Gonadotropin-releasing Hormone Receptor As A Molecular Target for Contraception in Feral Cats

Abstract

The global overpopulation of feral cats generates concern regarding their welfare, negative impact on public health, and adverse effects on the environment. In the U.S. and other Westernized countries, the most common method for managing feral cat populations is impoundment in animal shelters, where they undergo surgical sterilization procedures as part of a shelter adoption or trap-neuter-release programs. However, such programs are labor-intensive, time-consuming, and costly. Further, several million, un-adopted cats are euthanized each year. Thus, there is a need for a permanent, nonsurgical, and low-cost method for controlling feral cat populations. Immunocontraception has the potential to provide the practical approach needed to manage stray cat populations. Gonadotropin-releasing hormone receptor (GnRHR) is an attractive target for immunocontraceptive vaccine development because it is highly expressed by anterior pituitary gonadotropic cells, important components of the hypothalamic-pituitary-gonadal axis that regulates normal mammalian reproduction. Therefore, induction of immune responses against pituitary gonadotropic cells by targeting GnRHR may impair normal reproductive function. The goal of this proof-of-concept work was to use a murine model to investigate the potentials of GnRHR as a target for immunocontraception. DNA plasmids were selected for immunocontraceptive vaccine construction because they are cost-effective, safe, and can elicit long-term

cytotoxic and humoral immune responses in animals. In order to overcome murine self-tolerance to the GnRHR, the nucleic acid sequence of feline (f)GnRHR was cloned into a plasmid with expectations of the murine immune system to elicit a stronger response to the heterologous antigen (fGnRHR) than to the homologous protein (mouse GnRHR). As an additional strategy for enhancing the potency of the vaccine against the self-antigen GnRHR, the DNA sequence for fGnRHR fused to ubiquitin (Ub) was incorporated into a plasmid in order to enhance presentation of GnRHR epitopes from Ub-fGnRHR to circulating CD4+ T cells. This strategy may efficiently activate these T cells, producing GnRHR-specific antibodies that ultimately target GnRHR-expressing pituitary gonadotropic cells. After DNA plasmid construction, adult male mice were vaccinated once with DNA encoding fGnRHR or Ub-fGnRHR. Vaccines were administered via intramuscular (IM) needle injection; IM injection with DNA-loaded, superparamagnetic iron oxide nanoparticles followed by magnetofection; or intradermal (ID) needle injection. Mice vaccinated with DNA encoding fGnRHR or Ub-fGnRHR via IM injection or magnetofection had normal serum testosterone levels at necropsy (or 12 weeks post-immunization) compared to pretreatment values. However, mice vaccinated with plasmid encoding Ub-fGnRHR via ID needle injection had significantly low testosterone concentrations 12 weeks post-immunization. Moreover, anti-GnRHR antibodies were detected in sera from mice intradermally vaccinated with DNA encoding Ub-fGnRHR. Also, PCR analysis indicated lower GnRHR mRNA expression in these animals. In summary, ID vaccination of mice with DNA encoding Ub-fGnRHR, a heterologous and ubiquitinated protein, abated serum testosterone concentrations and pituitary expression of GnRHR mRNA, as well as induced anti-GnRHR antibody production in treated animals. The outcome of these parameters indicate induction of an immunological response to overcome self-tolerance to the endogenous GnRHR, resulting in impairment to anterior pituitary gonadotropes that affected testosterone production. Based on these findings, the Ub-fGnRHR DNA construct warrants further investigation, as such a vaccine profile would be beneficial as an immunocontraceptive for controlling the overpopulation of feral cats worldwide.