Impact of feline zona pellucida glycoprotein B-derived synthetic peptides on in vitro fertilization of cat oocytes

Abstract

Although immunocontraception based on porcine zona pellucida (ZP) proteins is widely applied in many species, it is not suitable for cat contraception due to the lack of cross-reactivity. Since the first ZP gene expressed during oocyte growth in domestic cats is ZPB, we assumed that immunization with feline ZPB (fZPB)-derived synthetic peptides may cause irreversible infertility, which would be preferable in stray cats. Thus, the present study evaluated the immunogenicity and the contraceptive potential of synthetic fZPB peptides. Antigenic epitope sequences were detected via epitope mapping using specific rabbit anti-fZP antibodies. Six peptides representing the recognized epitopes were synthesized subsequently. Two out of six peptides (ZPB amino acid residue 130 -149 5 P3 and 175-193 5 P6) cross-reacted with anti-fZP antiserum in dot blot analysis and ELISA. Coupled to BSA, both peptides were utilized to produce specific antibodies in rats. Despite several booster injections the antibody titers monitored by ELISA did not exceed 1:5000. Both rat antisera were tested for contraceptive potential in cat in vitro maturation/in vitro fertilization (IVF). Antiserum against peptide P3 significantly inhibited sperm binding and fertilization of cat oocytes in vitro (57.3% of sperm binding; 41.5% of fertilization), whereas the inhibition by anti-P6 was not significant. Pre-incubation of sperm cells with both peptides before IVF failed to affect either sperm binding or fertilization (22.3 6 3.7 sperm/egg vs 25.5 6 5.8 for P3 and 20.7 6 4.0 for P6, respectively). In conclusion, antibodies directed against one of the two identified antigenic determinants of fZPB inhibited sperm binding and IVF and therefore showed promising results as a contraceptive. However, the specific immune response and anti-fertile properties of this synthetic vaccine have to be examined in vivo to verify the suitability of its components.

Embryonic gene activation in in vitro produced embryos of the domestic cat (Felis catus)

“…In the past 20 years, numerous studies have been performed in domestic cats investigating in vitro oocyte maturation, IVF (Johnston et al 1989, Goodrowe et al 1991, Jewgenow et al 1997, Ringleb et al 2004, influences of ovarian status and season on the oocyte maturation or embryonic developmental potency (Spindler & Wildt 1999, Freistedt et al 2001, as well as in vitro embryo culture conditions (Herrick et al 2007) and embryo cryopreservation (Gomez et al 2003). Although dramatic improvements have been made for in vitro techniques, artificially produced embryos still demonstrate lower developmental competence than in vivo embryos (Roth et al 1994).…”

Contraceptive Vaccines for the Humane Control of Community Cat Populations

“…IHC studies demonstrated that pZP antibodies did not bind to cat oocytes, but fZP antibodies did. In a third study, antibodies raised in rats against a portion of the fZP protein containing an epitope recognized by rabbit anti-fZP serum (ZPB amino acid residue 130– 149) reduced in vitro sperm binding and fertilization of feline oocytes 37. These studies are contradictory, clouding the issue of whether ZP has contraceptive potential in cats.…”

High-Resolution Ultrasonography of Xenografted Domestic Cat Ovarian Cortex

“…All cumulus-oocyte complexes (COCs) were collected by gentle disruption of the antral follicles. Oocytes with complete compact cumulus and uniform, dark cytoplasm were cultured as described previously [37], with one variation in the method: about 20 companion COCs obtained from domestic cats ovariectomized the same day were added to each well in a Tissue Culture Insert (10 mm, NUNC) to keep them separated from the recovered eggs. After in vitro maturation for 24 h the oocytes were analysed under a phase contrast microscope for polar body extrusion and were stained with aceto-orcein to confirm nuclear maturation.…”