Progress 07/13/98 to 07/12/04
Outputs
The long-range aim of this project was to increase the understanding of the mechanisms regulating gonadotropin-releasing hormone (GnRH) induced luteinizing hormone (LH) secretion to ultimately improve the efficiency of reproductive function in cattle. Ovarian follicular growth during the dominance period of development is prerequisite for reproductive function in cattle and is dependent upon the pulsatile secretion of the luteinizing hormone (LH) from the anterior pituitary. The later, is in turn dependent upon the pulsatile release of GnRH from the hypothalamus. A collaborative study was conducted, during this period, with G. L. Williams and T. E. Spencer to determine how leptin enhances the secretion of LH in fasted cows. Leptin is believed to communicate an animal's nutritional status to the central nervous system centers that control the process of reproduction. Exogenous leptin increases mean concentrations of LH in the peripheral circulation of fasted cows. In
addition, leptin increases the release of LH from excised anterior pituitary tissue, from fasted cows, incubated in vitro. In this study, we tested the hypothesis that expression of leptin receptors in the anterior pituitary are increased by fasting. Ten ovariectomized, estradiol-replaced cows were assigned to one of two groups: Full-fed and Fasted (fasted for 72 hours). At the end of the fasting period, cows in both groups were killed and their anterior pituitaries excised. Real-time polymerase chain reaction indicated that normalized expression of the leptin receptor did not differ between full fed and fasted cows. In situ hybridization analysis for leptin receptor mRNA revealed no distinctive pattern of expression between the full-fed and fasted cows. These results do not support the hypothesis that expression of leptin receptors in the anterior pituitary is increased in fasted cows.
Impacts
These findings do not support involvement of increased leptin receptors in leptin induced LH release in fasted cows. Further work is warranted before the role of leptin in the secretion of LH in fasted cattle can be determined.
Publications
- Amstalden, M., Harms, P.G., Welsh Jr., T.H., Randel, R.D. and Williams, G.L. 2005. Effects of leptin on gonadotropin-releasing hormone release from hypothalamic-infundibular explants and gonadotropin release from adenohypophyseal primary cell cultures: further evidence that fully nourished cattle are resistant to leptin. Animal Reproduction Science 85: 41-52.
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Progress 01/01/03 to 12/31/03
Outputs
The aim of this project is to improve the reproductive efficiency of cattle by increasing the understanding of the mechanisms controlling secretion of the gonadotropin-releasing hormone (GnRH). Ovarian follicular growth during the dominance period of development is prerequisite for reproductive function in cattle and is dependent upon the pulsatile secretion of the luteinizing hormone from the anterior pituitary which is in turn dependent upon the pulsatile release of GnRH from the hypothalamus. This past year, a collaborative study was conducted with G. L. Williams and R. D. Randel on the involvement of Leptin in the secretion of GnRH in cattle. In rodents, leptin stimulates the secretion of GnRH from hypothalamic-infundibular (HYP) explants incubated in vitro. In our study, we determined if leptin could stimulate release of GnRH from bovine HYP explants using an in vitro culture procedure operational in my laboratory. In the first experiment, HYP explants collected
from 24 bulls and steers were incubated with Krebs-Ringer bicarbonate buffer (KRB) containing 0, 10, 100, or 1000 ng/ml recombinant ovine leptin (oleptin) for 30 minutes after a 3 hour period of equilibration. None of the doses of leptin affected (P>05) GnRH release into the media. In a second experiment, HYP explants collected from 6 steers were incubated with KRB containing 0 or 1000 ng/ml of oleptin for 2 consecutive 30 minute periods and challenged with 60 mM potassium afterwards. Leptin did not affect (P>0.05) basal or potassium-stimulated release of GnRH.
Impacts
These findings do not support involvement of leptin in GnRH release in cattle. Further work is warranted before the role of leptin in the secretion of GnRH in cattle can be determined.
Publications
- Amstalden, M., Zieba, D.A., Edwards, J.F., Harms, P.G., Welsh, Jr., T.H., Stanko, R.L., Williams, G.L. 2003. Leptin acts at the bovine adenohypophysis to enhance basal and GnRH-mediated release of LH: differential effects are dependent upon nutritional history. Biology of Reproduction 69:1539-1544.
- Zieba, D.A., Amstalden, M, Morton, S., Gallino, J.L., Edwards, J.F., Harms, P.G. and Williams, G.L. 2003 Effects of leptin on basal and GHRH-stimulated GH secretion from the bovine adenohypophysis are dependent upon nutritional status. Journal of Endocrinology 178:83-89
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Progress 01/01/02 to 12/31/02
Outputs
This project is intended to improve the reproductive efficiency of cattle by increasing the understanding of the mechanisms controlling secretion of the gonadotropin-releasing hormone (GnRH). Ovarian follicular growth during the dominance period of development is prerequisite for reproductive function in cattle and is dependent upon the pulsatile secretion of the luteinizing hormone (LH) from the anterior pituitary which is in turn dependent upon the pulsatile release of GnRH from the hypothalamus. During the past year attention was focused on mechanisms by which the nutritional status of cows may impact the secretion GnRH. It has been proposed that leptin may act at a hypothalamic level to stimulate the secretion of the GnRH. A collaborative study was conducted with Gary L. Williams (source of cows and treatment of the cows) to determine if leptin may act upon the hypothalamus to influence the secretion GnRH in cattle in two nutritional states. Ten mature,
ovariectomized cows in moderately thin body condition, each bearing an estradiol implant were used. The cows were randomly assigned to one of two dietary groups: 1. Normal-Fed (N=5), and 2. Fasted (72 hour fast N=5). At the end of the 72 hour period, the cows were euthanized and the hypothalami collected and midsagittal sectioned. The resulting hypothalamic explant halves (medialbasal area with the attached infundibulum) were incubated in Krebs-Ringer bicarbonate medium (KRB) in vitro to evaluate GnRH release in response to ovine leptin. We have previously demonstrated the utility of monitoring GnRH secretion from hypothalamic explants as a useful procedure for investigating mechanisms regulating the secretion of GnRH. After a period of equilibration, the hypothalamic explant halves were incubated in the presence of KRB alone or KRB with 1000 ng of ovine leptin. Neither fasting nor leptin affected the secretion of GnRH from the hypothalamic explants halves.
Impacts
These findings do not support involvement of leptin in GnRH release in cattle that are normally fed or fasted. However, further work is warranted before a definitive role of leptin in the secretion of GnRH in cattle can be determined.
Publications
- No publications reported this period
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Progress 01/01/01 to 12/31/01
Outputs
The long range goal of this project is to improve the reproductive efficiency of cattle by increasing the understanding of the mechanisms that control secretion of the gonadotropin-releasing hormone (GnRH) from the hypothalamic area of the brain. Ovarian follicular growth during the dominance phase of development is prerequisite for reproductive function in cattle and is dependent upon the pulsatile secretion of the luteinizing hormone from the anterior pituitary which is in turn dependent upon the pulsatile release of GnRH from the hypothalamus. In rats, Prostaglandin E2 (PGE2) is involved in the release of GnRH. Intraventricular administration of PGE2 released LH into the peripheral blood by stimulating GnRH release. Other evidence supporting a role of PGE2 in release of GnRH, is that incubation of rat hypothalamic explants in vitro with PGE2 released GnRH. The involvement of PGE2 in GnRH release in cattle is unknown. The objective of a study this past year was to
test the hypothesis that PGE2 induces release of GnRH from cattle infundibular explants incubated in vitro, as it does from rat infundibular explants. Infundibular explant halves from steers were incubated over 9 thirty-minute periods in Krebs-Ringer bicarbonate media with or without PGE2 (period 6) and 60 milli M potassium (period 8) at 37 degrees C and saturated with 95% oxygen and 5% carbon dioxide. GnRH released into the media was evaluated by radioimmunoassay. PGE2 at concentrations of 0, 100, 1000 micro M did not alter release of GnRH. Depolarization of the explant tissue with potassium, induced a clear increase in GnRH release. The potassium induced depolarization response was consistent with that previously observed from cattle and rat explants. These results indicate that PGE2 is unable to stimulate GnRH release from bovine infundibular explants halves incubated in vitro. Thus, the findings do not support a role for PGE2 in release of GnRH from the infundibulum of cattle.
Impacts
These findings suggest that PGE2 is not involved in GnRH release in cattle as is the case for rats. However, further work is warranted using other research approaches to rule out such a role for GnRH.
Publications
- Stephens, T.P. 2001. Effect of prostaglandin E2 (PGE2) on the release of gonadotropin-releasing hormone (GnRH) from infundibular explants of cattle. Master of Science Thesis. Texas A&M University. College Station. 93p.
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Progress 01/01/00 to 12/31/00
Outputs
This research effort is designed to enhance the reproductive efficiency of cattle by furthering the understanding of mechanisms that control secretion of the gonadotropin-releasing hormone (GnRH) from the hypothalamic area of the brain. The ovarian follicular development and ovulation that is prerequisite for reproductive function in cattle is dependent upon the pulsatile secretion of the luteinizing hormone from the anterior pituitary which is dependent upon the pulsatile release of GnRH from the hypothalamus. A specific objective of our effort was to evaluate the relationship of the content and releasable quantity of hypothalamic GnRH in cattle at varying reproductive states. Our hypothesis was that releasable GnRH is dependent on the reproductive status of the animal. Gonadal intact and gonadectomized cows and bulls and suckled cows at varying intervals (1-4, 10-13 and 30-33 days) postpartum were used for our studies. Hypothalami (specifically the infundibulum or
stalk median eminence) were collected immediately following routine slaughter. Releasable GnRH was determined for each group by radioimmunoassay (RIA) of the quantity of GnRH released in response to potassum induced depolarization of the infundibular explants incubated in vitro. Quantity of infundibular GnRH was determined by RIA of GnRH extracted from each infundibulum plus that released during culture. Our results indicate that: 1) releasable GnRH is greater in the infundibulia from intact compared with gonadectomized cows and bulls, 2) infundibular GnRH content was greater in non cycling-suckled (1-4 days postpartum) cows compared with that in cows with normal estrous cycles, 3) releasable GnRH from infundibular explants was greater in early (1-4 days) postpartum compared to later (10-13 days) postpartum cows. Since release of GnRH was not related to GnRH content in the infundibulum, we conclude that factors other than content affect the releasable pool of hypothalamic GnRH in
cattle.
Impacts
These findings support the hypothesis that the quantity of releasable GnRH varies with the reproductive status of cattle. Further work is warranted to determine how reproductive state influences the releasable pool of hypothalamic GnRH.
Publications
- No publications reported this period
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Progress 01/01/99 to 12/31/99
Outputs
This research is intended to enhance the reproductive efficiency of cattle by increasing the understanding of neural involvement in gonadotropin-releasing hormone (GnRH) secretion from the hypothalamus. Ovarian activity (follicular development and subsequent ovulation) prerequisite for reproductive function is dependent upon the pulsatile secretion of luteinizing hormone from the anterior pituitary which in turn is dependent upon the pulsatile secretion of GnRH from the hypothalamus. Intracellular calcium is involved in mediating the pulsatile release of GnRH. The objective of this year's effort was to investigate the relationship of GnRH release with intracellular calcium oscillations using the immortalized hypothalamic cell line, GT1-1, which synthetizes and releases GnRH. GnRH release was measured by radioimmunoassay of media incubated with GT1-1 cells for 1 hour with control or test substances to induce release of GnRH. Intracellular calcium oscillations were
analyzed by confocal microscopy of fluorescence intensities, at 3 second intervals, of GT1-1 cells loaded with flu0-3, AM. Both prostaglandin E2 (PGE2) and prostaglandin E1 (PGE1) released GnRH in a dose dependent manner, with PGE1 being more potent than PGE2. Over 90% of the GT1-1 cells exhibited intrinsic intracellular oscillations of calcium. The frequency or pattern of the oscillations was synchronized in cells within an aggregate of cells, but varied between unconnected aggregates of cells. Intrinsic calcium oscillations were absent when EGTA, a calcium binding agent, was present in the media. Both PGE2 and PGE1 induced an immediate increase in the frequency of calcium oscillations. In summary, GT1-1 neuronal cells exhibit intrinsic calcium oscillations whose frequency and pattern is synchronized between interconnected cells but vary between unconnected cells. Extracellular calcium appears important for the intracellular calcium oscillations. Both PGE2 and PGE1, at doses that
induced release of GnRH, induced a dramatic increase in the frequency of intracellular calcium oscillations. These results indicate that release of GnRH is associated with an increased frequency of intracellular calcium oscillations and suggest that intracellular calcium oscillations are involved in GnRH release.
Impacts
These findings suggest that intracellular calcium oscillations are involved in the release of GnRH. Thus, neural anomalies that interfere with the intracellular calcium oscillations may interfere with the release of GnRH and hence diminish reproductive function.
Publications
- Gilmore, C.D. 1999. Association of intracellular calcium oscillations with release of gonadotropin-releasing hormone (GnRH) from GT1-1 neuronal cells. Master of Science. Texas A&M University. College Station. 68p.
- Harms, P.G., Gilmore, C.D., Barhoumi, R., Chen, P.C. and Burghardt, R.C. 1999. Prostaglandin E2 induced release of gonadotropin-releasing hormone from GT1-1 neuronal cells is associated with increased frequency of intracellular calcium oscillations. Soc. Neurosci. Abstr, Vol. 25, Part 1, p. 1188 (Abstract 479.14).
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Progress 07/13/98 to 12/31/98
Outputs
The long term goal of this research is to enhance the reproductive efficiency of cattle by furthering the understanding of the neural control of gonadotropin-releasing hormone secretion (GnRH) from the hypothalamus. Ovarian function (follicular development, ovulation and luteal activity) prerequisite for reproduction is dependent upon the pulsatile secretion of GnRH. Nitric oxide appears to be involved in the secretion of GnRH from the hypothalamus of male rats. To provide evidence for nitric oxide involvement in the secretion of GnRH in cattle, a study was conducted to determine if exogenous nitric oxide would induce GnRH release from cattle median eminence (infundibular) explants incubated in vitro. Sodium nitroprusside, a donor of nitric oxide, increased GnRH release from steer median eminence explants at a dosage of 500 micromolar. Dosages of 250 and 750 micromolar did not increase GnRH release. Neither of the 3 doses of sodium nitroprusside increased the release
of GnRH from median eminence explants from heifers. These findings support involvement of nitric oxide in cattle and that the effect may be dependent upon the sex of the animal. Further work is warranted to verify the involvement of nitric oxide in GnRH release in cattle. We previously demonstrated that calcium / calmodulin dependent protein kinase II may be involved in GnRH secretion by showing that two different inhibitors of calcium / calmodulin dependent protein kinase II blocked depolarization induced release of GnRH from rat and cattle hypothalamic explants incubated in vitro. We also showed that inhibitors of calcium / calmodulin dependent protein kinase II blocked depolarization induced release of GnRH from cells of the immortalized GnRH secreting neuronal cell line GT1-1. Experiments were conducted to determine if the effects of the calcium / calmodulin dependent protein kinase II antagonists are unique for depolarization induced GnRH release or if they are capable of
blocking GnRH release in response to other agents known to induce GnRH release. To that end, cultured GT1-1 neuronal cells were incubated with 0.0, 0.5 and 5.0 micromolar KN-62, a specific inhibitor of calcium / calmodulin dependent protein kinase II, in the presence of prostaglandin E2 (PGE2). KN-62 inhibited PGE2 induced release of GnRH is a dosage dependent manner. These findings provide additional support for a role of calcium / calmodulin dependent protein kinase II in the release of GnRH.
Impacts
(N/A)
Publications
- Waters, W.W., Chen, P.L., McArthur, N.H., Moreno, P.A. and Harms. P.G. 1998. Calcium/calmodulin-dependent protein kinase II involvement in release of gonadotropin-releasing hormone. Neuroendocrinology 67:145-152.
- Lang, A.L., Vogelsang, M.M., Potter, G.D., Blanchard, T.L. and Harms, P.G. 1998. Semen parameters and hormone concentrations in stallions subjected to long-term estrogen administration. Journal of Equine Veterinary Science 18:114-117.
- Teague, S.R. 1998. The effect of nitric oxide on in vitro release of GnRH from the bovine infundibulum. Master of Science Thesis. Texas A&M University. College Station. 75p.
- Chen, P.L., Gilmore, C.D. and Harms, P.G. 1998. Calcium / calmodulin protein kinase II involvement in prostaglandin E2-induced LHRH release from GT1-1 neuronal cells. Biology of Reproduction 58: Supplement 1:97 (Abstract 83). l998.
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