STUDY OF CRISP PROTEINS AS A TARGET FOR A CANINE CONTRACEPTIVE VACCINE

SULZYK, VALERIA; WEIGE MUÑOZ, M; CUASNICU P S

Jornada; Jornadas Anuales de la Sociedad Argentina de Biología (SAB; 2022

The uncontrolled growth of dog populations causes a negative impact on both public health and animal welfare. Given that Cysteine Rich Secretory Proteins (CRISP proteins) are evolutionarily conserved and relevant for the fertilization process in different species, and considering the previous identification of at least one canine CRISP protein in our laboratory, the aim of this work was to characterize that protein as a possible target for the development of a canine contraceptive vaccine. From this perspective, the epididymis is considered an excellent male contraceptive target because it does not affect testicular function and its permeability of the blood-epididymal barrier. In order to study the epididymal origin of the identified canine CRISP protein, testes and epididymis from surgical sterilizations of 8 dogs were analyzed by Western Blot, using the human anti-CRISP3 antibody, the same used to identify the canine CRISP. The results showed the presence of canine CRISP in all epididymal extracts analyzed and its absence in the corresponding testicular extracts, confirming the epididymal origin of this protein. To investigate whether the CRISP protein is present in fresh sperm and remains there even after capacitation, ejaculated canine sperm were incubated for 4 hours at 37C and 5% CO2 in two different capacitation media. The results obtained confirmed the ability of both media to capacitate canine sperm, judging by the levels of protein tyrosine phosphorylation, progressive motility, acrosomal reaction and hyperactivity. On the other hand, indirect immunofluorescence studies using human anti-CRISP3 revealed the presence of canine CRISP on the surface of the acrosomal region of fresh canine sperm and its relocation towards the equatorial region segment of capacitated sperm with both media, indicating the accessibility of canine CRISP to the antibodies generated after the immunization. Furthermore, since the equatorial segment is the region through which gamete fusion occurs, these results support the possible involvement of canine CRISP in the fusion stage during fertilization. Together, these results indicating the epididymal origin of the canine CRISP protein and its localization on the surface of fresh and capacitated spermatozoa support the use of canine CRISP as a target for the development of a contraceptive vaccine for dogs of both sexes