Orexin-B modulates luteinizing hormone and growth hormone secretion from porcine pituitary cells in culture

To test the hypothesis that orexin-B acts directly on the anterior pituitary to regulate LH and growth hormone (GH) secretion, anterior pituitary cells from prepuberal gilts were studied in primary culture. On day 4 of culture, 10(5) cells/well were challenged with 0.1, 10 or 1000 nM GnRH; 10, 100 or 1000 nM [Ala15]-hGRF-(1-29)NH2 or 0.1, 1, 10 or 100 nM, orexin-B individually or in combinations with 0.1 and 1000 nM GnRH or 10 and 1000 nM GRF. Secreted LH and GH were measured at 4 h after treatment. Basal LH and GH secretion (control; n = 6 pigs) was 183 +/- 18 and 108 +/- 4.8 ng/well, respectively. Relative to control at 4 h, all doses of GnRH and GRF increased (P < 0.0001) LH and GH secretion, respectively. All doses of orexin-B increased (P < 0.01) LH secretion, except for the 0.1 nM dose. Basal GH secretion was unaffected by orexin-B. Addition of 1, 10 or 100 nM orexin-B in combinations with 0.1 nM GnRH increased (P < 0.001) LH secretion compared to GnRH alone. Only 0.1 nM (P = 0.06) and 100 nM (P < 0.001) orexin-B in combinations with 1000 nM GnRH increased LH secretion compared to GnRH alone. All doses of orexin-B in combination with 1000 nM GRF suppressed (P < 0.0001) GH secretion compare to GRF alone, while only 0.1 nM orexin-B in combination with 10 nM GRF suppressed (P < 0.01) GH secretion compared to GRF. These results indicate that orexin may directly modulate LH and GH secretion at the level of the pituitary gland.     

Role of leptin in modulating the hypothalamic-pituitary axis and luteinizing hormone secretion in the prepuberal gilt

Three experiments (EXP) were conducted to test the hypothesis that leptin modulates LH, GnRH, and neuropeptide Y (NPY) secretion. In EXP I, prepuberal gilts received intracerebroventricular (i.c.v.) leptin injections and blood samples were collected. In EXP II, anterior pituitary cells from prepuberal gilts in primary culture were challenged with 10(-14), 10(-13), 10(-12), 10(-11), 10(-10), 10(-9), 10(-8), 10(-7), or 10(-6) M leptin individually or in combinations with 10(-10), 10(-9), and 10(-8) M GnRH. In EXP III, hypothalamic-preoptic area (HYP-POA) explants were placed in perfusion system and exposed to 0 (n=5), 10(-12) M (n=4), 10(-10) M (n=4), 10(-8) M (n=4), or 10(-6) M (n=5) human recombinant leptin (LEP) for 30 min. In EXP I, serum LH concentrations were unaffected by leptin treatment. In EXP II, all doses of leptin increased LH secretion except for 10(-12) and 10(-7) M. Only 10(-7), or 10(-13) M leptin in combination with 10(-8) or 10(-9) M GnRH, respectively, suppressed LH secretion. In EXP III, prior to leptin, media GnRH concentrations were similar across treatments. Media GnRH concentrations increased after 10(-12), 10(-10), and 10(-8) M leptin compared to control. Leptin treatment failed to influence NPY secretion across treatments. These results indicate that components of the neuroendocrine axis that regulate GnRH and LH secretion are functional and leptin sensitive before the onset of puberty. Other neural peptides in addition to NPY may mediate the acute effects of leptin on the GnRH-LH system and lastly, the inability of i.c.v. leptin treatment to increase LH secretion may in part be related to stage of sexual maturation and associated change in negative feedback action of estradiol on LH secretion.     

N-methyl-d,l-aspartate stimulates growth hormone and prolactin but inhibits luteinizing hormone secretion in the pig.

The effects of n-methyl-d,l-aspartate (NMA), a neuroexcitatory amino acid agonist, on luteinizing hormone (LH), prolactin (PRL) and growth hormone (GH) secretion in gilts treated with ovarian steroids was studied. Mature gilts which had displayed one or more estrous cycles of 18 to 22 d were ovariectomized and assigned to one of three treatments administered i.m.: corn oil vehicle (V; n = 6); 10 micrograms estradiol-17 b/kg BW given 33 hr before NMA (E; n = 6); .85 mg progesterone/kg BW given twice daily for 6 d prior to NMA (P4; n = 6). Blood was collected via jugular cannulae every 15 min for 6 hr. Pigs received 10 mg NMA/kg BW i.v. 2 hr after blood collection began and a combined synthetic [Ala15]-h GH releasing factor (1-29)-NH2 (GRF; 1 micrograms/kg BW) and gonadotropin releasing hormone (GnRH; .2 micrograms/kg BW) challenge given i.v. 3 hr after NMA. NMA did not alter LH secretion in E gilts. However, NMA decreased (P < .02) serum LH concentrations in V and P4 gilts. Serum LH concentrations increased (P < .01) after GnRH in all gilts. NMA did not alter PRL secretion in P4 pigs, but increased (P < .01) serum PRL concentrations in V and E animals. Treatment with NMA increased (P < .01) GH secretion in all animals while the GRF challenge increased (P < .01) serum GH concentrations in all animals except in V treated pigs. NMA increased (P < .05) cortisol secretion in all treatment groups. These results indicate that NMA inhibits LH secretion and is a secretagogue of PRL, GH and cortisol secretion with ovarian steroids modulating the LH and PRL response to NMA.     

Luteinizing hormone and growth hormone secretion in ewes infused intracerebroventricularly with neuropeptide Y

Neuropeptide Y (NPY) provides an important hypothalamic link between nutritional status and neuroendocrine mechanisms regulating growth and reproduction. The objective of the following series of experiments was to determine the effects of single or continuous administration of NPY on secretion of luteinizing hormone (LH) and (or) growth hormone (GH). In experiment 1, four ovariectomized (OVX) ewes and four OVX + estrogen-treated ewes each received, in a 4 x 4 Latin Square arrangement of treatments, a single injection of 0, 0.5, 5, or 50 microg NPY via an intracerebroventricular (i.c.v.) cannulae to determine the effects on secretion of GH. NPY significantly elevated serum GH at the 50 microg dose regardless of estrogen exposure (P = 0.003). In experiment 2, eight OVX ewes were infused i.c.v. with NPY or saline (n = 4/trmt) continuously for 20 h in a linearly increasing dose, ending at 50 microg/h NPY. Blood samples were collected via jugular cannulae every 10 min during hour -4-0 (interval 1, pre-treatment), hour 6-10 (interval 2) and hour 16-20 (interval 3) relative to the initiation of infusion (0 h). Mean LH and LH pulse frequency were lower in NPY- versus saline-infused ewes during intervals 2 and 3 (P < 0.01), but NPY had no discernable effect on serum GH (P > 0.10). In experiment 3, four OVX ewes were continuously infused with NPY as in experiment 2, except that the maximum 50 microg/h dose was achieved after only 10 h of infusion. Blood samples were collected every 10 min, beginning 4 h before and continuing until 4h after the NPY infusion. Mean serum LH changed significantly over time (P = 0.0001), decreasing below pre-treatment levels by hour 3 of NPY infusion (P < 0.01), and returning to pre-treatment concentrations following the end of infusion (P > 0.15). Serum GH also changed significantly over time (P < 0.001). Mean GH levels tended to be greater than pre-treatment levels by hour 2 of infusion (P < 0.08), but thereafter returned to basal levels. Serum GH also increased following the end of NPY infusion (P < 0.03). From these data we conclude that NPY exerts a persistent inhibitory effect on secretion of LH, and may stimulate the secretion of GH during the initiation and cessation of infusion of NPY. These observations support a role for NPY in mediating the effects of undernutrition on both LH and GH, and also provide evidence for potential mechanisms by which leptin, acting through NPY, may stimulate the secretion of GH.     

Injection of neuropeptide Y into the third cerebroventricle differentially influences pituitary secretion of luteinizing hormone and growth hormone in ovariectomized cows.

Hypothalamic neurons that control the luteinizing hormone (LH) and growth hormone (GH) axes are localized in regions that also express neuropeptide Y (NPY). Increased hypothalamic expression of NPY due to diet restriction has been associated with suppressed secretion of LH and enhanced secretion of GH in numerous species. However, these physiological relationships have not been described in cattle. Thus, two studies were conducted to characterize these relationships using ovariectomized (Experiment 1) or ovariectomized estrogen-implanted (Experiment 2) cows. In Experiment 1, four well-nourished, ovariectomized cows received third cerebroventricular (TCV) injections of 50 and 500 micrograms of NPY in a split-plot design. Venous blood was collected at 10-min intervals from -4 hr (pre-injection control period) to +4 hr (postinjection treatment period) relative to TCV injection. NPY suppressed (P < or = 0.04) tonic secretion of LH irrespective of dose and tended to stimulate (P < or = 0.10) an increase in tonic secretion of GH. In Experiment 2, six ovariectomized cows that were well nourished and implanted with estradiol received TCV injections of 0, 50, or 500 micrograms of NPY in a replicated 3 x 3 Latin Square. Both doses of NPY suppressed (P < 0.06) mean concentration of LH relative to the 0-microgram dose. The 50-microgram dose of NPY tended (P < 0.10) to increase the amplitude of GH pulses. In conclusion, TCV injection of NPY suppressed pituitary secretion of LH and simultaneously tended to increase pituitary secretion of GH.     

Effect of GnRH and its antagonist (Antarelix) on LH release from cultured bovine anterior pituitary cells

In the following investigations, the LH secretion of cells from pituitaries in heifers on days 16-18 of their oestrous cycle (n = 14) was analysed. Cells were dissociated with trypsin and collagenase and maintained in a static culture system. For the estimation of LH release, the cells were incubated with various concentrations of mammalian GnRH (Lutrelef) for 6 h. To determine the action of Antarelix (GnRH antagonist), the cells were preincubated for 1 h with concentrations of 10(-5) or 10(-4) M Antarelix followed by 10(-6) M GnRH coincubation for a further 6 h. At the end of each incubation, the medium was collected for LH analysis. Parallel, intracellular LH was qualitatively detected by immunocytochemistry. Changes in the intensity of LH staining within the cells in dependence of different GnRH concentrations were not observed, but a significant increase LH secretion in pituitary cells was measured at 10(-6) M GnRH. Antarelix had no effect on basal LH secretion at concentrations of 10(-4) and 10(-5) M. After coincubation of pituitary cells with Antarelix and GnRH, Antarelix blocked the GnRH-stimulated LH secretion with a maximal effect of 10(-4) M, but the staining of immunoreactive intracellular LH was detected at approximately the same level compared to the pituitary cells treated with exogenous GnRH alone. These data demonstrate that Antarelix is effective in influencing the GnRH-stimulated LH secretion of pituitary cells in vitro. After administration of Antarelix in vivo, the GnRH-stimulated LH secretion of cultured pituitary cells was not inhibited.     

Influence of catecholamines, prostaglandins and thyroid hormones on growth hormone secretion by chicken pituitary cells in vitro

In young chickens plasma concentrations of growth hormone (GH) are depressed by prostaglandins (PG) E1 and E2, epinephrine, norepinephrine, alpha 2 and beta agonists or thyroid hormones. A primary culture of chicken adenohypophyseal cells was used to examine the direct effects of these agents at the level of the pituitary as evaluated by GH release in the presence and absence of growth hormone releasing factor (GRF). Following collagenase dispersion and culture (preincubation, 48 hr) cells were exposed (incubation, 2 hr) to test agents, except for thyroid hormones which were added during the preincubation, and incubation period. Growth hormone release was increased (P less than .05) in the presence of PGE1 (10(-8)M by 34%; 10(-7)M by 54%), PGE2 (10(-8)M by 29%; 10(-7)M by 29%), PGF2 alpha (10(-8)M by 28%), and the beta agonist isoproterenol (10(-7)M by 46%). Basal GH release from chicken pituitary cells was not affected by dopamine, norepinephrine, epinephrine, thyroxine (T4), triiodothyronine (T3), or alpha adrenergic agonists. Growth hormone releasing factor stimulated GH release was not affected by the presence of prostaglandins E1, E2 or F2 alpha in the incubation media. However, GRF stimulated GH release was reduced by high doses of catecholamines: dopamine (10(-6)M by 34%), norepinephrine (10(-6)M by 74%), epinephrine (10(-8)M by 47%; 10(-7)M by 41%; 10(-6)M by 89%), and by the alpha 1 adrenergic agonist, phenylephrine (10(-7)M by 52%), the alpha 2 agonist, clonidine (10(-8)M by 34%; 10(-7)M by 83%) and the beta agonist, isoproterenol (10(-7)M by 64%).(ABSTRACT TRUNCATED AT 250 WORDS)     

Androgens modulate growth hormone-releasing factor-induced GH release from bovine anterior pituitary cells in static culture.

Static primary cultures of bovine anterior pituitary (AP) cells were utilized to study the effect of sex steroids on basal growth hormone (GH) and GH-releasing hormone (GRF)-stimulated release of GH. The AP cells (5 x 10(5) cells/well) were allowed to attach for 72 hr and become confluent before treatments were imposed. Cells were incubated for an additional 24, 48 or 72 hr with either estradiol-17 beta (E2, 10(-11) to 10(-8) M), testosterone (T, 10(-8) to 10(-5) M), dihydrotestosterone (DHT, 10(-9) to 10(-6) M) or 5 alpha-androstane-3 alpha, 17 beta-diol (3 alpha-diol, 10(-11) to 10(-8) M). Media were collected every 24 hr and GH concentrations determined by RIA. Incubation of calf AP cells with gonadal steroids did not affect (P > 0.05) basal GH released at 24, 48, or 72 hr. In another experiment, calf AP cells were incubated with the same concentrations of the steroids for 24 hr, media harvested, cells washed and challenged in serum-free media for 1 hr with bovine GRF 1-44-NH2 (10(-8) M). In non-steroid treated wells, GRF increased (P < 0.05) GH from 58 to 134 ng/ml. Incubation with E2 or 3 alpha-diol did not affect (P > 0.05) GRF-induced GH release; however, preincubation with T (10(-5) M) and DHT (10(-9), 10(-8) and 10(-7) M) increased (P < 0.05) GRF-induced GH release above control concentrations (195, 235, 190 and 185 ng/ml, respectively). At the doses tested, sex steroids did not affect basal release of GH, but androgens increased responsiveness of somatotropes to GRF.     

Recombinant Porcine Leptin Reduces Feed Intake and Stimulates Growth Hormone Secretion in Swine

Two experiments (EXP) were conducted to test the hypothesis that porcine leptin affects GH, insulin-like growth factor-I (IGF-I), insulin, thyroxine (T₄) secretion, and feed intake. In EXP I, prepuberal gilts received intracerebroventricular (ICV) leptin injections. Blood was collected every 15 min for 4 hr before and 3 hr after ICV injections of 0.9% saline (S; n = 3), 10 μg (n = 4), 50 μg (n = 4), or 100 μg (n = 4) of leptin in S. Pigs were fed each day at 0800 and 1700 hr over a 2-wk period before the EXP. On the day of the EXP, pigs were fed at 0800 hr and blood sampling started at 0900 h. After the last sample was collected, feeders were placed in all pens. Feed intake was monitored at 4, 20, and 44 hr after feed presentation. In EXP II, pituitary cells from prepuberal gilts were studied in primary culture to determine if leptin affects GH secretion at the level of the pituitary. On Day 4 of culture, 10⁵ cells/well were challenged with 10⁻¹², 10⁻¹⁰, 10⁻⁸, or 10⁻⁶ M [Ala¹⁵]-h growth hormone-releasing factor-(1-29)NH₂ (GRF), 10⁻¹⁴, 10⁻¹³, 10⁻¹², 10⁻¹¹, 10⁻¹⁰, 10⁻⁹, 10⁻⁸, 10⁻⁷, or 10⁻⁶ M leptin individually or in combinations with 10⁻⁸ and 10⁻⁶ M GRF. Secreted GH was measured at 4 hr after treatment. In EXP I, before injection, serum GH concentrations were similar. Serum GH concentrations increased (P < 0.01) after injection of 10 μg (21 ± 1 ng/ml), 50 μg (9 ± 1 ng/ml), and 100 μg (13 ± 1 ng/ml) of leptin compared with S (1 ± 2 ng/ml) treated pigs. The GH response to leptin was greater (P < 0.001) in 10 μg than 50 or 100 μg leptin-treated pigs. By 20 hr the 10, 50, and 100 μg doses of leptin reduced feed intake by 53% (P < 0.08), 76%, and 90% (P < 0.05), respectively, compared with S pigs. Serum IGF-I, insulin, T₄, glucose, and free fatty acids were unaffected by leptin treatment. In EXP II, relative to control (31 ± 2 ng/well), 10⁻¹⁰, 10⁻⁸, and 10⁻⁶ M GRF increased (P < 0.01) GH secretion by 131%, 156%, and 170%, respectively. Only 10⁻⁶ M and 10⁻⁷ M leptin increased (P < 0.01) GH secretion. Addition of 10⁻¹¹ and 10⁻⁹ M leptin in combination with 10⁻⁶ M GRF or 10⁻¹¹ M leptin in combination with 10⁻⁸ M GRF-suppressed (P < 0.05) GH secretion. These results indicate that leptin modulates GH secretion and, as shown in other species, leptin suppressed feed intake in the pig.     

Effects of VIP and GRF on the release of growth hormone in perifused bovine adenohypophysis

The effects of vasoactive intestinal polypeptide (VIP) and growth hormone releasing factor (GRF:hpGRF(1–29)-NH₂) on the release of growth hormone (GH) from anterior pituitaries from cows were examined by using an in vitro superfusion system. The pituitaries were excised randomly from cycling cows, dissected to obtain medial portions, and minced to obtain cubes with approximate dimensions of 1.5mm on a side. For each perifusion setup, 5 pieces of pituitary tissues were chambered and flushed with modified KRB solution saturated with 95% O₂-5% CO₂ at 38C. Perifusion with media containing 10⁻⁶ and 10⁻⁷M VIP for 30 min induced a significant release of GH during the treatments (P<0.05). VIP (10⁻⁸M) increased GH levels significantly (P<0.05), but to a minor degree. Perifusion with the media containing 10⁻⁶, 10⁻⁷ and 10⁻⁸M GRF for 30 min markedly increased the GH concentration and the effects continued up to 90 min after termination of the perifusion of the peptide (P<0.05, P<0.01). The GH releasing effects of GRF could be seen at doses as low as 10⁻¹¹M GRF (P<0.05, P<0.01).

These findings indicate that the GH releasing effect of VIP is less potent that that of GRF in cows.     

The control of adenohypophysial hormone secretion by amino acids and peptides in swine

Several different amino acids and peptides control secretion of adenohypophysial hormones and this control may be indirect, via the modulation of hypothalamic hormone secretion. Indeed, classical hypothalamic hormones (e.g., gonadotropin-releasing hormone [GnRH], growth hormone-releasing hormone [GHRH], somatostatin, etc.) may be released into the hypothalamo-hypophysial portal vasculature, travel to the adenohypophysis and there stimulate or inhibit secretion of hormones. Alternatively, some amino acids and peptides exert direct stimulatory or inhibitory effects on the adenohypophysis, thereby impacting hormone secretion. In swine, the most extensively studied modulators of adenohypophysial hormone secretion are the excitatory amino acids (ExAA), namely glutamate and aspartate, and the endogenous opioid peptides (EOP). In general, excitatory amino acids stimulate release of luteinizing hormone (LH), follicle-stimulating hormone (FSH), growth hormone (GH), and prolactin (PRL). Secretion of adenohypophysial hormones induced by ExAA is primarily, but perhaps not exclusively, a consequence of action at the central nervous system. By acting primarily at the level of the central nervous system, EOP inhibit LH secretion, stimulate GH release and depending on the animal model studied, exert either stimulatory or inhibitory influences on PRL secretion. However, the EOP also inhibited LH release by direct action on the adenohypophysis. More recently, peptides such as neuropeptide-Y (NPY), orexin-B, ghrelin, galanin, and substance P have been evaluated for possible roles in controlling adenohypophysial hormone secretion in swine. For example, NPY, orexin-B, and ghrelin increased basal GH secretion and modulated the GH response to GHRH, at least in part, by direct action on the adenohypophysis. Secretion of LH was stimulated by orexin-B, galanin, and substance P from porcine pituitary cells in vitro. Because the ExAA and various peptides modulate secretion of adenohypophysial hormones, these compounds may play an important role in regulating swine growth and reproduction.     

Effects of pituitary adenylate cyclase-activating polypeptide (PACAP), prostaglandin E2 (PGE2) and growth hormone releasing factor (GRF) on the release of growth hormone from cultured bovine anterior pituitary cells in vitro

The effect of pituitary adenylate cyclase-activating polypeptide (PACAP) on growth hormone (GH) release was compared with that of prostaglandin E₂ (PGE₂) and growth hormone releasing factor (GRF) from cultured bovine anterior pituitary cells in vitro. Both PACAP and PGE₂ stimulated GH release at concentrations as low as 10⁻⁹ and 10⁻⁸ M, respectively, (P<0.01). However, GRF released GH at a concentration as low as 10⁻¹³ M (P<0.01). Percent increases of GH compared with controls were not significantly different among GRF, PACAP, and PGE₂ at 10⁻⁷ M; however, the increases of GH by the 10⁻⁸ M GRF, PACAP and PGE₂ were 196, 118, and 27%, respectively, (P<0.01), and 124, 65, and 1% in the 10⁻⁹ M media, respectively, (P<0.01). When GRF and somatostatin (SS) were added together, the GH releasing effect of GRF was blunted (P<0.01). Similar bluntness were observed in PACAP and PGE₂, when SS was added. The stimulatory effects of GRF and PGE₂ together were similar to that by either GRF or PGE₂ alone. When GRF and PACAP were added together, the GH released by both secretagogues was greater than that by PACAP alone (P<0.01); however, a synergistic effect was not clear when compared with GRF alone.

These findings suggest that PACAP and PGE₂ may modulate the release of GH in cattle.     

Serum growth hormone and nitrogen metabolism responses in young bull calves infused with growth hormone-releasing factor for 20 days

Intravenous infusion of growth hormone (GH)-releasing factor (GRF) sustains elevated serum GH for at least 5 days in young Holstein steers, but the effects of extended infusion of GRF on serum GH and nitrogen (N) metabolism have not been determined. Thirteen Dutch-Friesian bull calves (148 +/- 1.5 kg) were assigned randomly to receive daily either 0 or 3.6 mg GRF (hGRF1-44NH2; U-68420) in saline as a continuous infusion for 20 days. Calves were fed milk replacer twice daily while housed indoors in wooden-slatted floor box crates (metabolism cages). Nitrogen determinations were made on daily feed, feces, and urine samples which were pooled for days 9 to 14 of treatment. Concentrations of GH were quantified in blood samples collected at 20 min intervals for 8 hr on day 1, 10 and 20. The infusion of GRF increased baseline GH (P less than .07), the number of GH pulses (P less than .0001), the amplitude of the GH pulses (P less than .001), and area under the GH response curve (P less than .0002). Within GRF-infused calves baseline GH (P less than .0001) and area under the GH response curve (P less than .006) were greater on day 20 than on day 1 or 10 (day X treatment interaction, P less than .04). Area under the GH response curve was similar on each sampling day in saline-infused calves, but baseline GH was higher (P less than .03) on day 20 than either day 1 or 10. Infusion of GRF increased episodic GH secretion in spite of limited pulsatile activity in saline-infused calves.(ABSTRACT TRUNCATED AT 250 WORDS)     

Pituitary concentrations of gonadotropins and receptors for GnRH in suckled beef cows at various intervals after calving

Mature beef cows were slaughtered at 5 (n = 6), 10 (n = 6), 20 (n = 6) or 30 (n = 5) d after calving to identify endocrine events that may affect the duration of postpartum anestrus. Additional cows (n = 6) were slaughtered 12 to 14 d after their first postpartum estrus (luteal phase cows). Anterior pituitary concentrations of luteinizing hormone (LH) were low at d 5 (383 +/- 69 micrograms/g), averaged 445 +/- 103 and 682 +/- 207 micrograms/g at d 10 and 20, respectively, and were elevated (P less than .05) by d 30 (1,097 +/- 174 micrograms) to a concentration similar to luteal phase cows (1,208 +/- 148 micrograms/g). Concentrations of follicle-stimulating hormone (FSH) averaged 12.4 +/- 1.1, 9.6 +/- 2, 8.6 +/- 1.8 and 7.4 +/- 3.3 mg/g at d 5, 10, 20 and 30, respectively. Affinity (1.6 +/- .2 X 10(9) M-1) of anterior pituitary receptors for the GnRH (gonadotropin-releasing hormone) analog (DAla6; des-Gly10, [D-Ala6]-LH-RH ethylamide) and weights (2.1 +/- .1 g) of the anterior pituitaries did not differ among groups (P greater than .05). Number of receptors for GnRH averaged 37 +/- 7, 39 +/- 9, 25 +/- 5 and 23 +/- 5 X 10(-14) M/mg protein at d 5, 10, 20 and 30, respectively. Anterior pituitaries from luteal phase cows contained 22 +/- 2 X 10(-14) M/mg protein of receptors for GnRH.(ABSTRACT TRUNCATED AT 250 WORDS)     

Influence of dose, frequency, and duration of infused gonadotropin-releasing hormone on secretion of luteinizing hormone and follicle-stimulating hormone in nutritionally anestrous beef cows.

Nutritionally induced anovulatory cows were ovariectomized and used to determine the relationships between dose, frequency, and duration of exogenous gonadotropin-releasing hormone (GnRH) pulses and amplitude, frequency, and concentrations of luteinizing hormone (LH) and follicle-stimulating hormone (FSH) in serum. In Experiment 1, cows were given pulses of saline (control) or 2 micrograms of GnRH infused i.v. during a 0.1-, 1.25-, 5-, 10-, or 20-min period. Concentrations of LH and FSH during 35 min after GnRH infusion were greater than in control cows (P < 0.01), and FSH concentrations were greater when GnRH infusions were for 10 min or less compared with 20 min. In Experiment 2, the effect of GnRH pulse frequency and dose on LH and FSH concentrations, pulse frequency, and pulse amplitude were determined. Exogenous GnRH (0, 2, or 4 micrograms) was infused in 5 min at frequencies of once every hour or once every 4th hr for 3 d. There was a dose of GnRH x frequency x day effect on LH and FSH concentrations (P < 0.01), indicating that gonadotropes are sensitive to changes in pulse frequency, dose, and time of exposure to GnRH. There were more LH pulses when GnRH was infused every hour, compared with an infusion every 4th hr (P < 0.04). Amplitudes of LH pulses were greater with increased GnRH dose (P < 0.05), and there was a frequency x dose x day effect on FSH pulse amplitude (P < 0.0006). We conclude that LH and FSH secretion in the bovine is differentially regulated by frequency and dose of GnRH infusions.     

N-methyl-d,l-aspartate modulation of pituitary hormone secretion in the pig: Role of opioid peptides

Sixteen ovariectomized (OVX) mature gilts, averaging 139.6 ± 3.1 kg body weight (BW) were assigned randomly to receive either progesterone (P, 0.85 mg/kg BW, n=8) or corn oil vehicle (OIL, n=8) injections im twice daily for 10 d. On the day of experiment, all gilts received either the EAA agonist, N-methyl-d,l-aspartate (NMA; 10 mg/kg BW, iv) alone or NMA plus the EOP antagonist, naloxone (NAL, 1 mg/kg BW, iv), resulting in the following groups of 4 gilts each: OIL-NMA, OIL-NMA-NAL, P-NMA and P-NMA-NAL. Blood samples were collected via jugular cannula every 15 min for 6 hr. All pigs received NMA 5 min following pretreatment with either 0.9% saline or NAL 2 hr after blood collection began and a GnRH challenge 3 hr after NMA. Administration of NMA suppressed (P<0.03) LH secretion in OIL-NMA gilts and treatment with NAL failed to reverse the suppressive effect of NMA on LH secretion in OIL-NMA-NAL gilts. Similar to OIL-NMA gilts, NMA decreased (P<0.03) mean serum LH concentrations in P-NMA gilts. However, in P-NMA-NAL gilts, serum LH concentrations were not changed following treatment. All gilts responded to GnRH with increased (P<0.01) LH secretion. Additionally, administration of NMA increased (P<0.01) growth hormone (GH) and prolactin (PRL) secretion in both OIL-NMA and P-NMA gilts, but this increase in GH and PRL secretion was attenuated (P<0.01) by pretreatment with NAL in OIL-NMA-NAL and P-NMA-NAL gilts. Serum cortisol concentrations increased (P<0.01) in all gilts and the magnitude of the cortisol response was not different among groups. In summary, results of the present study confirmed previous findings that NMA suppresses LH secretion in both oil- and P-treated OVX gilts, but we failed to provide definitive evidence that EOP are involved in the NMA-induced suppression of LH secretion. However, NMA may, in part, activate the EOP system which in turn increased GH and PRL secretion in the gilt.     

Luteinizing hormone release after administration of the gonadotropin-releasing hormone agonist Fertilan (goserelin) for synchronization of ovulation in pigs

The generic GnRH agonist, Fertilan (goserelin), was tested for the ability to induce an LH surge and ovulation in estrus-synchronized gilts. Three experiments were performed to 1) examine the effect of various doses of Fertilan on secretion of LH in barrows, to select doses to investigate in gilts (Exp. 1); 2) determine doses of Fertilan that would induce a preovulatory-like rise of LH in gilts (Exp. 2); and 3) determine the time of ovulation after Fertilan treatment (Exp. 3). In Exp. 1, 10 barrows were injected on d 1, 4, 7, 10, and 13 with 10, 20, or 40 microg of Fertilan; 50 microg of Gonavet (depherelin; GnRH control) or saline (negative control); and sequential blood samples were collected for 480 min. There was a dose-dependent stimulation (P < 0.05) of LH release. Maximal plasma concentrations of LH (LH(MAX)) were 2.1 +/- 0.2, 4.1 +/- 0.3, 2.6 +/- 0.4, and 3.4 +/- 0.3 ng/mL after 10, 20, and 40 microg of Fertilan and 50 microg of Gonavet, respectively, and duration of release was 78 +/- 9, 177 +/- 12, 138 +/- 7, and 180 +/- 11 min, respectively. Fertilan doses of 10 and 20 microg were deemed to be the most suitable for testing in gilts. In Exp. 2, 12 gilts received (after estrus synchronization with Regumate and eCG) injections of 10 or 20 microg of Fertilan or 50 microg of Gonavet 80 h after eCG to stimulate a preovulatory-like LH surge and ovulation. An LH surge was induced in 3 of the 4 gilts in both of the Fertilan groups and in all of the Gonavet-treated gilts. Characteristics of induced release of LH did not differ among groups: LH(MAX), 5.0 +/- 0.9 vs. 4.6 +/- 1.8 vs. 6.6 +/- 1.1 ng/mL; duration, 11.7 +/- 2.0 vs. 12.3 +/- 2.2 vs. 14.3 +/- 0.5 h; interval from GnRH injection to LH(MAX), 4.0 +/- 2.0 vs. 6.7 +/- 1.3 vs. 5.8 +/- 1.6 h. In Exp. 3, estrus-synchronized gilts were injected with 20 microg of Fertilan (n = 8) or 50 microg of Gonavet (n = 4), and the time of ovulation was determined by repeated endoscopic examination. Time of ovulation ranged from 34 to 42 h postGnRH; however, ovulation occurred earlier in the Gonavet compared with the other groups (P < 0.05). Results of these experiments indicate that 1) barrows are an appropriate model for determining GnRH doses that can be effective in inducing a preovulatory-like LH surge in females; 2) the generic GnRH agonist Fertilan, at doses of 10 to 20 microg, can stimulate an LH surge in gilts, with subsequent ovulation; and 3) Fertilan at doses of 10 and 20 microg should be examined further for use in fixed-time insemination protocols.     

Comparison of luteinizing hormone and steroid hormone secretion during the peri- and post-ovulatory periods in Mangalica and Landrace gilts

The objective of this study was to determine plasma concentrations of luteinizing hormone (LH), progesterone (P4) and estradiol-17beta (E2) in Mangalica gilts (M), a Hungarian native breed, and compare them with Landrace gilts (L) during the peri- and post-ovulatory periods. The estrous cycle of gilts was synchronised by Regumate feeding, and ovulation was induced with a gonadotropin-releasing hormone (GnRH) agonist. Blood sampling was carried out via indwelling jugular catheters three times a day and in 2-h intervals during a 16-h period after the GnRH application. The concentrations of LH, E2 and P4 were determined by immunoassays. Gilts of both breeds showed a typical gonadotropin and gonadal hormone secretion pattern. Preovulatory E2 peaks were observed on day 2 (M) and day 4 (L) after the last Regumate feeding. Highest E2 concentration was different between M and L breeds (46.5 +/- 5.7 vs. 26.0 +/- 6.8 pg/ml, P < 0.05). Maximum LH levels measured up to 6 h after GnRH were not different between M and L breeds (11.5 +/- 4.1 vs. 6.6 +/- 2.3 ng/ml). Both LH amounts during surge (41.1 +/- 15.9 vs. 27.5 +/- 6.1 ng/ml) and total over LH release (73.4 +/- 22.2 vs. 50.0 +/- 8.7 ng/ml) did not differ significantly between M and L breeds. P4 concentrations started to rise on day 6 after Regumate feeding and increased significantly from 0.6 +/- 0.3 and 0.7 +/- 0.4 ng/ml to maximal 14.0 +/- 2.4 and 11.3 +/- 2.1 ng/ml in M and L breeds, respectively. Mean P4 secretion was higher in M on days 10-15 (12.9 +/- 2.6 vs. 9.3 +/- 2.2 ng/ml; P<0.05). At the same time the number of corpora lutea was lower in M compared to L (10.3 +/-1.5 vs. 17.8 +/- 5.0, P<0.05). In our experiment, there was no evidence that differences in the secretion of analysed hormones during the peri- and post-ovulatory periods are a possible cause of usually lower fecundity in Mangalica gilts.     

Modulation of growth hormone-releasing factor-induced release of growth hormone from bovine pituitary cells

Growth hormone (GH)-releasing factor (GRF) at concentrations of 10−¹² through 10−⁷M for 6 hr linearly increased GH release (b₁ = 10.4 ± .3) from bovine anterior pituitary cells in culture. Maximum release of GH (262% above controls) occurred at 10−⁷M GRF. In contrast, GH release-inhibiting factor (SRIF) at 10−¹² through 10−⁵M had no effect on basal concentrations of GH. In a second experiment, as the proportion of SRIF relative to GRF increased. SRIF suppression of GRF-induced GH release from anterior pituitary cells increased. In a third experiment, anterior pituitary cells cultured in media containing fetal calf serum (FCS) were treated with cortisol (0 or 10 ng/ml media) for 24 hr before exposure to 10−¹³ through 10−⁷M GRF. GRF linearly increased GH secretion (b₁ = 7.4 ± .3) and cortisol augmented this response (b₁ = 10.5 ± .6). However, when cells were cultured in media containing dextran-charcoal treated FCS, cortisol did not alter GRF-induced GH release. Our results demonstrate that GH response of bovine anterior pituitary cells to GRF was modulated negatively by SRIF. However, augmentation of GRF-induced GH release by cortisol was evident only when cells were cultured in media supplemented with untreated FCS.