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.

Adrenocorticotropin-induced changes in ovine pituitary gonadotropin secretion in vitro.

In vitro pituitary perifusion experiments were conducted to examine the effect of ACTH and related peptides on basal and GnRH-stimulated gonadotropin release. Treatments of 5 X 10(-7) M ACTH-(1-39), ACTH-(1-24), or ACTH-(18-39) were examined for their ability to influence basal gonadotropin secretion and the subsequent response to a 10(-9)- or 10(-8) M GnRH challenge. Administration of the 1-39 or 18-39 peptide sequences of ACTH similarly stimulated the release of LH and FSH (P less than 0.01). ACTH-(1-24) had no effect on basal gonadotropin secretion. Pretreatment with ACTH-(1-39) inhibited the LH and FSH responses to 10(-9) and 10(-8) M GnRH (P less than 0.05). Suppression of the LH response to 10(-8) M GnRH (P less than 0.05) and the FSH response to 10(-9) M GnRH (P less than 0.05) was observed after ACTH-(1-24) treatment. The administration of ACTH-(18-39) had no significant effect on GnRH-induced gonadotropin release. PRL concentrations were not affected by any of the ACTH peptides. Exposure to 10(-10) M GnRH or 5 X 10(-7) M synthetic ACTH-(1-39) produced an equivalent stimulation of LH secretion. GnRH pretreatment enhanced (P less than 0.05), while ACTH-(1-39) diminished (P less than 0.05), the subsequent response to GnRH. The GnRH receptor antagonist [D-pGlu1, D-Phe2, D-Trp3,6]GnRH attenuated the LH and FSH responses to GnRH and ACTH-(1-39) (P less than 0.05). The results obtained in this study indicate that certain portions of the ACTH molecule may affect gonadotropin secretion, perhaps by interacting with the GnRH receptor.

Luteinizing hormone synthesis in cultured fetal human pituitary cells exposed to gonadotropin-releasing hormone.

To investigate whether GnRH regulates LH synthesis during human development, pituitary cells from second trimester fetuses were incubated with [35S]methionine ([35S]met) and [3H]glucosamine ([3H]gln) for 48 h with 0, 10(-9), and 10(-7) mol/L GnRH. Immunoassayable (i) LH was measured in media and cellular lysates, and dual label scintillation analysis was used to quantitate incorporation of radiolabeled precursors into cells, trichloroacetic acid-precipitable proteins, and immunoprecipitated LH subjected to electrophoresis. Exposure of cells to GnRH did not affect cellular uptake or incorporation of precursors into proteins, but specifically increased total (secreted plus cellular) LH synthesis. Both GnRH concentrations significantly increased iLH release and enhanced secreted and cellular [3H]gln-LH. The secretion of [35S] met-LH was stimulated only by 10(-7) mol/L GnRH. The proportion of newly synthesized LH that was secreted and the 3H/35S ratio of secreted and cellular LH were uninfluenced by GnRH. Although basal LH synthesis was not sex dependent, total iLH content and GnRH-stimulated LH translation were greater in cells from females than in those from males. Therefore, GnRH regulates LH synthesis by second trimester fetal human gonadotrophs without influencing the proportion of total radiolabeled LH that is secreted. The existence of a sex difference in total iLH content and GnRH-stimulated LH translation is consistent with the sexual dimorphism in pituitary LH content occurring during human development.

Effect of cortisol or adrenocorticotrophin on release of luteinizing hormone induced by luteinizing hormone releasing hormone in the dairy heifer.

During treatment with cortisol or ACTH, dairy heifers were given two doses of LH releasing hormone (LH-RH) spaced 1.5 h apart. Serum concentrations of cortisol and LH were monitored during each treatment. Treatment with both ACTH and cortisol raised plasma cortisol levels above the respective saline controls (P less than 0.001). Neither treatment affected basal LH concentrations. A slight depression in LH response was seen in the cortisol-treated animals after the first LH-RH injection, as shown by a statistically significant depression at three of the sample times. There was no significant difference between treated and control LH values after the second LH-RH administration. Treatment with ACTH resulted in significantly reduced LH values at all sample times after both injections of LH-RH.

The effect of opioid peptides on ovine pituitary gonadotropin secretion in vitro.

Although a hypothalamic site of action has been firmly established for opiate-mediated gonadotropin regulation, there have been several reports which indicate the possibility of a direct influence on the pituitary gland. The objective of this study was to further investigate this possibility in an in vitro pituitary perifusion system utilizing ovine tissue. Treatment with gamma-endorphin (GE) or human beta-endorphin (hBE) resulted in elevated basal LH release (p less than 0.05), followed by an inhibition in the response to a subsequent GnRH challenge (p less than 0.05). The stimulatory effect of hBE was found to be dose-responsive (p less than 0.01). PRL secretion was not similarly stimulated. Ovine beta-endorphin (oBE) had no effect on LH secretion, even though it differs from hBE by only 2 amino acids and contains the active GE sequence. Met-enkephalin also did not influence gonadotropin secretion. Naloxone pretreatment did not reverse the effects of hBE on gonadotropin release. It was found, however, that [D-pGlu1, D-Phe2, D-Trp3,6]-GnRH, a specific GnRH receptor antagonist, did reduce hBE-induced LH and FSH release (p less than 0.05). Naloxone pretreatment alone suppressed the response to GnRH (p less than 0.05). These data indicate that certain opioid peptides can influence ovine gonadotropin secretion in vitro by activating the GnRH receptor. Furthermore, a facilitory role is suggested for endogenous opiates in the local regulation of pituitary gonadotropin secretion.

Gonadotroph function in 3-week-old gilts reared in a warm or cool thermal environment.

The present study evaluated the effect of a warm (cycling 27-32 degrees C, 50-70% RH) or cool (21 degrees C, 55% RH) thermal environment (TE) on gonadotroph function in 3-week-old gilts (females). Pituitary cells from twelve gilts reared in each TE were cultured at a density of 250,000 cells/1 ml well and exposed to vehicle (culture medium); 1,1, and 10 nM gonadotropin-releasing hormone (GnRH); 2 mM 8-Br-cAMP (cAMP); and 100 nM phorbol myristate acetate (PMA). Luteinizing hormone (LH) and follicle-stimulating hormone (FSH) secretion in culture were stimulated by GnRH and by pharmacological compounds (p < .0001). In vitro LH secretion was approximately three-fold higher in the warm, compared to the cool, TE group (p < .0001). Similarly, cellular LH content in the warm TE exceeded that in the cool TE (p < .005). The in vitro secretion of FSH and cellular FSH content were significantly elevated in the warm TE (p < .02). Serum LH concentrations in the warm and cool TE were 7.01 +/- 1.75 and 2.13 +/- .44 ng/ml, respectively (p < .02). Serum FSH concentrations were 6.38 +/- .53 and 4.59 +/- .28 ng/ml in the warm and cool TE, respectively (p < .01). The results of this study demonstrate that secretory responses of gonadotrophs in early postweaning pigs are influenced by chronic TE exposure. These differences in secretory activity may reflect levels of cellular gonadotropin available for release.

Lactotroph and somatotroph function in piglets reared in a constant hot environment.

The present study evaluated the effect of rearing in a constant hot (32 degrees C) or cool (21 degrees C) thermal environment (TE) on lactotroph and somatotroph secretory activity in 5-wk-old barrows (castrate males) and gilts (female). Pituitary cells from seven gilts and seven barrows from each TE were cultured at a density of 250,000 cells/1 ml well and exposed to vehicle (culture medium); .1, 1, 10, or 100 nM thyrotropin-releasing hormone (TRH); or .1, 1, or 10 nM growth hormone-releasing hormone (GHRH). Post-receptor cellular stimulation was induced pharmacologically with 2 mM 8-Br-cAMP (cAMP); 100 nM phorbol myristate acetate (PMA); or 59 mM KCl. Prolactin secretion in culture was stimulated by TRH and by pharmacological compounds (p < .0001). In vitro PRL secretion was increased approximately two-fold in the hot environment (p < .02). Similarly, cellular PRL content in the hot TE was twice that in the cool TE (p < .01). The in vitro secretion of growth hormone (GH) was increased by GHRH and by pharmacological compounds (p < .0001), but was unaffected by TE (p > .5). No effects of sex (p > .3) or sex x TE interactions (p > .2) were detected in any endpoint. The results of this study demonstrate that lactotroph, but not somatotroph, secretory activity is enhanced by a constant hot TE in early postweaning pigs. This increase in secretory activity does not appear to be dependent on receptor-mediated cellular activation, but may reflect enhanced levels of cellular PRL available for release.

Expression of luteinizing hormone-releasing hormone and its receptor in porcine immune tissues.

Luteinizing hormone-releasing hormone (LHRH) is the primary regulator of pituitary LH release. However, LHRH has also been identified in extrahypothalamic sites including immune tissues. Accordingly, immunomodulatory properties for LHRH have been suggested. We wanted to determine whether LHRH and its receptor are produced by immune tissues in the pig. First, a cDNA was cloned and sequenced from the porcine hypothalamus that showed 87.5% homology with the human LHRH gene. Internal primers were identified from this sequence for amplifying a 268 bp product by PCR. In addition to the hypothalamus, PCR products reflecting LHRH mRNA were amplified in porcine spleen, thymus, and peripheral blood lymphocyte (PBL) cDNA. LHRH mRNA was not detected in liver, cerebral cortex, or pituitary tissue samples. Primers were designed to amplify a 360 bp fragment of LHRH-receptor cDNA. PCR products reflecting LHRH-receptor mRNA were amplified in pig hypothalamus, pituitary, thymus, spleen and PBL cDNA samples. No such products were amplified in cortex and liver samples. In summary, we report the sequence of a cDNA coding for LHRH and Gonadotropin-RH associated peptide (GAP) in the pig hypothalamus. Additionally, we provide evidence that LHRH and its receptor are synthesized in porcine immune tissues. This leads us to speculate that LHRH may have local, immunomodulatory functions in pigs.

Somatotroph, lactotroph and thyrotroph function in three-week-old gilts reared in a hot or cool environment.

The influence of the thermal environment on the ability of the pituitary gland to secrete growth hormone (GH), prolactin (PRL), and thyroid-stimulating hormone (TSH) was examined in gilts reared under hot (H: 27-32 degrees C, 50-90% RH, n = 6) or cool (C: 20 degrees C, 50% RH, n = 6) conditions. Piglets were sacrificed at 3 wks of age. Pituitary cells from each animal were cultured and exposed to vehicle (culture medium); 1, 1, and 10 nM thyrotropin-releasing hormone (TRH); .1, 1, and 10 nM growth hormone-releasing hormone (GHRH); 59 mM KCl; 2 mM 8-Br-cAMP; and 100 nM phorbol myristate acetate. Rearing in the H, compared to the C, environment increased plasma PRL concentrations (p < .001), in vitro PRL secretion subsequent to all secretagogue treatments (p < .001), and cellular PRL content (p < .001). The stimulated release of TSH in culture was reduced (p < .001), but cellular TSH content was increased (p < .05) by exposure to the H environment. The total amount of TSH in culture (secreted + cellular) was not affected by thermal environment. The release of GH in vitro, cellular GH content, total GH in culture, and plasma GH concentrations were similar between H and C groups. The only dose-response curves that differed in slope between thermal groups were those produced by the TSH response to TRH (p < .001). The results of this study suggest that chronic exposure to a hot environment can 1) enhance PRL secretion by a mechanism which affects the quantity of releasable PRL rather than lactotroph sensitivity to secretagogues and 2) reduce TSH secretion by inhibiting thyrotroph secretory response to stimulation.

Somatotroph function in the neonatal pig.

The objective of this study was to evaluate developmental changes in somatotroph function and related gene expression in neonatal pigs. Male piglets were sacrificed at 1, 7, 14, 21, 28, 35, and 42 d of age (8/age group) for the collection of tissue and blood. Serum concentrations of GH were determined. Quantitations of mRNA were performed for pituitary Pit-1, GH, and GHRH receptor. Cultures of pituitary cells from each pig were stimulated with 0, 0.1, 1, or 10 nM GHRH; 2 mM 8-Br-cAMP; or 100 nM phorbol myristate acetate. Elevated serum concentrations of GH were observed at 1 d of age, followed by a pronounced decrease to basal levels thereafter (P < 0.0001). A mild transient increase in circulating GH occurred at Day 28. In vitro GH secretion was significantly stimulated by secretagogue treatments (P < 0.0001). Age-related declines in in vitro GH secretion were observed regardless of if the cells were stimulated by GHRH or by secretagogues that bypass the GHRH receptor (P < 0.001). Similarly, cellular GH content varied with age (P = 0.01). Levels of pituitary GH mRNA (P = 0.01) and GHRH receptor mRNA (P = 0.0002) decreased with age. The quantity of GHRH receptor mRNA was correlated with GH mRNA levels (r = 0.55, P = 0.02), serum GH concentrations (r = 0.55, P = 0.02), and in vitro GH secretion (r = 0.66, P = 0.001). Pituitary Pit-1 mRNA levels at 7 and 14 d of age were significantly elevated relative to all other sampling times (P = 0.0002). Levels of Pit-1 and GH mRNAs were significantly correlated (r = 0.64, P = 0.003). These results demonstrate a strong developmental regulation of somatotrophic function and related gene expression during the early neonatal period of the pig. Age-related decreases in secretory function may be mediated by concurrent mechanisms relating to the expression of the GHRH receptor and of GH.

Relationship between in vitro somatotroph function and growth in three-week-old barrows.

The relationship between in vitro somatotroph function and growth was examined in piglets demonstrating a continuous range of growth characteristics. Twenty barrows were sacrificed at 3 weeks of age for the collection of pituitary tissue and blood. Pituitary cells from each animal were cultured and exposed to vehicle (culture medium); .1, 1, and 10 nM growth hormone-releasing hormone (GHRH); 2 mM 8-Br-cAMP (cAMP); 100 nM phorbol myristate acetate (PMA); and 59 mM KCl. All secretagogue treatments stimulated growth hormone (GH) secretion (p < .0001). Basal and stimulated GH secretion in culture, intracellular GH content (icGH), and serum insulin-like growth factor-1 (IGF-1) concentrations were all positively correlated with 3-week weight gain (p < .05). Concentrations of GH in the serum sample taken at sacrifice were not related to growth (p > .3). Intracellular GH content was correlated with in vitro GH secretion (p < .01) and serum IGF-1 concentrations (p < .001). Somatotroph function was contrasted in the 7 largest and 7 smallest piglets (large, 8.3 +/- .3 kg, n = 7; small, 4.5 +/- .2 kg, n = 7). Treatment with GHRH produced a dose-related increase in GH secretion in both experimental groups (p < .0001). No significant size x GHRH interaction was detected (p = .09), When contrasted with the small group, the large group demonstrated elevated GH secretion in culture (p < .01), icGH content (p < .001), and circulating IGF-1 (p < .001). The results of this study raise the possibility of a functional relationship between porcine somatotroph secretory activity and growth, mediated by IGF-1, which may be regulated by the quantity of GH available for release.

Endocrine responses to weaning and changes in post-weaning diet in the young pig.

The effects of weaning and changing post-weaning diet composition on growth patterns and growth-related hormonal profiles were evaluated in neonatal pigs. Forty-eight crossbred piglets were assigned to two groups (n = 24/group) based on weaning at 2 or 3 wk of age (2W and 3W groups. respectively). At weaning, piglets were removed from the sow and placed on a commercial starter ration for the first 11 d post-weaning (Phase I diet). At Day 12 post-weaning, pigs were placed on a growing ration for the remainder of the study (Phase II diet). Body weights and blood samples were collected twice weekly from birth until 42 d of age. Serum insulin-like growth factor (IGF)-1, IGF-2, and average daily gain (ADG) were reduced (P < 0.05) in both groups as a result of weaning, whereas serum growth hormone (GH) was elevated (P < 0.05). Earlier weaning resulted in a greater reduction in growth rate and serum IGF-2 values (P < 0.05). Mild reductions in ADG occurred after the Phase I to II dietary change in both weaning groups (P < 0.05), but serum IGF-1 decreased only in the 2W group (P < 0.05). Growth hormone concentrations tended to increase after the change in post-weaning diets (P = 0.07 and 0.16 in 2W and 3W, respectively). Serum thyroxine (T4) and triiodothyronine (T3) levels were unaltered by weaning but declined in both groups after the change in starter diets (P < 0.05). Changes in cortisol concentrations were not associated with weaning or the change in post-weaning diets. With the exception of serum IGF-1 concentrations, which were elevated in the 2W group, growth and endocrine endpoints were equivalent between experimental groups at the end of the study (42 d of age). These results indicate that earlier weaning and changing solid diets can more severely affect patterns of early growth and related hormone secretion, but effective compensatory mechanisms restore normal physiological and physical development.

Somatotroph and lactotroph function in relation to growth in six-week-old pigs reared in a hot or cool environment.

The influences of thermal environment and individual growth rate on somatotroph and lactotroph function were examined in 6-week-old barrows reared entirely in a hot (H: 27-32 degrees C, n = 8) or cool (C: 21 degrees C, n = 10) environment. Growth hormone (GH) and prolactin (PRL) cell contents and responses to growth hormone-releasing hormone (GHRH) or thyrotropin-releasing hormone (TRH) were evaluated in cultured pituitary cells from each animal. Plasma GH, PRL, and insulin-like growth factor-1 (IGF-1) concentrations also were monitored. Thermal environment did not affect in vitro GH secretion, cellular GH content, or plasma GH concentrations. Stimulated in vitro GH release (GHRH-basal) and plasma GH were inversely related to average daily gain (ADG, r = -.76, p < .005 and r = -.51, p < .05, respectively). Cellular GH content also declined as ADG increased (r = -.57, p < .05). Plasma IGF-1 concentrations were not affected by thermal environment and were not related to ADG. Pituitary cells from H animals secreted and contained more PRL than cells from C animals (p < .05). Plasma PRL values were correlated with ADG (r = .54, p < .05), but did not differ between thermal groups. Stimulated in vitro PRL (TRH-vehicle) secretion was positively related with ADG only in the H group (r = .97, p < .001). In contrast, cellular PRL content decreased with ADG in cells from the H barrows (r = -.8, p < .05). Lactotroph function was not related to growth in cells from C pigs. In summary, 1) heat enhanced PRL secretion and cell content; 2) growth and somatotroph function were inversely related; and 3) serum PRL and the PRL response to TRH in cells from H barrows were positively related to growth.

Effects of weaning on somatotrophic gene expression and circulating levels of insulin-like growth factor-1 (IGF-1) and IGF-2 in pigs.

The present study evaluated somatotrophic gene expression in liver, muscle and adipose tissue 4 d after weaning, a time point corresponding to greatly reduced serum concentrations of insulin-like growth factor (IGF)-1 and IGF-2 in pigs. Two-week-old barrows were either cross-fostered to a sow (SOW, n = 8) or weaned and fed a phase 1 diet containing either 0 or 7% spray-dried plasma (NP, n = 8 and SDP, n = 8; respectively). Piglets were allocated such that two size groups were equivalently represented in each experimental group (small, 3.5-4.3 kg and large, 4.6-5.7 kg). Animals were weighed daily and sacrificed 4 d after weaning for blood and tissue collection. Daily gains of the SOW piglets were significantly greater than those of the weaned pigs for the first 3 d of the experiment (P < 0.0001). Weight gains in the SOW and SDP pigs between d 3 and 4 were equivalently elevated relative to the NP pigs (P < 0.0001). Serum IGF-1 and IGF-2 concentrations were decreased in both NP and SDP compared to SOW (P < 0.0001). Serum IGF-2 levels were significantly lower in small piglets (P = 0.006). A Weaning Group X Size interaction was noted for liver IGF-2 mRNA (P < 0.03), reflecting a higher level of expression in large SOW piglets relative to small SOW piglets. Weaning did not affect IGF-1, IGF-2, or growth hormone (GH) receptor mRNA levels in liver, muscle, or fat (P > 0.05). Liver IGF-binding protein (IGFBP)-3 and acid-labile subunit (ALS) mRNA levels also were unaffected by weaning. Small pigs had lower levels of liver ALS (P = 0.0003), muscle IGF-2 (P = 0.02), and muscle GH receptor (P = 0.006) mRNAs. In contrast, adipose tissue IGF-1 and IGF-2 mRNA levels were greatest in the small piglets (P = 0.001 and 0.029, respectively).

Partial nucleotide sequence of the porcine corticosteroid-binding globulin (CBG) cDNA and specification of CBG expression sites in postnatal pigs.

The purpose of this study was to develop a porcine CBG cDNA probe in order to examine the porcine CBG mRNA expression in major tissues from the postnatal pig. The reverse transcriptase-polymerase chain reaction (RT-PCR) was conducted to develop the porcine CBG cDNA probe using total RNA extracted from liver of 40-day-old pig. The RT-PCR product was subcloned into the pGEM vector (Promega, Madison, WI) and subjected to restriction enzyme treatments and DNA sequencing. Northern blot analysis was conducted using total RNA extracted from samples (approximately 200 mg) of liver, lung, kidney, and whole adrenal tissue that were collected from pigs on day 3 (n = 2) or day 40 (n = 2) postpartum. A 500 bp partial porcine CBG cDNA encoded 166 amino acids and had 83, 78, and 77% homology to a 494-nucleotide sequence of CBG from sheep, human, and rabbit, respectively. The deduced peptide sequence of the partial porcine CBG showed 77, 62, 60, and 51% homology to sheep, human, rabbit, and rat CBG sequences, respectively. An approximately 1.53 kb CBG mRNA was detected only in the liver tissue. In conclusion, the development of a partial CBG cDNA for swine makes it possible to study the ontogeny and the regulation of CBG synthesis at the molecular level and, based on tissues examined in this study, the liver appears to be the primary source of CBG biosynthesis in the postnatal pig.

Characterization of a monoclonal antibody which detects luteinizing hormone from diverse mammalian species.

The present study describes the development and characterization of a monoclonal antibody (518B7) generated against bovine LH (bLH). Although 518B7 was extremely specific for LH, very low species specificity was observed. A RIA using this antibody and radioiodinated equine LH (eLH) showed good sensitivity for all mammalian LH preparations tested, with the exception of human LH (15%, relative to the eLH reference standard). Activities of most mammalian LH's ranged between approximately 50-200%. Much less activity was detected with reptilian LH (less than 1.5%). Amphibian and avian LH fractions were essentially inactive. The reactivities of LH alpha and beta subunits from a variety of mammals clearly showed that the antibody reacts with the beta subunit. Sensitive RIAs were also developed utilizing 125I-bovine and 125I-rat LH. Interestingly, all hormone preparations which showed sufficient reactivity for statistical analysis within the dose ranges used in the present study (0.01-1000 ng/tube) produced a displacement curve parallel to the reference standard. We have also validated the use of 518B7 in detecting LH in serum. Parallel dilution curves relative to purified LH reference standards were observed with equine and bovine serum samples and equine pituitary extract. High (average 94%) recoveries were also seen with bovine serum with known amounts of exogenously added bLH. Similar patterns of LH secretion were detected with a RIA based upon 125I-bLH and 518B7 and a previously described polyclonal antibody-based RIA in bovine serum samples during estrus. Thus, a monoclonal antibody for LH has been produced which can be used to develop sensitive and specific RIAs in many different mammalian species.(ABSTRACT TRUNCATED AT 250 WORDS)

cDNA cloning and tissue-specific gene expression of ovine leptin, NPY-Y1 receptor, and NPY-Y2 receptor.

The physiological regulation of food intake is a critical factor in both the rate at which an animal grows and its reproductive activity. Recently, progress has been made in elucidating a complex system in which insulin, leptin, and neuropeptide Y function to monitor an animal's energy balance and regulate feed intake and fertility. RNA was extracted from ovine hypothalamic, anterior pituitary, pancreas, and adipose tissue. Using the reverse transcription-polymerase chain reaction. cDNAs were cloned and sequenced for leptin (350 base pairs [bp], GenBank accession number U62123 and 441 bp, GenBank accession number U84247), NPY-Y1 receptor (350 bp, GenBank accession no. U62122) and NPY-Y2 receptor (440 bp, GenBank accession no. U83458). Probes generated from these clones were used to detect mRNA expression within tissues thought to be involved in the coregulation of feed intake and reproduction. Leptin was found to be expressed in sheep adipose tissue. The ovine NPY-Y1 receptor mRNA was detected within the arcuate nucleus and paraventricular nucleus of the hypothalamus, the dentate gyrus of the hippocampus and in pancreatic, anterior pituitary, and adipose tissues. Expression of ovine NPY-Y2 receptor mRNA was detected in the hippocampus and within pancreatic tissue. These observations provide evidence of potential mechanisms that exist for mediating communication between peripheral and central tissues within the insulin-leptin-NPY pathway.

Effects of an intravenous injection of NPY on leptin and NPY-Y1 receptor mRNA expression in ovine adipose tissue.

Neuropeptide Y (NPY) is highly expressed in hypothalami of undernourished and genetically obese animals, and is a potent regulator of food intake and reproduction. Leptin, a protein expressed by adipocytes, has been reported to reduce hypothalamic NPY expression. We recently detected (by ribonuclease protection assay [RPA]) expression of the NPY receptor subtype Y1 (but not Y2) mRNA in adipose tissue. Based on these observations we hypothesized that NPY-Y1 receptors in adipose may represent a peripheral mechanism by which NPY can regulate leptin expression in a direct and rapid manner. To test this hypothesis, adipose samples were biopsied from the tailhead region of 48 +/- 3 kg wether lambs immediately before and 30 min after a single intravenous injection of 50 micrograms porcine NPY ("treated" animals, n = 5), or vehicle ("control" animals, n = 4). Injection of NPY resulted in an increase in expression (P = 0.013; as measured by RPA) of both leptin and NPY-Y1 mRNA. In treated animals, negative correlations were found between response in leptin expression and body weight (r = -0.82, P = 0.092), and between leptin response and initial leptin mRNA levels (r = -0.81, P = 0.097). These data provide evidence of a peripheral mechanism by which NPY may regulate adipocyte expression of both leptin and NPY-Y1 receptor mRNA.

Transcriptional regulation of pituitary synthesis and secretion of growth hormone in growing wethers and the influence of zeranol on these mechanisms.

This experiment evaluated relationships between pituitary messenger RNA levels of the transcription factor Pit-1, the growth hormone releasing-hormone receptor (GHRHR), and synthesis and secretion of GH in growing wethers. The experiment also evaluated the influence of the estrogenic compound, zeranol, on these relationships. Seventy wethers that were 9.5 +/- 1 day of age were randomly assigned to a control group or to one of three zeranol treatment groups that were implanted (12 mg, Ralgro) at 0, 45, and (or) 90 days of age. Twenty-eight days after implantation (i.e., Days 28, 73, 118) and on Day 135, sera were collected serially from wethers (n > or = 5) from each group and then their pituitary was collected. As wethers gained weight with age, the pituitary increased in size and so did the relative message levels of Pit-1 and GH (effect of time, P < 0.01). However, as wethers reached 135 days of age, serum concentrations of GH had declined while concentrations of IGF-I had increased (linear contrast, P < 0.01). Additionally, zeranol increased serum concentrations of GH and IGF-I and this effect on GH appeared to be a consequence of increased pulse amplitude, particularly at 73 and 118 days of age (treatment x time, P

Postnatal function of the somatotrophic axis in pigs born naturally or by Caesarian section.

This study was designed to determine what effects the birth process would have on development of the somatotrophic axis in neonatal pigs. Eight crossbred sows were selected (n = 4 natural birth and n = 4 Caesarian section) for the present study. Blood and tissue samples from 38 pigs were collected at birth. Twenty pigs were maintained with natural birth sows until sacrificed for blood and tissue collection at 2 wk of age. Gestational age at birth did not differ (P > 0.16) between natural birth and C-section pigs. Average daily gain (ADG) from birth until 2 wk of age was reduced (P < 0.0001) by 39.3% in the C-section pigs as compared to the natural birth pigs. Serum growth hormone (GH) did not differ (P > 0.86) at birth, but was greater (P < 0.024) at 2 wk in C-section pigs. Serum insulin-like growth factor 1 (IGF-1) was greater at birth (P < 0. 0025) and at 2 wk of age (P < 0.035) in the natural birth pigs. Serum concentration of IGF-2 did not differ at birth (P > 0.8) but was greater (P < 0.043) in natural birth pigs at 2 wk of age. Pituitary content of GH mRNA and GH-releasing hormone receptor mRNA did not differ (P > 0.90) between groups regardless of age; however, expression of both mRNAs declined (P < 0.0003) from birth until 2 wk of age. There tended to be a birth type X age interaction (P < 0. 082) for liver IGF-1 mRNA such that C-section pigs had a greater expression at 2 wk of age. Liver IGF-1 mRNA expression increased (P < 0.0001) in both groups from birth to 2 wk of age. Liver expression of GH receptor mRNA was greater in C-section pigs at birth (P < 0. 04) and 2 wk of age (P < 0.03). These data provide evidence that the natural birth process affects postnatal development and/or function of the somatotrophic axis in the neonatal pig.