Evaluation of kisspeptin in the mare
Date
2010
Authors
Magee, Christianne, author
Clay, Colin M., advisor
Tobet, Stuart A., committee member
Nett, Torrance M., committee member
Goodrich, Laurie R., committee member
Duval, Dawn L., committee member
Journal Title
Journal ISSN
Volume Title
Abstract
Identified in 2003 for their role in reproductive physiology, kisspeptins have become major players in the field of reproductive neuroendocrinology. With the ability to act as a central regulator for the onset of reproductive function in prepubertal and seasonal animals, the possibility that kisspeptin signaling could be used to modify seasonal reproductive function in the horse held great promise. My hypothesis was that kisspeptin, acting via a hypothalamic signaling mechanism to stimulate the GnRH neuron, could initiate reproductive function in the horse. The initial objectives of these studies were to (1) establish biological and physiological evidence for kisspeptin signaling in the hypothalamus of the mare, (2) demonstrate peripheral administration of kisspeptin could elicit a rise in serum luteinizing hormone (LH) concentrations in the diestrous mare, and (3) demonstrate that kisspeptin, acting via LH, could induce ovulation in the estrous mare. The diestrous mare has kisspeptin immunoreactive neurons in the hypothalamus that are in close proximity to Gonadotropin Releasing Hormone (GnRH) neurons. At the time of these initial studies, the equine sequence for the kisspeptin decapeptide (Kp-10) was not yet available; therefore, I utilized the rodent Kp-10 (rKp-10, YNWNSFGLRY-NH2). Even though I was using a heterologous ligand, the diestrous mare was responsive to administration of rKp-10 (0.5 and 1.0 mg) such that there was a short (< 1 hour), but significant (2-fold) rise in circulating levels of LH and follicle stimulating hormone (FSH) after kisspeptin administration. I was also able to establish a threshold dose for kisspeptin responsiveness in the diestrous mare as there was no change in serum gonadotropin levels following a 1.0 μg dose of rKp-10. In the estrous mare, a single injection of 1.0 mg rKp-10 IV was unable to induce ovulation (173), presumably due to the short duration of the kisspeptin induced LH surge as compared to the 3-5 day endogenous peri-ovulatory LH surge (306). To understand the dynamic of kisspeptin signaling to the hypothalamus and the anterior pituitary gland, I sought to determine the effect of treating mares with repeated injection of kisspeptide in diestrus and estrus. If the future of kisspeptin in the horse involves the use of modified agonists or antagonists, it will be necessary to understand how the mare responds to repeated stimulation with kisspeptin. Before beginning these studies, the equine sequence for Kp-I0 (eKp-10, YRWNSFGLRY-NH2) had become available. Therefore, I used the homologous peptide for these studies. By treating mares with eKp-10 (0.5 mg IV every 4 hours), the hypothalamus and pituitary gland were repeatedly stimulated to elicit a GnRH and gonadotropin response. Repeated administration of kisspeptin in the diestrous mare is not able to sustain a 2-fold increase in LH concentration for 48 hours following the initial injection. Interestingly, kisspeptin caused a decrease in basal LH, but not FSH levels, indicating a decrease in LH synthesis or secretion via a pituitary effect. Although the mare does not exhibit a change in peripheral LH levels following eKp-10 if a GnRH antagonist (e.g. Antide) has been administered, I sought some evidence for kisspeptin signaling directly to the anterior pituitary. To support the idea of a direct pituitary effect of kisspeptin, I challenged primary pituitary cells in culture with 100 nM GnRH and 100 nM of eKp-10. Surprisingly, I identified three populations of cells that respond with a change in intracellular calcium concentration and grouped them as follows: cells that responded to (1) both GnRH and eKp-l0, (2) only GnRH, or (3) only eKp-10. The identification of gonadotrope and non-gonadotrope kisspeptin responsive pituitary cells is the first evidence for a direct mechanism for kisspeptin signaling at the level of the equine pituitary gland. In the estrous mare, repeated administration of eKp-10 is not able to shorten the interval to ovulation whether it is administered before or after the development of a dominant follicle. Another surprising finding was a significant decrease in sexual receptivity in mares within 48 hours of beginning treatment with kisspeptin, which is likely due to a decrease in estradiol synthesis by the maturing follicle. Given the lack of ovulation induction in the estrous mare and the changes in behavioral receptivity, I do not recommend the use of kisspeptin as an ovulation inducing agent at this time. However, there was no decrease in basal LH levels in the estrous mares. Thus, kisspeptin may be signaling via different mechanisms in the estrous vs. diestrous mare. In summary, these studies do provide evidence for kisspeptin signaling in the mare, but they reveal that the signaling mechanism in the horse may be more complex than my original hypothesis of a simple, linear process that is working only through the GnRH neuron.
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Subject
Animal sciences
Molecular biology
Physiology