Progress 10/01/02 to 09/30/08
Outputs
OUTPUTS: The studies in this project conducted by myself, graduate students, technicians and colleagues required developing new animal and laboratory techniques. The studies were a learning experience for those involved. Cannulation of the facial vein to access the intercavernous-sinus near the pituitary was developed in our laboratory. The study was supported by NIH for 3 years in collaboration with Dr. JD Veldhuis. Our studies on changes in cortisol output before and after metyrapone treatment (inhibits cortisol treatment) followed by cortisol treatment were used as preliminary studies to determine if the horse could act as a model to develop an analytical construct for humans. The preliminary results of our studies were used to set up the final studies that were published by Veldhuis and colleagues in 2008. The unique collection of blood samples taken from the pituitary and jugular vein for the cortisol study were used in our lab to investigate the interactions between the adrenal gland and the ovary by way of changes in circulating luteinizing hormone (LH) and follicle stimulating hormone (FSH). Final statistical analysis indicated no difference in jugular or pituitary LH and FSH levels or their pulse frequency and amplitude during the experimental period before and after treatment with metyrapone or cortisol. Therefore, it was decided to change direction in our lab and develop a unique single chain recombinant equine LH (reLH) which could be used to treat mares and stallions to examine changes in cortisol production. With the support of several granting agencies and in collaboration with Dr. Irv Boime from Washington U, the recombinant eLH was cloned in his laboratory and a biotech company scaled up the protocol to supply us with enough reLH to conduct stallion and mare studies. To determine the efficacy of the reLH, two experiments were carried out to demonstrate that: 1. ReLH treatment increased testosterone levels in stallions and 2. ReLH induced ovulation in mares. Both studies were presented at veterinary meetings and basic science symposiums from 2006-2008. Both studies have been published. ReLH has been on the market for veterinarians to induce ovulation in mares and available to researchers for further scientific studies. A series of experiments to determine the effects of recombinant eLH on the adrenal gland will be conducted in the future. PARTICIPANTS: Geraldine Meyers-Brown organized mares, collected blood samples. Lisa Bidstrup: Technician in Dr. Roser's lab. Collected blood samples. Data collection and graphs. Lil Sibley: Technician: Taught Jennifer how to run radioimmunoassay for LH and FSH. Jennifer Winnings: MS graduate student in Dr. Roser's lab at UC Davis. The LH and FSH levels and amplitude and frequency were, in part, Jennifer's MS project. TARGET AUDIENCES: For the development and efficacy of recombinant equine LH there were PowerPoint presentations at four symposiums for equine veterinarians and researchers: 1. Bluegrass Symposium in Lexington, KY, 2. American Association for Equine Practitioners, International Symposium on Equine Reproduction and the West Coast Equine Reproduction Symposium. There was a poster presentation at the annual meeting of the Society for the Study of Reproduction. PROJECT MODIFICATIONS: Because there were no changes in the levels of LH and FSH during the metyrapone and cortisol treatment, it was decided to go in a different direction. The direction was to develop a recombinant equine LH (reLH) that could be injected into horses to investigate changes in cortisol. The development and efficacy of reLH were carried out. Future studies will include the effects of reLH on cortisol release from the adrenal gland.
Impacts
Cortisol is an important mediator of physiological stress responses. Hypothalamic corticotrophin-releasing hormone (CRH), arginine vasopressin (AVP) and pituitary adrenocorticotropic hormone ( ACTH), in addition to hypothalamic and pituitary cortisol feedback to regulate cortisol secretion. Importantly, joint interactions among the four, rather than the signal of any one hormone, govern this life-preserving axis. The objective was to estimate endogenous feed-forward and feedback dynamics in a tetrapartite (4-hormone) nonlinearly coupled system. The hypothesis was that ensemble dynamics exhibit quantifiable (a) time evolution after an endogenous stressor, such as acute cortisol depletion or hypoglycemia, (b) individual and joint feedback of cortisol on ACTH, CRH and AVP outflow, (c) synergy between CRH and AVP in driving ACTH secretion and (d) possible in vivo autofeedback by hypothalamic CRH and AVP. Preliminary studies in our lab resulted in a new protocol whereby the pituitary blood and jugular blood could be accessed and collected every minute in live horses with minimum restraint and minimum depletion of the total blood volume. The protocol was used by colleagues in New Zealand to carry out more intense studies. An analytical construct of the interactions of the hormones was generated by Veldhuis and his co-workers. Previously no model existed in the literature in any species to assess the timing of the interactions of the four hormones . The work in these studies resulted in a manuscript which frames an analytically tractable model of the CRH-AVP-ACTH-cortisol ensemble as a prototype of complex reciprocal interactions. This study will be helpful in analyzing pathophysiology of the adrenal axis in other species including the cow and sheep as well as provide insight to drugs that might alleviate stress-induced symptoms in clinical cases. The second part of this project was to investigate the dynamic interactions between the adrenal gland and the ovary in the mare. By measuring LH and FSH hormone profiles in blood taken from the pituitary cannula and the jugular vein during the cortisol study, it was determined that manipulating cortisol levels did not affect changes in LH and FSH levels or their amplitude and frequency. Therefore, to determine if gonadotropins had a direct effect on the adrenal gland, it was decided to clone equine LH in large amounts for injections. A unique single chain recombinant equine LH (reLH) was cloned. A company scaled it up for research purposes. The initial research was done in our lab to determine the efficacy of the recombinant in the stallion to stimulate testosterone production from the testis and the mare to induce ovulation. Our laboratory demonstrated that the reLH was very effective in stimulating a biological response in the stallion and mare. ReLH has been available to veterinarians to induce ovulation in mares and researchers to study reproductive events. This new technology will improve knowledge on the reproductive biology of the mare's ovary and its interaction with the adrenal gland.
Publications
- Yoon, M.J., Boime, I., Colgin, M., Niswender, K.D., King, K.D., King, S.S., Alvarenga, M., Jablonka-Shariff, A., Pearl, C.A., and Roser, J.F. (2007). The efficacy of a single chain recombinant equine luteinizing hormone (reLH) in mares: induction of ovulation, hormone profiles and inter-ovulatory intervals. Domestic Animal Endocrinology, 33:470-479.
- Keenan, D.M., Alexander, S., Irvine, S., and Velduis, J.D. (2008). Quantifying nonlinear interactions within the hypothalamo-pituitary adrenal axis in the conscious horse. Endocrinology, Nov 20 [Epub ahead of print].
- Jablonka-Shariff, A., Roser, J.F., Bousfield, G.R., Wolfe, M.W., Sibley, L.E., Colgin, M., and Boime, I. (2007). Expression and bioactivity of a single chain recombinant equine luteinizing hormone (reLH). Theriogenology, 67:311-320.
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Progress 01/01/07 to 12/31/07
Outputs
OUTPUTS: Studies in the literature indicate that the mechanism by which stress affects reproduction is through the hormonal interaction between the HPA and the HPG, but the specific mechanisms involved are unclear. The objective of this ongoing research is to determine what mechanisms are involved in stress-related alterations in reproductive function by investigating dynamic changes in pituitary gonadotropin secretion after suppression of cortisol and subsequent cortisol replacement in the mare. Specific Aim: To determine the dynamics between HPA and HPG by determining GnRH-LH and FSH pulses (frequency and amplitude). Previously we reported that metyrapone treatment in mares during their diestrus period caused a significant decrease in cortisol along with changes in the pulsatility of LH. Ongoing studies have involved further analysis of the pulsatility of LH and FSH. A cannula was surgically inserted into the facial vein of nine cycling mares during their diestrus period. The
cannula was placed into the intercavernous-sinus near the pituitary via the facial vein. Six mares were used as controls, two of which were treated the next day with a continuous 3-hour infusion of metyrapone. Three mares were given metyrapone on the first day and metyrapone and cortisol replacement on the second day. Blood from the cannula was collected at 1 min intervals starting at 30 minutes prior to treatment, during the 3-hour metyrapone and or cortisol replacement and 1 hr post treatment. A GnRH bolus was given at the end of treatment. Results: Systemic cortisol levels declined 50% from baseline within the 3-hour treatment period. It appears that there was an increase in LH and FSH pulse frequency and amplitude during treatment with metyrapone and that after cortisol treatment pulsatility was similar to controls. GnRH-induced LH and FSH responses were not different among the control and treated mares. In conclusion, results indicate that the drug metyrapone is a potent
inhibitor of cortisol production in diestrous mares. In addition, it appears that metyrapone treatment via a decrease in cortisol production may increase LH and FSH pulsatility. The inhibitory effects of cortisol appear to be at the level of the hypothalamus on secretion of GnRH. Plasma is being analyzed for progesterone.
PARTICIPANTS: Three lab technicians, Lil Sibley, Geri Meyers-Brown and Lisa Bidstrup, an MS graduate student, Jennifer Winnings, and several undergraduate students worked on this project under the supervision of Dr. Jan Roser. In addition, one veterinarian, Dr. Mary Scott, helped with the surgical placement of the facial vein cannulas. This project provided practical experience for the lab techs, graduate student and undergraduate students working with mares to determine their estrous cycles, learning surgical techniques, learning how to take blood and analyzing the blood for hormone profiles. The team also gained scientific knowledge on the interactions between the HPA axis and the HPG axis on reproductive function.
TARGET AUDIENCES: The target audience is the animal breeding industry. Domestic species as well as horses can experience stressful situations whether it be performance, disease, physical trauma or shipping. Stress can impact changes in reproductive hormones resulting in subfertility. The knowledge gained from these studies will be helpful to breeders and veterinarians when treating stress related reproductive dysfunction.
PROJECT MODIFICATIONS: There have been no changes in approach to investigating the inter-relationship between the HPA axis and the HPG axis on reproductive function.
Impacts
Results from these studies will enhance our understanding of the dynamic mechanisms involved in the hormonal interplay between the HPA and the HPR. Such information may clarify new approaches to the treatment and prevention of stress-related alterations in reproductive function which may interfere with pregnancy rates.
Publications
- No publications reported this period
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Progress 01/01/06 to 12/31/06
Outputs
Furthering our understanding of gonadotropin function in pathologies of equine reproduction requires the availability of purified hormone. A chimera of equine α and β subunits was constructed to avoid inefficient assembly previously observed with human and bovine molcules. This chimeric construct was formed by overlap PCR in which the carboxy terminal end of the eLHβ subunit was genetically fused to the amino end of the α subunit. The chimeric gene construct was transfected into CHO cells and the recombinant product was purified through multiple steps, including a Fractogel resin separation. Dilutions of pituitary derived native eLH and the single chain recombinant LH were compared in an eLH radioimmunoassay (RIA); the concentration curves between the single chain recombinant eLH and the native eLH standard were parallel. The biological activity of the analog was determined in vitro and in vivo using homologous equine models. Testicular tissue from
five colts was processed for Leydig cell cultures. Increasing doses of recombinant LH were incubated with equine Leydig cells for 24 h in vitro and testosterone production was determined by RIA. Recombinant eLH stimulated a greater than 15-fold increase in testosterone production in a dose-dependent manner. Quarter Horse breeding stallions were treated with either recombinant LH (n = 5) or saline (n = 3) and plasma testosterone concentrations were measured by RIA. Recombinant eLH stimulated a four-fold increase in circulating testosterone concentrations compared with the saline control. Efficacy of this recombinant LH in shortening the interval to ovulation and effects on endogenous hormones were tested in cycling mares. A total of 114 mares of light horse breeds were treated with either a vehicle, various doses of recombinant LH, or human chorionic gonadotropin in four separate studies. Cycling mares were examined by palpation and ultrasound per rectum daily or every 12 h from the
time of treatment to ovulation. Ovulation rates within 48 h following recombinant LH treatment were significantly higher when mares were treated with doses ranging between 0.6 mg and 0.9 mg recombinant LH. The most effective dose was 0.75 mg, which induced an ovulation rate of 90% within 48 hours; similar to the ovulation rate of 86% after hCG. Hormone profiles appeared similar between control and treated animals. Inter-ovulaory intervals were similar between control and treatment cycles. In conclusion, recombinant LH is a reliable and effective ovulatory agent that does not have detrimental effects on endogenous hormone profiles or the inter-ovulatory intervals. Therefore, the single chain recombinant should be effective for a variety of structure-function studies.
Impacts
An effective, available equine LH molecule will contribute significantly to future studies on the role of this gonadotropin in normal and pathological states. This will enhance our understanding of the dynamic mechanisms involved in the hormonal interplay between the HPA and the HPR. This understanding may elucidate new approaches to the treatment and prevention of stress-related alterations in reproductive function which interfere with pregnancy.
Publications
- King, S.S, Campbell, A.G., Dille, E.A. Roser, J.F., Murphy, L.L., Jones, K.L. 2005 . Dopamine receptors in equine ovarian tissues. Domestic Animal Endocrinology, 28: 405-415.
- Zelli, R., Sylla, L., Monaci, M., Stradaioli, G., Sibley, L.E., Roser, J.F., Munro, C., Liu, . 2006 . I.K. Gonadotropin secretion and pituitary responsiveness to GnRH in mares with granulose-theca cell tumor. Theriofenology, 66:1210-1218.
- Hess, M.F., Roser, J.F. 2005. A comparison of the effects of equine luteinizing hormone (eLH), equine growth hormone (eGH) and human recombinant isulin-like growth factor (hrIGF-I) on steroid production in cultured equine Leyding cells during sexual maturation. Animal Reprod Science, 8-: 7-19
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Progress 01/01/05 to 12/31/05
Outputs
Studies in the literature indicate that the mechanism by which stress affects reproduction is through the hormonal interaction between the HPA and the HPG, but the specific mechanisms involved are unclear. The objective of this ongoing reserach is to determine what mechanisms are involved in stress-related alterations in reproductive function by investigating dynamic changes in pituitary gonadotropin secretion after suppression of cortisol and subsequent cortisol replacement in the mare. Specific Aim: To determine the dynamics between HPA and HPG by determining GnRH-FSH pulses (frequency and amplitude). Previously we reported that metyrapone treatment in mares during their diestrus period caused a significant decrease in cortisol along with changes in the pulsatility of LH. Ongoing studies have involved analysis of the pulsatility of FSH. A cannula was surgically inserted into the facial vein of nine cycling mares during their diestrus period. The cannula was placed
into the intercavernous-sinus near the pituitary via the facial vein. Six mares were used as controls, two of which were treated the next day with a continuous 3-hour infusion of metyrapone. Three mares were given metyrapone on the first day and metyrapone and cortisol replacement on the second day. Blood from the cannula was collected at 1 min intervals starting at 30 minutes prior to treatment, during the 3-hour metyrapone and or cortisol replacement and 1 hr post treatment. A GnRH bolus was given at the end of treatment. Partial results: Systemic cortisol levels declined 50% from baseline within the 3-hour treatment period. In the three mare analyzed so far, it appears that there was a decrease in FSH pulse frequency and amplitude during treatment and after GnRH treatment. However, pulse amplitude increased back to control levels with cortisol replacement. In conclusion, preliminary results indicate that the drug metyrapone is a potent inhibitor of cortisol production in diestrous
mares. In addition, it appears that metyrapone treatment may have an effect on FSH pulsatility as well as GnRH - induced FSH release. All animal studies are complete. Plasma is being analyzed for FSH in the 6 other mares.
Impacts
Results from these studies will enhance our understanding of the dynamic mechanisms involved in the hormonal interplay between the HPA and the HPR. Such information may clarify new approaches to the treatment and prevention of stress-related alterations in reproductive function which may interfere with pregnancy rates.
Publications
- No publications reported this period
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Progress 01/01/04 to 12/31/04
Outputs
Previous studies in the literature indicate that the mechanism by which stress affects reproduction is through the hormonal interaction between the HPA and the HPG, but the specific mechanisms involved are unclear. The objective of this ongoing study is to determine what mechanisms are involved in stress-related alterations in reproductive function by investigating dynamic changes in pituitary gonadotropin secretion after suppression of cortisol and subsequent cortisol replacement in the mare. Specific Aim 1: To determine time and dose of metyrapone (drug suppresses cortisol release) that may affect the reproductive hormones in the mare. Various treatment regimes (Trial A, B, C, D and E) were carried out on 3 mares each trial during diestrus (7-12 days post ovulation). Blood was collected via a jugular vein every 30 min from -0.5 through + 8 hours. Plasma was assayed for LH by RIA. Partial results: A bolus of metyrapone of 1.25 gm did not affect LH release (Trial A)
nor did a bolus of 1.25 gm plus a 4 hours infusion of metyrapone at 0.05g/min (Trial C). However, a 38% increase in LH release was observed after withdrawl of a 2.5 hr metyrapone infusion that was preceded by a 1.25 gm bolus injection (Trial B). At the time of withdrawal, cortisol levels had declined by 73% from baseline. This data suggests that cortisol has an effect on GnRH pulses (frequency or amplitude) or LH stores in the pituitary. Specific Aim 2: To determine the dynamics between HPA and HPG by determining GnRH-LH pulses (frequency and amplitude). A cannula was surgically inserted into the facial vein of nine cycling mares during their diestrus period. The cannula was placed into the intercavernous-sinus near the pituitary via the facial vein. Six mares were used as controls, two of which were treated the next day with a continuous 3-hour infusion of metyrapone. Three mares were given metyrapone on the first day and metyrapone and cortisol replacement on the second day. Blood
from the cannula was collected at 1 min intervals starting at 30 minutes prior to treatment, during the 3-hour metyrapone and or cortisol treatment and 1 hr post treatment. Partial results: Systemic cortisol levels declined 50% from baseline within the 3-hour treatment period. It appears that there was a decrease in LH pulse amplitude during treatment. However, pulse amplitude increased back to control levels with cortisol replacement. In conclusion, preliminary results indicate that the drug metyrapone is a potent inhibitor of cortisol production in diestrous mares. In addition, it appears that metyrapone treatment may have an effect on basal secretion of pituitary LH as well as GnRH-induced LH release as previously reported in the last progress report. All animal studies are complete. Plasma is now being analyzed for LH, FSH and progesterone.
Impacts
Results from these studies will enhance our understanding of the dynamic mechanisms involved in the hormonal interplay between the HPA and the HPR. Such information may clarify new approaches to the treatment and prevention of stress-related alterations in reproductive function.
Publications
- No publications reported this period
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Progress 01/01/03 to 12/31/03
Outputs
Stress affects reproductive function in a variety of domestic species including the horse. Previous studies indicate that the mechanism by which stress affects reproduction is through the hormonal interaction between the HPA and the HPG, but the specific mechanisms involved are unclear. The objective of this ongoing study is to determine what mechanisms are involved in stress-related alterations in reproductive function by investigating dynamic changes in pituitary gonadotropin secretion after suppression of cortisol and subsequent cortisol replacement. A cannula was surgically inserted into the facial vein of six cycling mares during their diestrus period (7-12 days post ovulation) when plasma concentrations of luteinizing hormone (LH) is relatively low and constant. The cannula was threaded up the facial vein into the intercavernous-sinus, which collects both hypothalamic and pituitary blood near the pituitary. Of the 6 mares, two were used as their own controls and
treated the next day with a continuous 3-hour infusion of metyrapone (I.V.; a drug that suppresses adrenal cortisol production). At the end of the treatment, mares were given an injection of gonadotropin releasing hormone (GnRH, I.V.), which stimulates the pituitary to secrete LH. Blood from the cannula was collected at 1 min intervals starting at 30 minutes prior to treatment, during the 3-hour metyrapone treatment period and up to 1 hour after the injection of GnRH. Systemic cortisol levels declined 70% within the 3-hour treatment period. However, there appears to be no difference in basal secretion levels of LH from the cannula between controls and mares treated with meytrapone. When treated with GnRH, the two treated mares responded differently. One mare responded with a higher concentration of LH from the cannulated vein compared to her own control levels while the other mare responded with secreting the same or lower levels. Further studies will be carried out with a larger
number of mares to determine the effects of treatment on LH and follicle stimulating hormone (FSH) levels from the pituitary during treatment with metyrapone and after treatment with GnRH. In addition, mares will be given cortisol replacement therapy to determine if dynamic changes in the cortisol levels will affect pituitary output of gonadotropins. In conclusion, preliminary results indicate that the drug metyrapone is a potent inhibitor of cortisol production in diestrous mares. In addition, it appears that metyrapone treatment may not have an effect on basal secretion of pituitary LH but may affect GnRH - induced LH release.
Impacts
Results from these studies will enhance our understanding of the dynamic mechanisms involved in the hormonal interplay between the HPA and the HPR. Such information may clarify new approaches to the treatment and prevention of stress-related alterations in reproductive function.
Publications
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Progress 01/01/02 to 12/31/02
Outputs
The long term goal is to identify mechanisms involved in the interaction between the HPA (brain and adrenal gland) and HPG (brain and ovary. Specific Aim 1: To determine the effects of cortisol suppression and replacement therapy on circulating concentrations of reproductive hormones in the mare. Specific Aim 2: To determine the dynamics between the HPA and the HPG after cortisol suppression and replacement.
Impacts
Stress affects the brain (hypothalus and pituitary) and adrenal gland output. How stress may have an effect on reproductive events is unknown but part of the problem may lie in the hormone interplay between adrenal and ovarian function. By experimentally altering the hormones involved in adrenal function, we hope to determine the extent to which ovarian function is affected. This will give us further insight in to how stress affects reproduction.
Publications
- No publications reported this period
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