Progress 11/01/05 to 07/31/09
Outputs
OUTPUTS: Fertility of several agriculturally important species has declined significantly in recent years. Swine producers in the U.S. routinely replace approximately 50% of breeding age females each year. The majority of females are removed for reproductive failure; caused in part by inadequate gonadotropin secretion leading to lack of estrus and reduced farrowing rates. Therefore, methods that increase secretion of gonadotropins (luteinizing hormone, LH; follicle stimulating hormone, FSH) should improve reproductive performance and increase the biological efficiency of pork production. Our goal is to identify how gonadotropin secretion from the pituitary gland is controlled by the hypothalamus. Gonadotropin-releasing hormone (GnRH) is secreted from neuroendocrine cells in the hypothalamus and is transported through the hypophyseal portal vasculature to the anterior pituitary gland where it binds high affinity receptors located on gonadotrope cells to stimulate the synthesis and secretion of LH and FSH. The tonic release of GnRH, which is necessary for normal reproduction, reflects the net difference in the sum total of inhibitory and excitatory neural signals which relay critical information about important factors (e.g. lactation, environment, stress, nutrition, ect) to the reproductive axis. An important step in accomplishing our goal is to establish the central pathways in the hypothalamus that regulate GnRH and, consequently, LH secretion. To this end, we have sought to identify the role of RFamide-like peptides in regulating LH release. To achieve this objective we cloned portions of two genes for RFamide-like peptides and deduced the amino acid sequence for the porcine specific peptides. Using frozen sections of hypothalamic tissue collected from prepubertal gilts, we performed immunocytochemistry to determine the spatial organization neurons that produce RFamide-related peptide (RFRP) and the RFamide-like peptide kisspeptin. We have conducted studies in which we injected kisspeptin and RFRP-3 into the peripheral circulation and the cerebroventricles of gilts. PARTICIPANTS: C.R. Barb, USDA-ARS, Swine Physiology, Richard Russell Research Center, Athens GA G.J. Hausman, USDA-ARS, Swine Physiology, Richard Russell Research Center, Athens, GA J.J. Ford, USDA-ARS, MARC, Clay Center NE Krzysztof Czaja, Washington State University, Pullman WA Neely Heidorn, Graduate Student, Ph.D. Candidate, The University of Georgia TARGET AUDIENCES: Animal Scientists, University Extension personnel, Swine producers PROJECT MODIFICATIONS: This project was awarded to the PD as a Post-doctoral award at Colorado State University. The PD is currently an Assistant Professor at the University of Georgia and the award moved to Georgia with him. Due to lack of sufficient number of sheep to perform the original aims, the PD requested and was granted a modification of objectives for this award. The objective was modified to identifying central pathways that regulate gonadotropin secretion in the pig because of the agricultural importance and project relevance of this species as well as the need to improve reproductive efficiency swine in the U.S.
Impacts
We identified a population of kisspeptin immunoreactive neurons in the arcuate nucleus (ARC) of the porcine hypothalamus. Location of kiss neurons in the ARC of the pig hypothalamus, an area that controls GnRH release, indicates that kisspeptin could activate GnRH secretion in the gilt. Intracerebroventricular injection (i.c.v.) of kisspeptin (10-100 micrograms) to prepubertal gilts resulted in a dramatic dose-dependent, surge-like release of LH and FSH from the anterior pituitary gland. Further, the stimulatory effect of kisspeptin on LH, but not FSH, was retained when it was administer into the peripheral circulation (1-5 mg). The failure of kisspeptin to stimulate FSH release when it is injected in the peripheral vasculature is probably indicative of a central cite action rather than a direct effect on the pituitary gland. Unlike ruminant animals, kisspeptin does not activate secretion of growth hormone from the pituitary gland of the gilt. This may reflect differences between ruminants and pigs in the central pathways that regulate growth hormone. Kisspeptin can be considered a stimulatory input for GnRH secretion in the pig. Ostensibly, however, GnRH release is also negatively controlled by specific neural mechanisms. Because RFRPs are thought to produce a specific gonadotropin-inhibitory hormone (GnIH), we cloned a portion of the coding sequence for the porcine RFRP gene. This cDNA sequence encodes an open-reading frame that reveals that the pig, unlike other mammalian species, can potentially produce three different RFRP neuropeptides (termed RFRP-1, RFRP-2, and RFRP-3) from the RFRP pro-hormone. Furthermore, we identified RFRP immunoreactive neurons in the ARC and paraventricular nucleus of the porcine hypothalamus. This places RFRP neurons in areas of the pig hypothalamus that control reproduction and growth. We next tested the effect of RFRP-3 on LH secretion. Secretion of LH from the anterior pituitary gland was unchanged when RFRP-3 was administered into the peripheral circulation of ovariectomized gilts. This is in contrast to the recent report for both sheep and cattle and highlights the importance of species specific investigation into central regulatory mechanisms. We interpret this observation as evidence that RFRP-3 may not act directly at the pituitary gland to inhibit LH secretion in the pig, but that it has a central site of action to inhibit GnRH release. In support of this hypothesis, we found that the integrated secretory pattern of LH release in the ovariectomized gilt following i.c.v. injection of 10 micrograms of RFRP-3 was reduced when compared with saline injected controls (n=3 per treatment). We are cautious in defining RFRP-3 definatevely as a gonadotropin-inhibitory hormone in the pig until a full range of doses are tested in more animals. Nonetheless, it indicates that RFRP-3 could act as an inhibitory signal to the GnRH neuronal network in the pig. We do not know, at present, the biological function of RFRP-1 or RFRP-2 in the pig. This will be a focus of ongoing research in our laboratory.
Publications
- No publications reported this period
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Progress 11/01/07 to 10/31/08
Outputs
OUTPUTS: Fertility of several agriculturally important species has declined significantly in recent years. Swine producers in the U.S. routinely replace 40-60% of sows each year. The majority of females removed for reproductive failure caused, in part, by inadequate gonadotropin secretion leading to lack of estrus and reduced farrowing rates. Therefore, methods that increase secretion of gonadotropins should improve reproductive performance and increase the biological efficiency of pork production in the U.S. Factors such as ovarian steroids and nutrition are important regulators of reproduction. These factors act at the level of the hypothalamus to control the release of gonadotropin-releasing hormone (GnRH) and secretion of luteinizing hormone (LH) from the anterior pituitary gland, but how this occurs is not well understood. Our long-term goal is to identify how gonadotropin secretion from the pituitary gland is controlled by the hypothalamus. An important step in accomplishing our goal is to establish the central pathways in the hypothalamus that regulate GnRH and LH secretion. To this end, we have established that the recently identified neuropeptide kisspeptin (Kiss) activates LH release in the prepubertal gilt, presumably by a GnRH dependent mechanism. At issue then is whether kiss neurons are localized within the porcine hypothalamus in such a way that they could interact with GnRH neurons to control the release of GnRH. Prepubertal gilts, with ovaries intact, were perfused transcardially with 4% paraformaldehyde. We collected the hypothalamus of each gilt and performed immunocytochemistry for Kiss to determine the spatial organization of Kiss neurons in the porcine hypothalamus. In other studies, we sought to identify potential negative regulatory pathways controlling GnRH secretion. RFamide-related peptide-3 had been found to inhibit LH release in rodents. We collected fresh hypothalamic tissue from two prepubertal gilts and extracted total RNA. Using PCR and RFamide specific primers, we amplified a portion of the porcine RFamide gene and performed and open reading frame analysis to deduce the amino acid sequence of the porcine RFamide precursor protein. We conducted in vitro experiments using primary cultures of porcine anterior pituitary cells to determine if mammalian RFamide-related peptide-3 inhibits LH release directly from the gonadotrope cells. Using frozen twenty-micron sections of hypothalamic tissue collected from prepubertal gilts, we performed immunocytochemistry for RFamide protein to determine the spatial organization of RFamide neurons in the porcine hypothalamus. PARTICIPANTS: C.R. Barb, USDA-ARS, Swine Physiology, Richard Russell Research Center, Athens GA J.J. Ford, USDA-ARS, MARC, Clay Center NE Krzysztof Czaja, Washington State University, Pullman WA Neely Heidorn, Graduate Student, Ph.D. Candidate, The University of Georgia TARGET AUDIENCES: Animal Scientists, University Extension personnel, Swine producers PROJECT MODIFICATIONS: This project was awarded to the PD as a Post-doctoral award at Colorado State University. The PD is currently an Assistant Professor at the University of Georgia and the award moved to Georgia with him. Due to lack of sufficient number of sheep in Georgia to perform the original aims, the PD requested and was granted a modification of objectives for this award. The objective was modified to identifying central pathways that regulate gonadotropin secretion in the pig because of the agricultural importance of this species and the need to improve reproductive efficiency swine in the U.S.
Impacts
We identified a population of Kiss neurons in the arcuate nucleus (ARC) of the porcine hypothalamus. Location of kiss neurons in the ARC of the pig hypothalamus, an area that controls GnRH release, indicates that Kiss stimulates LH release from the anterior pituitary gland of prepubertal gilts by activating secretion of GnRH from the hypothalamus. RFamide neurons are thought to produce a gonadotropin-inhibitory hormone (GnIH). We identified a population of RFamide neurons in the paraventricular nucleus and preoptic areas of the porcine hypothalamus. These areas of the hypothalamus control GH and LH release in the pig. Thus RFamide neurons in the pig are located in such a way to inhibit GnRH/LH secretion. The porcine specific RFamide cDNA that we cloned revealed an open reading frame that codes for a precursor protein that yields at least two RFamide peptides, RFamide-related peptide-1 (RFRP-1) and -3 (RFRP-3). The latter is thought to be the mammalian GnIH. Unlike other mammals, the porcine precursor protein contains the appropriate amino motif between RFRP-1 and RFRP-3 to produce an RFamide-related peptide-2 (RFRP-2). This is significant because in avian species, it is the second intervening peptide that inhibits LH release. Interestingly, RFRP-3 did not inhibit GnRH stimulated LH release from culture porcine pituitary cells. Thus we postulate that in the pig, RFRP-2 rather than RFRP-3 may act as a gonadotropin inhibitory hormone. This is significant because it indicates the GnIH system may be distinctly unique in the pig. Further investigation into the mechanisms controlling Kiss and RFamide peptides in the pig are expected to lead to the development of novel strategies or therapies that target these pathways to improve fertility and facilitate more efficient pork production.
Publications
- Lents, C. A., N. L. Heidorn, C. R. Barb, and J. J. Ford. 2008. Central and peripheral administration of kisspeptin activates gonadotropin but not somatotropin secretion in prepubertal gilts. Reproduction 135:879-887.
- Barb, C. R., G. J. Hausman, and C. A. Lents. 2008. Energy metabolism and leptin: effects on neuroendocrine regulation of reproduction in the gilt and sow. Reprod. Domest. Anim. 43(Suppl. 2):324-330.
- Heidorn, N. L., C. A. Lents, K. Czaja, C. R. Barb, K. Tsutsui. 2008. RFamide-related peptide-like immunoreactivity in the porcine hypothalamus indicates the presence of a gonadotropin-inhibitory system in the pig. Abstract 14, page 46, Proceedings of the 6th International congress on Farm Animal Endocrinology, Roanoke, VA.
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Progress 11/01/06 to 10/31/07
Outputs
OUTPUTS: Normal reproductive function in mammals is dependent on the continued pulsatile release of gonadotropin hormones; luteinizing hormone (LH) and follicle-stimulating hormone (FSH). Kisspeptin is a newly identified neurohormone that been implicated in the regulation of LH and FSH. Kisspeptin signaling is necessary for the onset of puberty in laboratory animals such as rodents and monkeys. Some data are available on the effects of kisspeptin on LH secretion in the adult ewe; however, the possible role of kisspeptin in mediating the onset of puberty in larger domestic species, in particular the pig, has yet to be determined. We hypothesize that kisspeptin is an important regulator of gonadotropin secretion in the pig. Our laboratory has established that kisspeptin when administered in the lateral ventricles of the brain of the prepubertal pig stimulates both LH and FSH secretion. Of particular importance is the fact that it does so to the same extent as gonadotropin-releasing
hormone (GnRH). This supports the concept that kisspeptin plays a role in regulating gonadotropin secretion in the pig and that it might be an important determinant of the onset of puberty in swine. In a similar fashion, peripheral administration of kisspeptin to prepubertal gilts stimulates an LH surge, but did not alter FSH secretion. Because the occurrence of puberty in the pig is directed by mechanisms in which the hypothalamic-pituitary axis regulates both gonadal function and growth we tested the hypothesis that kisspeptin would modulate growth hormone (GH) secretion in the prepubertal gilt. However, the stimulatory actions of either centrally or peripherally administered kisspeptin did not extend to GH. Research will focus on understanding the temporal and spatial relationship of kisspeptin neurons with the GnRH neural network in gilts.
PARTICIPANTS: Collaborators/partners: C. R. Barb, Animal Physiology, USDA-ARS, Richard Russell Research Center, Athens, GA. J. J. Ford, USDA-ARS, MARC, Clay Center, NE. Training: Jason Segerf, Undergraduate Student, UGA Neely Heidorn, Graduate Student, UGA Carl Rogers, Staff, UGA
Impacts
Investigation has demonstrated a role of kisspeptin in controlling gonadotropin secretion and possibly the onset of puberty in the pig. Therapies to enhance kisspeptin secretion during the peripubertal period may increase the number of gilts that express estrus and become pregnant. The fact that kisspeptin can be administered peripherally as a potent stimulator of LH secretion suggest that kisspeptin itself could possibly be used to induce ovulation and estrus in the pig and possibly other domestic livestock.
Publications
- Lents, C.A., and Barb, C.R. 2007. Stimulatory actions of kisspeptin on gonadotropin secretion in prepubertal swine. Biol. Reprod. Special Issue pg 105.
- Lents, C.A., and Barb, C.R. 2007. Emerging concepts regarding the integration of neuroendocrine signals that regulate gonadotropin secretion in domestic livestock. J. Anim. Sci. 85(Suppl. 1):648
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Progress 11/01/05 to 11/01/06
Outputs
In the past year the PI has moved from Colorado State University to the University of Georgia. This transition has slowed progress on this project this year. Experiments in this project use mice that are transgenic for the proximal 9100 bp of the ovine gonadotropin-releasing hormone receptor (GnRHR) gene promoter directing expression of luciferase. Transfer of these mice from CSU to UGA has been slowed due to serological reports. Currently, the PI is pursuing avenues to rederive this line from frozen embryos. Embryos have been collected, frozen, and stored for shipment to a commercial vendor which will rederive this strain of transgenic mice. Previous work on this project had identified a cAMP response element (CRE) in the proximal 115 bp of the ovine GnRHR promoter that potentially underlies estrogen regulation of the promoter. Mice which are transgenic for the proximal ovine GnRHR promoter containing a mutation in the CRE have been created and are currently being
screened. Transfer of these two lines of mice to UGA will facilitate renewed progress in the coming year.
Impacts
This research uses novel approaches (transgenic mice, adenoviral-mediated gene transfer and primary cultures of ovine pituitary cells) to identify the molecular mechanisms of estrogens regulation of the GnRH receptor gene. Estrogens regulation of the GnRH receptors is an important regulatory step in gonadotropin secretion during the preovulatory period. Increasing our understanding of how this occurs will improve our ability to manipulate the pituitary gland and improve its sensitivity to GnRH analogs used in reproductive management of domestic livestock.
Publications
- Lents, C. A., J. D. Cantlon, T. M. Nett, and C. M. Clay. 2006. Regulation of the GnRH receptor promoter by estradiol 17-beta is revealed using adenoviral mediated gene transfer and cultured pituitary cells. J. Anim. Sci. 84(Suppl 2):29 (Abstract).
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