Endocrine, ovulatory and reproductive characteristics of sows treated with an intravaginal GnRH agonist.

The present study examined the effectiveness of intravaginal administration of a GnRH agonist, triptorelin, on the induction of the preovulatory LH surge; synchronization of time of ovulation; and reproductive characteristics in weaned sows. Sows were given 100microg of triptorelin in 0.9, 1.2, or 1.5% methylcellulose gel intravaginally 96h after weaning and then bred at 8 and 32h post-treatment. Untreated sows inseminated once each day of estrus served as the positive controls, while females treated with only the methyl cellulose gel and also bred once each day of estrus were negative controls. Sows treated intravaginally with triptorelin exhibited preovulatory LH surges with magnitudes comparable to those that occurred spontaneously in the negative controls. Preovulatory LH surges were initiated over a narrow and well-defined time interval that occurred 4-12h after treatment in sows receiving triptorelin in 1.2 or 1.5% methyl cellulose gel. As a result, the majority of the sows in these two treatments had ovulations within a 12h time frame 36-48h after treatment. In contrast, both the LH surge and ovulation occurred over extended periods of time after weaning in negative controls and sows given triptorelin in 0.9% methylcellulose gel. Farrowing rates and litter size were similar between untreated controls and triptorelin-treated sows that were bred with two fixed timed inseminations. Insemination of sows induced to have ovulations and bred at least once while not in estrus did not have any overt negative effects on reproductive characteristics. These results demonstrate that 100microg of triptorelin administered intravaginally in a least 1.2% methyl cellulose gel induced a normal preovulatory LH surge and synchronized time of ovulation in weaned sows. Furthermore, there were no obvious changes in reproductive performance when these sows were bred with two fixed time inseminations regardless of whether they exhibited a standing reflex.

Growth hormone releasing factor decreases long form leptin receptor expression in porcine anterior pituitary cells.

Pituitary cells from six pigs, 180-200 days of age, were studied in primary culture to determine if growth hormone releasing factor (GRF) affects long form leptin receptor (Ob-Rl) expression. On Day 4 of culture, 10(5) live cells per well were challenged with either 0, 10(-6), 10(-7) or 10(-8)M [Ala15]-hGRF-(1-29)NH(2) (3 wells per treatment per pig). Secretion of growth hormone (GH) into the media and pituitary Ob-Rl mRNA expression were determined at 4 h after treatment. Media were analyzed for GH by radioimmunoassay, and total RNA was isolated from cells for determination of Ob-Rl expression by semi quantitative reverse transcription PCR. Basal GH concentration was 32+/-2 ng per 10(5) cells per well (n=18 wells) for 4 h. Relative to control at 4 h, 10(-6),10(-7) and 10(-8)M GRF increased (P<0.01) GH secretion by 151, 129 and 120%, but decreased (P<0.05) Ob-Rl expression by 32, 50 and 38%, respectively. These results indicate that GRF directly modulates Ob-Rl expression at the level of the pituitary, and thereby playing a role in regulating pituitary sensitivity to leptin.

Distribution of neurons containing leptin receptors in the hypothalamus of the pig.

Leptin, secreted by white adipocytes, has profound feeding, metabolic, and neuroendocrine effects. Leptin acts on the brain, but specific anatomical sites and pathways responsible for mediating these effects are still unclear. We have systematically examined the distribution of leptin receptor containing neurons in the porcine hypothalamus by means of immunohistochemical staining methods. Leptin receptor immunoreactivity (OBR-IR) was observed in both the preoptic area and anterior hypothalamic area. No immunoreactive structures were found in the median eminence. Only single, small neurons were observed in the arcuate nucleus. The most abundant OBR-IR cell bodies were located in the supraoptic nucleus. In the paraventricular nucleus, OBR-IR neurons were moderate in number. Single, dispersed neurons were found in the ventromedial nucleus. These findings indicate that there are distinct OBR-IR neuronal populations in the porcine hypothalamus and leptin not only plays an integrative role in feeding behavior, but also in neuroendocrine activity.

Leptin receptors, NPY, and tyrosine hydroxylase in autonomic neurons supplying fat depots in a pig.

The goal of this study was to determine the immunohistochemical characteristics of peripheral adrenergic OBR-immunoreactive (OBR-IR) neurons innervating adipose tissue in a pig. The retrograde tracer, Fast Blue (FB), was injected into either the subcutaneous, perirenal, or mesentery fat tissue depots of three male and three female pigs each with approximately 50 kg body weight. Sections containing FB(+) neurons were stained for OBR, tyrosine hydroxylase (TH) or neuropeptide Y (NPY) using a double labeling immunofluorescence method. OBR, TH, and NPY immunoreactivities were present in the thoracic (T) and lumbar (L) ganglia of the sympathetic chain, as well as in the coeliac superior mesenteric ganglion (CSMG), inferior mesenteric ganglion (IMG), intermesenteric ganglia (adrenal-ADG, aorticorenal-ARG, and ovarian-OG or testicular-TG ganglion). These results indicate that, in addition to neuroendocrine functions, leptin may affect peripheral tissues by acting on receptors located in sympathetic ganglion neurons.

Metabolic regulation of the neuroendocrine axis in pigs.

Transition from the fed to fasted state occurs more rapidly in prepubertal pigs than in mature pigs. Insulin-like growth factor I (IGF-I), leptin and specific metabolites, such as glucose and free fatty acids (FFA), play a role in signalling metabolic status to the brain-pituitary axis. High serum concentrations of FFA or glucose suppressed the growth hormone (GH) response to GH releasing hormone (GHRH), whereas glucose inhibited and FFA enhanced pituitary LH response to GnRH. The site of action of FFA and glucose on LH and GH secretion is at the pituitary cell. In cultured pig pituitary cells, IGF-I suppressed the GH response to GHRH and enhanced basal LH secretion. However, intracerebroventricular injection of IGF-I failed to affect LH or GH secretion in prepubertal gilts. Acute fasting suppressed leptin secretion without affecting LH or GH secretion. Metabolic fuel restriction by decreasing glucose availability increased GH secretion, but decreased LH secretion in prepubertal gilts. However, serum leptin concentrations were unchanged. Thus, the effects of acute energy deprivation on LH and GH secretion are independent of changes in leptin secretion. Serum leptin concentrations, hypothalamic leptin receptor mRNA and oestrogen-induced leptin gene expression in fat increased with age and adiposity in pigs. This increase occurred at the time of expected puberty in intact pigs. An intracerebroventricular injection of leptin suppressed feed intake, but stimulated GH secretion in prepubertal gilts. Leptin stimulated GnRH release from hypothalamic tissue in vitro. These results identify putative signals that link metabolic status and neuroendocrine control of appetite, growth and reproduction and have implications for nutrition-related human health problems due to high fat diets.

Developmental changes in the long form leptin receptor and related neuropeptide gene expression in the pig brain.

The hypothalamus is the key site of central regulation of energy homeostasis, appetite, and reproduction. The long form leptin receptor (Ob-Rl) is localized within the hypothalamus along with several neuropeptides that are involved in regulation of the neuroendocrine axis. In the present study, developmental changes in gene expression of the Ob-Rl, preproorexin, proopiomelanocortin (POMC), corticotropin releasing factor (CRF), somatostatin, and GnRH in the hypothalamus was studied. Expression of Ob-Rl and neuropeptide mRNA was examined by semiquantitative reverse transcription-polymerase chain reaction in hypothalami collected from 106-day-old fetus (n = 3) and 7-day-old (n = 3), 3.5-mo-old (n = 3), and 6-mo-old (n = 2) gilts. In addition, leptin mRNA expression in the first three ages was examined in back fat. Leptin mRNA expression increased (P < 0.05) by 7 days postnatal, but Ob-Rl mRNA expression increased (P < 0.01) by 3.5 mo. Expression of preproorexin (P < 0.05), somatostatin, and GnRH (P < 0.01) mRNA peaked by 3.5 mo of age while POMC mRNA expression increased markedly (P < 0.01) by 6 mo of age. The CRF mRNA expression did not change across ages. These findings suggest a possible relationship among Ob-Rl and a number of hypothalamic and peripheral peptides in the development of the neuroendocrine axis. These peptides may serve as messengers that link mechanisms that regulate reproduction and energy balance.

Serum leptin concentrations, luteinizing hormone and growth hormone secretion during feed and metabolic fuel restriction in the prepuberal gilt.

Two experiments were conducted to determine 1) the effect of acute feed deprivation on leptin secretion and 2) if the effect of metabolic fuel restriction on LH and GH secretion is associated with changes in serum leptin concentrations. Experiment (EXP) I, seven crossbred prepuberal gilts, 66 +/- 1 kg body weight (BW) and 130 d of age were used. All pigs were fed ad libitum. On the day of the EXP, feed was removed from four of the pigs at 0800 (time = 0) and pigs remained without feed for 28 hr. Blood samples were collected every 10 min from zero to 4 hr = Period (P) 1, 12 to 16 hr = P 2, and 24 to 28 hr = P 3 after feed removal. At hr 28 fasted animals were presented with feed and blood samples collected for an additional 2 hr = P 4. EXP II, gilts, averaging 140 d of age (n = 15) and which had been ovariectomized, were individually penned in an environmentally controlled building and exposed to a constant ambient temperature of 22 C and 12:12 hr light: dark photoperiod. Pigs were fed daily at 0700 hr. Gilts were randomly assigned to the following treatments: saline (S, n = 7), 100 (n = 4), or 300 (n = 4) mg/kg BW of 2-deoxy-D-glucose (2DG), a competitive inhibitor of glycolysis, in saline iv. Blood samples were collected every 15 min for 2 hr before and 5 hr after treatment. Blood samples from EXP I and II were assayed for LH, GH and leptin by RIA. Selected samples were quantified for glucose, insulin and free fatty acids (FFA). In EXP I, fasting reduced (P < 0.04) leptin pulse frequency by P 3. Plasma glucose concentrations were reduced (P < 0.02) throughout the fast compared to fed animals, where as serum insulin concentrations did not decrease (P < 0.02) until P 3. Serum FFA concentrations increased (P < 0.02) by P 2 and remained elevated. Subcutaneous back fat thickness was similar among pigs. Serum IGF-I concentration decreased (P < 0.01) by P 2 in fasted animals compared to fed animals and remained lower through periods 3 and 4. Serum LH and GH concentrations were not effected by fast. Realimentation resulted in a marked increase in serum glucose (P < 0.02), insulin (P < 0.02), serum GH (P < 0.01) concentrations and leptin pulse frequency (P < 0.01). EXP II treatment did not alter serum insulin levels but increased (P < 0.01) plasma glucose concentrations in the 300 mg 2DG group. Serum leptin concentrations were 4.0 +/- 0.1, 2.8 +/- 0.2, and 4.9 +/- 0.2 ng/ml for S, 100 and 300 mg 2DG pigs respectively, prior to treatment and remained unchanged following treatment. Serum IGF-I concentrations were not effected by treatment. The 300 mg dose of 2DG increased (P < 0.0001) mean GH concentrations (2.0 +/- 0.2 ng/ml) compared to S (0.8 +/- 0.2 ng/ml) and 100 mg 2DG (0.7 +/- 0.2 ng/ml). Frequency and amplitude of GH pulses were unaffected. However, number of LH pulses/5 hr were decreased (P < 0.01) by the 300 mg dose of 2DG (1.8 +/- 0.5) compared to S (4.0 +/- 0.4) and the 100 mg dose of 2DG (4.5 +/- 0.5). Mean serum LH concentrations and amplitude of LH pulses were unaffected. These results suggest that acute effects of energy deprivation on LH and GH secretion are independent of changes in serum leptin concentrations.

Nutritional regulators of the hypothalamic-pituitary axis in pigs.

Nutritional signals are detected by the central nervous system (CNS) and translated by the neuroendocrine system into signals that alter secretion of LH and growth hormone (GH). Furthermore, these signals directly affect the activity of the pituitary gland independently of CNS input. Insulin-like growth factor I (IGF-I), insulin, leptin and specific metabolites, such as glucose and free fatty acids (FFA), are potential signals of the metabolic status to the brain-pituitary axis. Intravenous injection of a lipid emulsion or glucose suppressed the GH and LH response to GH releasing hormone (GHRH) and GnRH, respectively. Insulin and IGF-I regulation of LH and GH secretion occur at the pituitary gland. Feed deprivation for 24 h suppressed leptin secretion without affecting LH or GH secretion, whereas central administration of leptin resulted in a decrease in feed intake and an increase in GH secretion. Oestrogen-induced leptin gene expression in adipose tissue increased with age and adiposity in pigs. Leptin stimulated GnRH release from hypothalamic tissue in vitro. These results identify putative signals that link metabolic status and neuroendocrine control of growth and reproduction by altering endocrine function during periods of fasting, feed restriction and lactation.

Feed intake and serum GH, LH and cortisol in gilts after intracerebroventricular or intravenous injection of urocortin.

In three experiments (Exp), ovariectomized gilts received intracerebroventricular (ICV; Exp 1 - with restraint, Exp 2 - without restraint) or intravenous (i.v.; Exp 3) injections of urocortin or saline to assess effects on feed intake and serum GH, LH, and cortisol. Following a 20-hr fast, feed was presented at 1 hr (Exp 1) or 30 min (Exp 2 and 3) after injection (time = 0 hr) of saline or 5 (U5) or 50 (U50) microg/pig (Exp 1 and 2) or 5 microg/kg BW (Exp 3) of urocortin. Blood samples were collected every 15 min from -2 to 6 hr relative to injection and hormone data pooled 2 hr before and hourly after treatment. Treatment with U50 decreased feed intake, relative to saline (treatment x time interaction; P < 0.05), when delivered ICV but not i.v. A treatment by time interaction was detected for GH (Exp 1, 2, 3) and LH (Exp 1 and 2; P < 0.01). Serum GH increased over time (relative to -2 hr; P < 0.05) following treatment with urocortin but not saline regardless of route of administration. Conversely, in Exp 1 (U5 and U50) and Exp 2 (U50), LH decreased relative to -2 hr with a delayed decrease during Exp 1. Serum cortisol was not affected by treatment in Exp 1, but increased following urocortin in Exp 2 and 3 (treatment by time interaction, P < 0.01). These data provide evidence that urocortin modulates GH and LH concentrations and suppresses feed intake in gilts via mechanisms which may be independent of cortisol and may depend upon dose and route of administration.

Luteinizing hormone response to controlled-release deslorelin in estradiol benzoate primed ovariectomized gilts.

Development of a controlled release formulation of gonadotropin releasing hormone that would stimulate a LH surge capable of reducing the time span of ovulations would greatly benefit reproductive management because a single timed insemination could be used. A dose-response study was conducted to determine if Deslorelin, a potent gonadotropin releasing hormone analogue, delivered via the SABER system, a biodegradable controlled release system, would stimulate an ovulatory-like LH surge in the pig. Twenty ovariectomized gilts, approximately 200 d old and 100 kg body weight (BW), received estradiol benzoate (15 microg/kg BW im) and 48 h later, the gilts were given deslorelin at 0, 12.5, 25.0, 50.0 or 100.0 microg im (n = 4 each treatment group). Compared to controls, mean blood deslorelin concentrations were still elevated at 30 h after deslorelin. Mean deslorelin magnitude, area under the curve and duration were sequentially greater (P<0.05) in a dose-dependent sequence. Compared to controls, serum LH concentrations were elevated (P<0.05) for 6 to 12 h after deslorelin. A dose-response relationship was absent for all parameters of LH secretion. Magnitude of the serum LH response was greatest (P<0.05) in the 12.5 microg and 50.0 microg groups, whereas area under the curve was lower (P<0.05) after 25.0 microg of deslorelin than after 12.5, 50.0 and 100.0 microg, which were not different from each other. Thus, no more than 12.5 microg of deslorelin is necessary to obtain maximum LH release in the model studied and doses less than 12.5 microg may also be effective.

Long form leptin receptor mRNA expression in the brain, pituitary, and other tissues in the pig.

Much effort has focused recently on understanding the role of leptin, the obese gene product secreted by adipocytes, in regulating growth and reproduction in rodents, humans and domestic animals. We previously demonstrated that leptin inhibited feed intake and stimulated growth hormone (GH) and luteinizing hormone (LH) secretion in the pig. This study was conducted to determine the location of long form leptin receptor (Ob-Rl) mRNA in various tissues of the pig. The leptin receptor has several splice variants in the human and mouse, but Ob-Rl is the major form capable of signal transduction. The Ob-Rl is expressed primarily in the hypothalamus of the human and rodents, but has been located in other tissues as well. In the present study, a partial porcine Ob-Rl cDNA, cloned in our laboratory and specific to the intracellular domain, was used to evaluate the Ob-Rl mRNA expression by RT-PCR in the brain and other tissues in three 105 d-old prepuberal gilts and in a 50 d-old fetus. In 105 d-old gilts, Ob-Rl mRNA was expressed in the hypothalamus, cerebral cortex, amygdala, thalamus, cerebellum, area postrema and anterior pituitary. In addition, Ob-Rl mRNA was expressed in ovary, uterine body, liver, kidney, pancreas, adrenal gland, heart, spleen, lung, intestine, bone marrow, muscle and adipose tissue. However, expression was absent in the thyroid, thymus, superior vena cava, aorta, spinal cord, uterine horn and oviduct. In the 50 d-old fetus, Ob-Rl mRNA was expressed in brain, intestine, muscle, fat, heart, liver and umbilical cord. These results support the idea that leptin might play a role in regulating numerous physiological functions.

Evidence for a luteinizing hormone surge center in the hypothalamus of the pig.

Studies were conducted to determine whether there is an LH surge generator in the hypothalamus of the pig. In experiment 1, 157-day-old ovariectomized (OVX) gilts received 1.5 microg estradiol benzoate (EB)/kg BW i.m. every 12 h from 0 through 24, 48, 72, or 96 h. Blood was sampled every 6 h from 3 to 36 h and every 3 h from 36 through 144 h. One of 3, 4 of 4, 4 of 4, and 2 of 3 gilts displayed an LH surge after treatment for 24, 48, 72, and 96 h, respectively. With the exception that time to maximum LH concentration was greater in gilts treated for 96 h than in those treated for 72 h (p < 0.05), parameters of the surge were similar among all gilts. In experiment 2a, an Alzet osmotic pump containing EB or vehicle was inserted s.c. behind an ear of 124-day-old OVX gilts, resulting in the following daily doses of EB: 0, 0.75, 1.50, or 3.00 microg/kg BW. Blood was sampled at 0, 2, 4, 6, and 8 h and every 8 h thereafter through 168 h to evaluate surge secretion of LH, and every 15 min for 8 h starting at 168 h to evaluate pulsatile LH secretion. Zero of 3, 0 of 2, 3 of 3, and 3 of 3 gilts displayed an LH surge after 0, 0.75, 1.50, and 3.00 microg EB/kg BW, respectively. Parameters of the surge were similar among gilts. Pulsatile LH secretion, evaluated 7 days after pump insertion, was significantly suppressed for estradiol-treated gilts compared to controls. In experiment 2b, at 182 days of age, 10 gilts used in experiment 2a plus 2 additional gilts in the original group prepared but not used for experiment 2a, were randomly assigned in groups (n=3) to the following daily doses of EB: 0, 0.19, 0.38, or 0.75 microg/kg BW, administered again by osmotic pump. Treatment and blood-sampling schedules were the same as in experiment 2a. Zero, 0, 1, and 2 gilts displayed an LH surge after treatment with 0, 0.19, 0.38, and 0.75 microg EB/kg BW, respectively. Parameters of the surge were similar among gilts that displayed an LH surge. Pulsatile LH secretion was significantly suppressed for estradiol-treated gilts compared to controls. Thus, the LH surge resulted from positive feedback stimulation of a specific surge generator rather than attenuation or dissipation of negative feedback inhibition of estradiol on a pulse generator.

Delay of estradiol-induced surge secretion of LH in gilts by intracerebroventricular injection of morphine.

In Experiment 1, ovariectomized (OVX) gilts, 143 d old and 58.5 +/- 1.8 kg BW, received 10 micrograms estradiol benzoate (EB)/kg BW i.m. and either 500 micrograms of the endogenous opioid peptide (EOP) agonist, morphine (MOR), in saline (SAL; n = 5), or SAL (n = 4) intracerebro-ventricularly at 40 and 48 hr after EB. With the exception of one MOR-treated gilt, which was deleted from Experiment 1, LH secretion was suppressed for at least 50 hr in all gilts. Timing of the LH surge was similar among gilts. However, total LH secreted was greater (P < 0.05) after SAL than MOR. The experiment was repeated at 179 d of age and 78.6 +/- 1.2 kg BW, except that treatments were reversed among gilts. Emergence of the LH surge was delayed (P < 0.005) by 10.8 hr and time to maximum LH concentration (P < 0.05) by 6.8 hr after MOR than after SAL. Magnitude and total LH secreted were not different among gilts. In Experiment 2, gilts which had displayed estrous cycles of 18-22 d were OVX and treated as in Experiment 1, except MOR (n = 3) or SAL (n = 4) were injected 10 hr later than in Experiment 1, i.e., at 50 and 58 hr after EB. Secretion of LH was suppressed for at least 60 hr in both groups. Time to emergence of the LH surge was delayed by 27 hr (P < 0.05) after MOR compared to after SAL. However, other parameters of the surge were not different among gilts. Thus, EOP modulate LH surge secretion negatively in the pig.

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 (i.c.v.) leptin injections. Blood was collected every 15 min for 4 hr before and 3 hr after i.c.v. injections of 0.9% saline (S; n = 3), 10 micrograms (n = 4), 50 micrograms (n = 4), or 100 micrograms (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, 10(5) 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 micrograms (21 +/- 1 ng/ml), 50 micrograms (9 +/- 1 ng/ml), and 100 micrograms (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 micrograms than 50 or 100 micrograms leptin-treated pigs. By 20 hr the 10, 50, and 100 micrograms doses of leptin reduced feed intake by 53% (P < 0.08), 76%, and 90% (P < 0.05), respectively, compared with S pigs. Serum IGF-1, 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.

Metabolic changes during the transition from the fed to the acute feed-deprived state in prepuberal and mature gilts.

The present study compared the metabolic response to acute feed deprivation in ovariectomized prepuberal (P; n = 6), 62 +/- 2 kg BW, and mature (M; n = 6) gilts, 124 +/- 4 kg BW. Blood was collected at 1, 2, 3, 4, 6, 8, 12, 16, 20, 24, 28, 32, 40, and 48 h after initiation of feeding. Samples were quantified for glucose, insulin, free fatty acids (FFA), beta-hydroxybutyrate (HBA), IGF-I, IGF binding proteins (BP)-1 to -4, cortisol, and amino acids (AA). At 24 h, blood samples were collected every 15 min for 8 h and an additional 1 h after i.v. injection of GnRH (.2 microgram/kg BW) and growth hormone-releasing factor (GRF; 1 microgram/kg BW). Samples were assayed for growth hormone (GH) and LH. Serum insulin concentrations were lower (P < .05) in P gilts. Plasma glucose concentrations were similar. Serum FFA concentrations were greater (P < .05) in P gilts. Serum concentrations of HBA were greater (P < .001) in P gilts at 48 h. Serum IGF-I concentrations were lower (P < .05) in P gilts by 16 h. Serum IGFBP-1-4 levels were similar. Serum cortisol concentrations were similar. Serum concentrations of the essential AA, isoleucine, lysine, threonine, valine, and phenylalanine were greater (P < .05) in P gilts at 40 h. Serum LH concentrations and response to GnRH were similar. Basal serum GH concentrations and peak response to GRF were greater (P < .05) in P than in M gilts. The transition from fed to unfed state occurs more rapidly in P than in M gilts.

The role of excitatory amino acids in pulsatile secretion of luteinizing hormone in gilts and barrows.

The relationship of excitatory amino acid (EAA) activity to LH secretion was investigated in ovariectomized crossbred prepuberal gilts (93 +/- 1 kg BW) and Yorkshire barrows (94 +/- 2 kg BW) in two experiments. In Exp. 1, eight gilts received, i.m., saline (S) or 20 mg of Ketamine (K)/kg BW, a noncompetitive EAA receptor antagonist. Within these groups, four then received 10 mg of N-methyl-DL-aspartate (NMA)/kg BW, an EAA agonist, or S i.v. Mean serum LH concentrations were similar among groups before treatment, did not change after S+S, but decreased (P < .05) by 1 h after S+NMA, 3 h after K+S, and 2 h after K+NMA. Serum cortisol concentrations did not change after S+S, but were increased (P < .05) from 30 to 90 min after S+NMA, at 120 min after K+S, and from 30 to 120 min after K+NMA. In Exp. 2, barrows received 2.5 mg of NMA/kg BW i.v. immediately after i.m. injection of S (n = 7) or 19.9 mg of K/kg BW (n = 8). Mean serum LH concentrations for the 2 h before treatment were similar among barrows, but decreased (P < .05) by 2 h after K+NMA and was not altered after S+NMA. Serum cortisol concentrations were increased at 30 min after S+NMA and from 60 to 90 min after K+NMA. We suggest that EAA both inhibit and stimulate LH secretion, with the inhibitory effects lying within the basal hypothalamus and the stimulatory effects lying within higher brain centers.

Immunocytochemical distribution of catecholamine-synthesizing neurons in the hypothalamus and pituitary gland of pigs: tyrosine hydroxylase and dopamine-beta-hydroxylase.

This study describes the distribution of catecholaminergic neurons in the hypothalamus and the pituitary gland of the domestic pig, Sus scrofa, an animal that is widely used as an experimental model of human physiology in addition to its worldwide agricultural importance. Hypothalamic catecholamine neurons were identified by immunocytochemical staining for the presence of the catecholamine synthesizing enzymes, tyrosine hydroxylase and dopamine-beta-hydroxylase. Tyrosine hydroxylase-immunoreactive perikarya were observed in the periventricular region throughout the extent of the third ventricle, the anterior and retrochiasmatic divisions of the supraoptic nucleus, the suprachiasmatic nucleus, the ventral and dorsolateral regions of the paraventricular nucleus and adjacent dorsal hypothalamus, the ventrolateral arcuate nucleus, and the posterior hypothalamus. Perikarya ranged from parvicellular (10-15 microns) to magnocellular (25-50 microns) and were of multiple shapes (rounded, fusiform, triangular, or multipolar) and generally had two to five processes with branched arborization. No dopamine-beta-hydroxylase immunoreactive perikarya were observed within the hypothalamus or in the adjacent basal forebrain structures. Both tyrosine hydroxylase- and dopamine-beta-hydroxylase-immunoreactive fibers and punctate varicosities were observed throughout areas containing tyrosine hydroxylase perikarya, but dopamine-beta-hydroxylase immunoreactivity was very sparse within the median eminence. Within the pituitary gland, only tyrosine hydroxylase fibers, and not dopamine-beta-hydroxylase immunoreactive fibers, were located throughout the neurohypophyseal tract and within the posterior pituitary in both pars intermedia and pars nervosa regions. Generally, the location and patterns of both catecholamine-synthesizing enzymes were similar to those reported for other mammalian species except for the absence of the A15 dorsal group and the very sparse dopamine-beta-hydroxylase immunoreactive fibers and varicosities in the median eminence in the pig. These findings provide an initial framework for elucidating behavioral and neuroendocrine species differences with regard to catecholamine neurotransmitters.

Influence of stage of the estrous cycle on insulin-like growth factor-I modulation of luteinizing hormone secretion in the gilt.

Follicular phase (FOL; Days 17-19; n = 8), luteal phase (LUT; Days 7-9; n = 6), and ovariectomized (OVX; n = 5) crossbred gilts were used (Day 0 = onset of estrus). Blood samples were collected via jugular vein cannula every 15 min for 2 h the day before pituitary collection. Serum was assayed for insulin-like growth factor (IGF)-I, IGF-I binding proteins (IGFBP), LH, estradiol (E2), and progesterone (P4). Anterior pituitary cells were dispersed, cultured, and challenged on Day 4 of culture (Day 0 = day of seeding) with 10(-7) M GnRH or IGF-I (10(-11), 10(-10), 10(-9), 10(-8), or 3 x 10(-8) M) individually or in combination. Serum E2 and P4 concentrations indicated that the animals were in the appropriate stage of the estrous cycle. Mean serum LH concentrations were greater (p < 0.0004) for OVX animals compared to FOL and LUT animals. Mean serum IGF-I concentrations were lower (p < 0.05) for OVX compared to FOL and LUT animals, while serum IGFBP were not different among animals. Basal LH secretion (control) was greater (p < 0.04) in OVX than in FOL or LUT cultures. Relative to control, 10(-11) M IGF-I increased (p < 0.02) LH secretion in FOL, LUT, and OVX cultures, and this response was greater (p < 0.05) in FOL and OVX than in LUT cultures. Only the 10(-11) M IGF-I enhanced basal LH secretion in LUT cultures. In addition, 10(-10) M IGF-I increased (p < 0.05) LH secretion in OVX cultures, and 10(-10) and 10(-9) M IGF-I stimulated (p < 0.05) LH secretion in FOL cultures, whereas basal LH secretion in all groups was unaffected (p > 0.05) by 10(-8) or 3 x 10(-8) M IGF-I. The GnRH-induced LH secretion was unaltered by IGF-I treatment. Results indicate that in vitro IGF-I treatment increased basal LH secretion, with reproductive status modulating LH response to IGF-I.

Glucose and free fatty acid modulation of growth hormone and luteinizing hormone secretion by cultured porcine pituitary cells.

The influence of glucose and free fatty acids (FFA) on LH and GH secretion from porcine pituitary cells was examined in culture. Cells were challenged with 10(-9), 10(-8), or 10(-7) M GnRH; 10(-9), 10(-8), or 10(-7) M [Ala15]-h growth hormone-releasing factor-(1-29)NH2 (GRF); 10(-3) or 10(-5) M forskolin; glucose (100, 300, or 600 mg/dL); and oleic or linoleic acid (10(-11) to 10(-5) M) individually or in combinations of glucose, oleic, and linoleic acid with GnRH, GRF, or forskolin. Relative to control (medium alone), GnRH increased (P < .003) LH secretion. Glucose at 100 mg/dL enhanced (P < .05) basal LH release, whereas the 600-mg/dL dose suppressed (P < .05) LH response to GnRH. Except for 10(-7) M, all doses of linoleic acid increased (P < .05) basal LH release, whereas only 10(-5) M linoleic acid suppressed (P < .02) LH response to GnRH. All doses of oleic acid enhanced (P < .05) basal LH release and suppressed (P < .03) the LH response to GnRH. In contrast, neither FFA suppressed the forskolin-induced LH secretion compared to forskolin alone. Growth hormone-releasing factor stimulated (P < .02) GH release in a dose-dependent manner. Glucose did not alter basal GH release but suppressed (P < .01) GRF-induced GH release. Only 300 mg/dL of glucose suppressed (P < .05) forskolin-induced GH release.(ABSTRACT TRUNCATED AT 250 WORDS)

Biogenic amines in the hypothalamus of rats after diethyldithiocarbamate or AIMAX treatment, an alternative for norepinephrine depletion.

The objective of this study was to determine whether AIMAX (a dithiocarbamoylhydrazine derivative) is suitable for determining the effects of norepinephrine (NE) depletion on reproduction in domestic animals. Therefore, the effect of AIMAX (n = 6) on concentrations of biogenic amines in the medial basal hypothalamus (MBH) and anterior hypothalamic area (AHA) of ovariectomized (OVX) rats primed with ovarian steroids was compared to that of diethyldithiocarbamate (DDC; n = 5), a potent dopamine-beta-hydroxylase (DBH) inhibitor, which is chemically similar to AIMAX. Rats that received only ovarian steroids and saline injections served as controls (n = 6). Treatment with DDC resulted in sedation and reduced body temperature. In contrast, rats behaved normally after AIMAX treatment. AI-MAX reduced (P < .05) NE but increased (P < .05) dopamine (DA) concentrations in MBH and AHA compared with controls. Similar changes in NE and DA concentrations were observed in DDC-treated rats. However, elevated epinephrine (EPI) levels were measured in MBH and AHA of only DDC-treated rats. Serum LH concentrations were suppressed (P < .005) in both AIMAX- and DDC-treated rats compared with control animals. Because AIMAX, like DDC, suppressed hypothalamic NE content and LH secretion, AIMAX should be useful in studying effects of NE depletion on gonadotropin secretion in domestic animals.