It’s hard to believe that the time has passed so quickly, but this October marked the 7th anniversary of the Michelson Prize & Grants program! As we celebrate this milestone, we’d like to share with our readers the important progress that has been made since then in the field of nonsurgical sterilization. Through the tireless efforts of our grantees and their research teams, the dedication of our diverse and talented Scientific Advisory Board, and the generosity of our funder, Dr. Gary Michelson, we have made significant steps toward the development of a noninvasive spay/neuter method for companion animals and we hope that you will share our enthusiasm after reading this update.
A major goal of the Found Animals Foundation is to develop a single-dose, permanent (10-20 year), nonsurgical sterilant that renders male and female dogs and cats infertile. Our desired product must induce sterility as well as suppress reproductive hormones and behavior (such as estrus, matings) in treated animals. If made available to the ~3500 shelters in the U.S.A., such a sterilant could guarantee that every animal adopted would be incapable of reproducing, thereby eliminating the birth of unwanted litters that ultimately end up back in the shelter system. Nationally and internationally, such a product could also be administered by paraprofessionals to feral and free-roaming dog and cat colonies. We envision that this nonsurgical sterilant will be a total game-changer for the care and management of shelter, feral, and free-roaming dogs and cats all over the world and could eventually eliminate the euthanasia of shelter animals altogether.
Thirty-five Michelson Grants in Reproductive Biology, totaling about $15 million in committed research funds, have been awarded to scientists around the world since 2009 to perform research leading to the development of this sterilant. Grants have been awarded to scientists in the U.S.A., Canada, Argentina, Australia, the Netherlands, New Zealand, and Scotland.
To date, scientists performing this research have targeted one of three sites in the body that are essential for reproduction. These are:
- the hypothalamus, the part of the brain that contains cells which initiate reproduction by secreting the hormones kisspeptin and gonadotropin releasing hormone (GnRH);
- the pituitary gland, a pea-sized organ at the base of the brain that responds to secretion of GnRH by secreting luteinizing hormone (LH) and follicle stimulating hormone (FSH); and
- the reproductive organs in the abdomen (ovaries) or scrotum (testes) that respond to LH and FSH by producing eggs, sperm, and hormones such as estrogen and testosterone that cause reproductive behaviors.
Scientists have endeavored to block normal reproductive function at these sites using one or more of the approaches described below. Some of their findings are bulleted under each approach.
Genes in all body cells encode for, or express, proteins/peptides that are active building blocks or enzymes. In the gene silencing approach, scientists inject an inactive virus (either a lentivirus or adeno-associated virus) intravenously that has a nucleic acid fragment inserted that is an exact mirror image of the gene that they want to silence. The mirror image (interference RNA) attaches to this gene and prevents its ability to express its protein. One of the first Michelson Grants awarded was to silence genes in cells of the hypothalamus that express kisspeptin and neurokinin B (NKB). These proteins/peptides normally induce GnRH secretion.
- A gene silencing construct to silence kisspeptin and NKB genes in the hypothalamus disrupted female rat estrous cycles but did not completely ablate fertility; studies are now underway to enhance specific delivery of this construct to the hypothalamus in high concentrations.
- Genes that express dog and cat kisspeptin and NKB in the hypothalamus were cloned and respective genomic structures were determined. It was discovered that the dog kiss1 gene is the most divergent of all mammalian species known.
- Genes expressing kisspeptin and the kisspeptin receptor in the hypothalamus were cloned in the dog. Investigators demonstrated that estrous cycle stage influences kisspeptin signaling in dogs.
- Silencing of the genes that express androgen receptor proteins in the testes induced long-term spermatogenic arrest and sterility in male mice.
- Dog testes (seminiferous tubules) were shown to express functional piRNA binding proteins called “PIWI proteins” that are essential to spermatogenesis; in normal reproduction, these are critical mediators of egg and sperm maturation. They could be a good target to silence in efforts to induce sterility.
Gene therapy also uses a lentivirus or adeno-associated virus, but the nucleic acid fragment inserted is a functional gene that expresses a protein. This approach is used to treat a variety of human diseases and has been able to express a blood clotting factor for more than 10 years in dogs with hemophilia. The Foundation has approved studies to use gene therapy to express antibodies to GnRH, to express gonadotropin inhibiting hormone (GnIH), and to express Mullerian Inhibiting Substance (MIS).
- Gene therapy delivering antibodies to GnRH causes sterility in rodents by blocking hypothalamic GnRH.
- Human GnIH inhibits secretion of reproductive steroid hormones in cat ovarian tissue.
Immunocontraception describes induction of antibodies to essential reproductive hormones so that the antibodies bind to and block these essential reproductive elements. The mechanism is similar to that of inducing antibodies by injecting inactivated bacteria or viruses that cause disease, except that immunocontraception relies on creating antibodies to “self” antigens. Dogs and cats have been immunized against GnRH for decades and shown to be infertile for 1 to 3 years after a single administration. Work funded by our program is in progress to provide slow or repeated release of vaccines against GnRH in the hypothalamus, the GnRH receptor in the pituitary, or other reproductive proteins.
- Administration of a GnRH vaccine on a subcutaneous three-dimensional adjuvant matrix that mimics the body’s response to infection resulted in recruitment and activation of (antigen presenting) dendritic cells and long-term persistence of antibodies to GnRH secreted by the hypothalamus.
- A study is in progress in mice to release GnRH vaccine from a slow-release subcutaneous implant “smart” device that releases the antigen when circulating antibodies to GnRH fall.
Targeted Delivery of Cytotoxins
Some scientists are trying to destroy cells in one of the three sites that are essential to reproduction by delivering toxins exclusively to the target cell, and not to other body tissues. This methodology also is used to destroy cancer cells in patients receiving chemotherapy.
- Investigators discovered that the gene sequence expressing the kisspeptin receptor protein (reported in public databases) in rat hypothalamic cells was incorrect and nonfunctional. The correct sequence was determined and confirmed, and the National Center for Biotechnology Information corrected the database based on that funded research.
- In a study of attempted toxin ablation of pituitary cells secreting LH and FSH, our investigators have found that gonadotropes are resistant to toxin ablation. Others have demonstrated that toxin degradation within structures called “endosomes” may be limiting efficacy. In fact, it has been estimated that only about 1 in 10,000 internalized toxin (ribosome inactivating proteins) escape endosomal destruction. A research team of Michelson Grant awardees is now adding “endosome escape” substances to the toxin ablation constructs to enhance efficacy.
- Administration of a GnRH agonist implant (deslorelin) to kittens within 24 hours of birth blocked receptors on pituitary cells from access to native GnRH. This resulted in delayed onset of puberty, to 42-91 weeks of age, compared to normal onset of puberty at 15.5 +/- 1.7 weeks in control kittens. Investigators hypothesize that neonatal use of a higher dose of deslorelin might prevent puberty altogether. A similar study in dogs is ongoing.
- Scientists discovered that Sertoli cells in the testes require a different mechanism of inducing cell death than do the primordial germ cells that produce sperm because of different phagocytic/endocytic functions.
- Administration of (toxic) reactive oxygen species linked to FSH that target the ovaries and testes caused germ cell death in mice.
Our grantees’ work is frequently published in peer-reviewed journals and presented at international conferences. If you would like to learn more about any of our funded projects, be sure to check out the full Michelson Grants Research Findings listing on our website!