretory Protein 1) as a good immunocontraceptive target, the aim of this work has been the identification of a homologue CRISP protein in the dog that could be used for immocontraceptive development in this species.  Using degenerated primers against conserved CRISP motives, we obtained a RT-PCR band of the expected size (aprox. 120 bp) from epididymal but not testicular samples.DNA sequencing of this band showed a high(>80%)homology to CRISP proteins. Western blotting results using antibodies against different CRISP proteins revealed the presence of 25 kDa band in epididymal but not testicular extracts when an anti-mouse CRISP3 antibody was used. As expected for a CRISP molecule, theepididymal band shifted itselectrophoretic mobility under reducing conditions. Moreover, the protein precipitates by exposure to a 50% saturated ammonium sulfatesolution as previously observed for rodent CRISP1. Altogether, these observations support the identification of a CRISP-like molecule in the dog epididymis which is at present being further characterized for future purification and functional studies.