Adipose tissue and the reproductive axis: biological aspects.

The discovery of leptin has clearly demonstrated a relationship between body fat and the neuroendocrine axis since leptin influences appetite and the reproductive axis. Since adipose tissue is a primary source of leptin, adipose tissue is no longer considered as simply a depot to store fat. Recent findings demonstrate that numerous other genes, i.e. neuropeptides, interleukins and other cytokines and biologically active substances such as leptin and insulin-like growth factors I and II, are also produced by adipose tissue, which could influence appetite and the reproductive axis. Targets of leptin in the hypothalamus include neuropeptide Y, proopiomelanocortin and kisspeptin. Transsynaptic connection of hypothalamic neurons to porcine adipose tissue may result in a direct influence of the hypothalamus on adipose tissue function. Nutritional signals such as leptin are detected by the central nervous system and translated by the neuroendocrine system into signals which ultimately regulates luteinizing hormone secretion. Furthermore, leptin directly affects gonadotropin-releasing hormone release from the hypothalamus, luteinizing hormone from the pituitary gland and ovarian follicular steroidogenesis. Although leptin is identified as a putative signal that links metabolic status and neuroendocrine control of reproduction, other adipocyte protein products may play key roles in regulating the reproductive axisin the pig.

Patterns of gene expression in pig adipose tissue: insulin-like growth factor system proteins, neuropeptide Y (NPY), NPY receptors, neurotrophic factors and other secreted factors.

Although cDNA microarray studies have examined gene expression in human and rodent adipose tissue, only one microarray study of adipose tissue from growing pigs has been reported. Total RNA was collected at slaughter from outer subcutaneous adipose tissue (OSQ) and middle subcutaneous adipose tissue (MSQ) from gilts at 90, 150, and 210 d (n=5 age(-1)). Dye labeled cDNA probes were hybridized to custom porcine microarrays (70-mer oligonucleotides). Gene expression of insulin-like growth factor binding proteins (IGFBPs), hormones, growth factors, neuropeptide Y (NPY) receptors (NPYRs) and other receptors in OSQ and MSQ changed little with age in growing pigs. Distinct patterns of relative gene expression were evident within NPYR and IGFBP family members in adipose tissue from growing pigs. Relative gene expression levels of NPY2R, NPY4R and angiopoietin 2 (ANG-2) distinguished OSQ and MSQ depots in growing pigs. We demonstrated, for the first time, the expression of IGFBP-7, IGFBP-5, NPY1R, NPY2R, NPY, connective tissue growth factor (CTGF), brain-derived neurotrophic factor (BDNF) and ciliary neurotrophic factor (CNTF) genes in pig adipose tissue with microarray and RT-PCR assays. Furthermore, adipose tissue CTGF gene expression was upregulated while NPY and NPY2R gene expression were significantly down regulated by age. These studies demonstrate that expression of neuropeptides and neurotrophic factors in pig adipose tissue may be involved in regulation of leptin secretion. Many other regulatory factors were not influenced by age in growing pigs but may be influenced by location or depot.

Energy metabolism and leptin: effects on neuroendocrine regulation of reproduction in the gilt and sow.

It is well established that reproductive function is metabolically gated. However, the mechanisms whereby energy stores and metabolic cues influence appetite, energy homeostasis and fertility are yet to be completely understood. Adipose tissue is no longer considered as only a depot to store excess energy. Recent findings have identified numerous genes, several neurotrophic factors, interleukins, insulin-like growth factor binding protein-5, ciliary neurotrophic factor and neuropeptide Y (NPY) as being expressed by adipose tissue during pubertal development. These studies demonstrated for the first time the expression of several major adipokines or cytokines in pig adipose tissue which may influence local and central metabolism and growth. Leptin appears to be the primary metabolic signal and is part of the adipose tissue-hypothalamic regulatory loop in the control of appetite, energy homeostasis and luteinizing hormone (LH) secretion. Leptin's actions on appetite regulation are mediated by inhibition of hypothalamic NPY and stimulation of proopiomelanocortin. Its effects on gonadotropin-releasing hormone (GnRH)/LH secretion are mediated by NPY and kisspeptin. Thus, leptin appears to be an important link between metabolic status, the neuroendocrine axis and subsequent fertility in the gilt and sow.

Hypothalamic neurons innervating fat tissue in the pig express leptin receptor immunoreactivity.

While leptin receptors have been found in both the autonomic ganglion neurons and the hypothalamic nuclei, studies dealing with the projections from the central nervous system to the adipose tissue have been conducted mainly in laboratory animals. Therefore, the purpose of our study was to establish whether hypothalamic neurons are transsynaptically connected to adipose tissue depots in the pig, and if these neurons express leptin receptor immunoreactivity. Pseudorabies virus (PRV; Bartha's K strain) was introduced in perirenal or subcutaneous adipose tissue depots in domestic pigs. On day 9, animals were euthanized and hypothalami were collected and processed immunohistochemically with primary antisera against PRV and leptin receptor (OBR). PRV-labeled neurons were localized in paraventricular nucleus, supraoptic nucleus and arcuate nucleus following injections in both the perirenal and the subcutaneous adipose tissue depots. Ventromedial nucleus, dorsomedial nucleus and preoptic area-labeled neurons were observed after injection of the PRV into the perirenal adipose tissue, while in the lateral hypothalamic area-labeled neurons projected only to the subcutaneous adipose tissue. The majority of the PRV-labeled neurons simultaneously expressed OBR-immunoreactivity. Our results provide the morphological data on multisynaptic projections from hypothalamus to the fat tissue in the pig and demonstrate that these neurons, located in areas involved in reproductive processes and feeding behavior, may regulate fat tissue metabolism.

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.

Neuropeptide Y modulates growth hormone but not luteinizing hormone secretion from prepuberal gilt anterior pituitary cells in culture.

Pituitary cells, from seven 160- to 170-day-old pigs, were studied in primary culture to determine the affects NPY on LH and GH secretion at the level of the pituitary. On day 4 of culture, medium was discarded, plates were rinsed twice with serum-free medium and cells were cultured in 1 ml fresh medium without serum and challenged individually with 10(-10), 10(-8) or 10(-6) M [Ala(15)]-h growth hormone-releasing factor-(1-29)NH(2) (GRF); 10(-9), 10(-8) or 10(-7) M GnRH or 10(-9), 10(-8), 10(-7) or 10(-6) M NPY individually or in combinations with 10(-9) or 10(-8) M GnRH or 10(-8) or 10(-6)M GRF. Cells were exposed to treatment for 4 h at which time medium was harvested and quantified for LH and GH. Basal LH secretion (control; n = 7 pituitaries) was 12 +/- 6 ng/well. Relative to control at 4 h, 10(-9), 10(-8) and 10(-7) M GnRH increased (P < 0.01) LH secretion by 169, 176 and 197%, respectively. Neuropeptide-Y did not alter (P > 0.4) basal LH secretion nor 10(-8) M GnRH-induced increase in LH secretion but 10(-9) M GnRH-stimulated LH secretion was reduced by NPY and was not different from control or GnRH alone. Basal GH secretion (control; n = 7 pituitaries) was 56 +/- 12 ng/well. Relative to control at 4 h, 10(-10), 10(-8) and 10(-6) M GRF increased GH secretion by 111%, 125% (P < 0.01) and 150% (P < 0.01), respectively. Only 10(-6) M (134%) and 10(-7) M (125%) NPY increased (P < 0.04) basal GH secretion. Addition of 10(-9), 10(-8) and 10(-7) M NPY in combination with 10(-8) M GRF suppressed (P < 0.04) GRF-stimulated GH secretion. However, 10(-9) M NPY enhanced (P < 0.06) the GH response to 10(-6) M GRF. These results demonstrate that NPY may directly modulate GH secretion at the level of the pituitary gland.

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.

Leptin: a metabolic signal affecting central regulation of reproduction in the pig.

The discovery of the obesity gene and its product, leptin, it is now possible to examine the relationship between body fat and the neuroendocrine axis. A minimum percentage of body fat may be linked to onset of puberty and weaning-to-estrus interval in the pig. Adipose tissue is no longer considered as only a depot to store excess energy in the form of fat. Recent findings demonstrate that numerous genes, i.e., relaxin, interleukins and other cytokines and biologically active substances such as leptin, insulin-like growth factor-I (IGF-I), IGF-II and Agouti protein are produced by porcine adipose tissue, which could have a profound effect on appetite and the reproductive axis. Hypothalamic neurons are transsynaptically connected to porcine adipose tissue and may regulate adipose tissue function. In the pig nutritional signals such as leptin are detected by the central nervous system (CNS) and translated by the neuroendocrine system into signals, which regulate appetite, hypothalamic gonadotropin-releasing hormone (GnRH) release and subsequent luteinizing hormone (LH) secretion. Furthermore, leptin directly affects LH secretion from the pituitary gland independent of CNS input. Changes in body weight or nutritional status are characterized by altered adipocyte function a reduction in adipose tissue leptin expression, serum leptin concentrations and a concurrent decrease in LH secretion. During pubertal development serum leptin levels, hypothalamic leptin receptor mRNA and estrogen-induced leptin gene expression in fat increased with age and adiposity in the pig and this occurred at the time of expected puberty. In the lactating sow serum and milk leptin concentrations were positively correlated with backfat thickness and level of dietary energy fed during gestation as well as feed consumption. Although, these results identify leptin as a putative signal that links metabolic status and neuroendocrine control of reproduction, other adipocyte protein products may play an important role in regulating the reproductive axis in the pig.

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.

Opioid modulation of FSH, growth hormone and prolactin secretion in the prepuberal gilt.

The role of endogenous opioid peptides (EOP) in modulating GH, prolactin (PRL) and FSH secretion was evaluated in prepuberal (P) gilts. In experiment I, P gilts received 1 (n = 2), 3 (n = 3) or 6 (n = 3) mg naloxone (NAL)/kg body weight i.v. Blood was collected every 15 min for 2 h prior to and 2 h after NAL and an additional 1 h after 100 micrograms gonadotrophin-releasing hormone (GnRH) i.v. In experiment II, P and mature (M) gilts were ovariectomized. Three weeks after ovariectomy, P and M gilts were injected twice a day for 10 days with either 0.85 mg progesterone (P4)/kg body weight or oil vehicle (V), resulting in the following groups: PP4 (n = 11), PV (n = 10), MP4 (n = 11) and MV (n = 10). All gilts received 1 mg NAL/kg body weight on the last day of treatment. Blood samples were collected every 15 min for 4 h before and 2 h after NAL and an additional 1 h after 100 micrograms GnRH i.v. In experiment III, six P and five M gilts were ovariectomized and surgically implanted with intracerebroventricular (i.c.v.) cannulae. Blood was collected every 15 min for 3 h before and 3 h after i.c.v. injection of 500 micrograms morphine in artificial cerebrospinal fluid (CSF) or 250 microliters CSF. In experiment I, all doses of NAL failed to alter PRL secretion, while NAL increased (P less than 0.05) GH secretion in three out of eight gilts. However, NAL suppressed (P less than 0.05) FSH concentrations.(ABSTRACT TRUNCATED AT 250 WORDS)

The role of melanocortin-3 and -4 receptor in regulating appetite, energy homeostasis and neuroendocrine function in the pig.

A recently discovered class of receptors, melanocortin-3 and -4 receptor (MC3/4-R), are located within the brain and modulate feed intake in rodents. Stimulation of the receptor (agonist) inhibits feed intake whereas blockade (antagonist) of the receptor increases intake. Our knowledge of factors regulating voluntary feed intake in humans and domestic animals is very limited. i.c.v. administration of an MC3/4-R agonist, NDP-MSH, suppressed (P<0.05) feed intake compared with controls at 12, 24, 48 and 72 h after treatment in growing pigs. Fed pigs were more responsive to the MC3/4-R agonist then fasted animals. However, i.c.v. treatment with MC3/4-R antagonist, SHU9119, failed to stimulate intake. The failure of MC3/4-R antagonist to stimulate feed intake suggests involvement of other brain hormone(s) which antagonize the action of SHU9119 at the MC3/4-R, blocking its stimulatory effect on intake. Treatment with NDP-MSH or SHU9119, across a wide dose range, failed to affect LH and GH secretion, except for the 10 micro g dose of NDP-MSH, which exhibited both a stimulatory and an inhibitory effect on GH secretion in fasted animals. Treatment with agouti-related peptide, a natural brain hormone that blocks the MC3/4R, failed to stimulate feed intake. These results do not support the idea that endogenous melanocortin pays a critical role in regulating feed intake and pituitary hormone secretion in the pig. SHU9119 blocked the NDP-MSH-induced increase in cAMP in HEK293 cells expressing the porcine MC4-R sequence without the missense mutation. The EC(50) and IC(50) values were similar to the human MC4-R, confirming that SHU9119 is a pig MC4-R antagonist. However, pigs were heterozygous for an MC4-R gene missense mutation. It is possible that the MC4-R mutation alters function and this may explain the failure to demonstrate MC3/4-R involvement in modulating feeding behavior and LH and GH secretion in the pig.

Catecholaminergic region A15 in the bovine and porcine hypothalamus.

Magnocellular perikarya within the retrochiasmatic division of the supraoptic nucleus of bovine and porcine hypothalami were immunoreactive (ir) with antiserum against tyrosine hydroxylase (TH), but not dopamine-beta-hydroxylase (DBH). Few cells in this region were also immunoreactive for vasopressin (VP) or oxytocin (OT). In contrast, the main division of the supraoptic nucleus contained numerous perikarya immunoreactive for VP and OT, but not TH nor DBH. Both the retrochiasmatic and principal divisions of the supraoptic nuclei contained TH- and DBH-ir fibers and varicosities. This region in bovine and porcine hypothalami corresponds to the ventral A15 catecholaminergic (dopamine-producing) cell group.

Effect of testosterone on n-methyl-d, l-aspartate-induced growth hormone secretion in male swine.

Previous research from our laboratory demonstrated that n-methyl-d, l-aspartate (NMA), a potent agonist of glutamate, increased growth hormone (GH) secretion in barrows and boars. To determine if testosterone modulates NMA-induced GH secretion, Poland China x Yorkshire swine were challenged with NMA in a model that compared GH responses in boars with those of barrows or barrows treated with testosterone propionate (TP). Boars and barrows weighing 112.6+/-1.4 kg (mean +/- SE) were fitted with indwelling jugular vein catheters. Barrows (n = 16) were given i.m. injections of TP (25 mg in corn oil) twice daily from d 0 to d 6. Boars (n = 16) and control barrows (n = 15) received twice daily injections of corn oil. On d 6, blood was sampled every 15 min for 4 h. Two h after sampling began, all animals received an i.v. injection of NMA at a dose of 2.5 mg/kg body weight. Mean testosterone concentrations (ng/ml serum) were similar (P > .1) for boars (8.1+/-0.8) and barrows receiving TP (7.3+/-0.3), but were greater in both cases (P < .05) than for barrows receiving corn oil (.2+/-.01). Prior to NMA injections, mean GH concentrations were similar (P > .1) among groups and averaged 2.7+/-.2 ng/ml serum across treatments. Serum concentrations of GH after NMA increased (P < .05) similarly among groups and averaged 6.3+/-0.3 ng/ml across treatments during the 2-h period after injection. These results were not supportive of a role for testosterone as a modulator of NMA-induced GH secretion in male swine.

Effect of cysteamine hydrochloride on secretion of growth hormone in male swine.

The objective of this study was to determine the effect of cysteamine hydrochloride (CSH) on growth hormone (GH) secretion in male swine. Twelve Poland China x Yorkshire boars, weighing 103.4 +/- 3.0 kg and fitted with indwelling jugular vein catheters, were individually penned in an environmentally controlled room. Boars received i.v. injections of either 0, 25, 50, or 75 mg CSH/kg body weight (BW) at h 0 (n = 3/treatment). Blood samples were collected every 15 min from h 0 to h 4. Serum concentrations of GH were determined by radioimmunoassay. There was an effect of treatment (P < .05) on mean GH concentrations. Mean GH concentrations (ng/ml) were 1.97 +/- .46, 2.24 +/- .59, .91 +/- .06, and .62 +/- .08 for boars receiving 0, 25, 50, and 75 mg CSH/kg BW, respectively. The dose of CSH-mean GH response had a linear (P < .01) component. Cysteamine hydrochloride at the 75 mg/kg BW dose decreased mean GH concentrations (P < .05) compared to the 0 and 25 mg/kg BW groups. The frequency and amplitude of GH pulses were similar (P > .1) among treatments. Overall, GH pulse amplitude was 2.35 +/- .58 ng/ml and GH pulse frequency was .75 +/- .07 pulses/h. Results from this experiment indicate that CSH suppresses circulating GH concentrations in a dose dependent fashion in boars.

Luteinizing hormone secretion following intracerebroventricular administration of morphine in the prepuberal gilt.

Antagonism of endogenous opioids with naloxone stimulates luteinizing hormone (LH) release in mature but not prepuberal gilts. The present report demonstrates that the opiate agonist morphine (500 micrograms), administered intracerebroventricularly (ICV), reduced LH secretion in both ovariectomized mature and prepuberal gilts. We suggest that opioid receptors are functionally coupled to the GnRH secretory system in prepuberal gilts even though endogenous opioid peptide modulation of LH secretion was not demonstrable in our previous studies.

On the site of action of naloxone-stimulated cortisol secretion in gilts.

The increase in serum cortisol concentrations following naloxone administration to female pigs was abolished by hypophysial stalk-transection, even though CRH and ACTH stimulated cortisol release in these animals. We suggest that the opioid antagonist enhances cortisol secretion primarily by a central action in pigs.

Prolactin secretion after bromocriptine in the hypophysial stalk-transfected gilt.

Blood samples were collected by jugular vein cannula from 6 hypophysial stalk-transected (HST) and 4 intact (INTACT) crossbred gilts at 0800, 0830 and 0900 h. Immediately after the 0900 h sample, thyrotropin releasing hormone (TRH) was injected i.v. to determine anterior pituitary gland response via release of prolactin (PRL). Blood was collected every 15 min for 1 h and every 30 min for an additional 2 h. All gilts received the dopamine (DA) agonist, bromocriptine (CB-154), s.c. at 1600 h and blood sampling and TRH challenge was repeated beginning at 0800 h the next day. Mean serum PRL concentration at 0800, 0830 and 0900 h was termed basal PRL for each day. Before CB-154, basal PRL for HST gilts was greater (P < .01) than for INTACT gilts, whereas, after CB-154, basal PRL were similar among HST and INTACT gilts. Before CB-154, TRH caused peak secretion of PRL of similar magnitude within 15 minutes in both HST and INTACT gilts; PRL decreased to basal concentration by 120 min after TRH in both groups. However, after CB-154, PRL response to TRH was blunted similarly in all gilts. These results indicate that suppression of DA pathways is an antecedent to the physiological mechanism by which a secretagogue, such as TRH, stimulates PRL secretion in the intact pig.

N-methyl-D,L-aspartate modulation of luteinizing hormone and growth hormone secretion from pig pituitary cells in culture.

Administration of n-methyl-d, l-aspartate (NMA) to pigs in vivo increased GH and suppressed LH secretion. Cultures of anterior pituitary cells from pigs in the follicular phase (FOL; n = 3) and luteal phase (LUT; n = 3) of the estrous cycle, and ovariectomized (OVX; n = 10) pigs were treated with NMA (10(-4), 10(-6) or 10(-8) M) or the NMA antagonist, 2-amino-5-phosphonopentanoic acid (AP5; 10(-4), 10(-6) or 10(-8) M), to determine if NMA affects the pituitary directly. Secreted LH and GH were measured at 4 h after treatment. Basal LH and GH secretion (control; C) were 1.1 +/- 0.6, 4.4 +/- 2.1 and 5.6 +/- 1.3 ng/well and 5.2 +/- 1.2, 7.5 +/- 1.2 and 5.2 +/- 1.7 ng/well for FOL, LUT and OVX, respectively. Relative to C, 10(-4) M NMA increased (P < 0.001) LH secretion 2.4-, 2.2- and 5.1-fold in FOL, LUT and OVX cultures, respectively. The effect of 10(-4) M NMA was inhibited by 10(-4) M AP5 (P < 0.05) in FOL cultures, but not in OVX cultures. GnRH increased (P < 0.001) LH levels 3.1-, 2.3- and 3.8-fold in FOL, LUT and OVX cultures, respectively. Relative to C, 10(-4), 10(-6) and 10(-8) M NMA increased (P < 0.03) GH secretion 1.5-, 1.5- and 2.3-fold in LUT and 1.7-, 2.3- 2.0-fold in OVX cultures, respectively. AP5 alone or in combination with NMA failed to alter basal GH secretion.(ABSTRACT TRUNCATED AT 250 WORDS)

Central nervous system peptide and amino acid modulation of luteinizing hormone and prolactin secretion in the pig.

We recently demonstrated that pulsatile LH secretion is associated with pulsatile gonadotropin releasing hormone (GnRH) in the pig. Endogenous opioid peptide (EOP) inhibition of pulsatile LH and prolactin (PRL) secretion is dependent on reproductive status and development of this EOP system is a brain maturational process independent of the ovary. Once sexual maturation has occurred, EOP then become part of a progesterone dependent system and EOP inhibit a noradrenergic component of this system. During lactation, EOP also inhibit pulsatile LH, but stimulate PRL secretion. N-methyl-d,l-aspartate (NMA), an agonist of the excitatory amino acids (EAA), aspartate and glutamate, suppressed LH secretion in gilts pretreated with progesterone or vehicle. Both the EOP agonist, morphine (MOR), and the EOP antagonist, naloxone (NAL), delayed emergence and time to maximum serum LH concentration of the estradiol-induced LH surge in prepuberal and mature gilts, respectively. Therefore, EOP may normally have both a permissive as well as an inhibitory role in the LH surge mechanism. Although a norepinephrine synthesis inhibitor failed to alter basal PRL secretion, the PRL increase after NAL was suppressed in progesterone-treated ovariectomized (OVX) gilts. NAL suppressed the PRL response to NMA in OVX gilts pretreated with oil vehicle or progesterone, indicating that NMA stimulation of PRL secretion is mediated through the EOP system.

Biology of leptin in the pig.

The recently discovered protein, leptin, which is secreted by fat cells in response to changes in body weight or energy, has been implicated in regulation of feed intake, energy expenditure and the neuroendocrine axis in rodents and humans. Leptin was first identified as the gene product found deficient in the obese ob/ob mouse. Administration of leptin to ob/ob mice led to improved reproduction as well as reduced feed intake and weight loss. The porcine leptin receptor has been cloned and is a member of the class 1 cytokine family of receptors. Leptin has been implicated in the regulation of immune function and the anorexia associated with disease. The leptin receptor is localized in the brain and pituitary of the pig. The leptin response to acute inflammation is uncoupled from anorexia and is differentially regulated among swine genotypes. In vitro studies demonstrated that the leptin gene is expressed by porcine preadipocytes and leptin gene expression is highly dependent on dexamethasone induced preadipocyte differentiation. Hormonally driven preadipocyte recruitment and subsequent fat cell size may regulate leptin gene expression in the pig. Expression of CCAAT-enhancer binding proteinalpha (C/EBPalpha) mediates insulin dependent preadipocyte leptin gene expression during lipid accretion. In contrast, insulin independent leptin gene expression may be maintained by C/EBPalpha auto-activation and phosphorylation/dephosphorylation. Adipogenic hormones may increase adipose tissue leptin gene expression in the fetus indirectly by inducing preadipocyte recruitment and subsequent differentiation. Central administration of leptin to pigs suppressed feed intake and stimulated growth hormone (GH) secretion. Serum leptin concentrations increased with age and estradiol-induced leptin mRNA expression in fat was age and weight dependent in prepuberal gilts. This occurred at the time of expected puberty in intact contemporaries and was associated with greater LH secretion. Further work demonstrated that leptin acts directly on pituitary cells to enhance LH and GH secretion, and brain tissue to stimulate gonadotropin releasing hormone secretion. Thus, development of nutritional schemes and (or) gene therapy to manipulate leptin secretion will lead to practical methods of controlling appetite, growth and reproduction in farm animals, thereby increasing efficiency of lean meat production.