Does obestatin modulate the hypothalamic appetite-regulating network in peripubertal sheep?

The participation of peripheral peptides in the processes regulating the food intake (energy homeostasis) at the central nervous system level remains unclear. This study focuses on the role of obestatin in neuronal activity within the hypothalamic appetite-regulating network in ruminants. The animals (n = 28) were randomly divided into two groups. The sheep in the control group received intracerebroventricular infusions of the Ringer-Locke solution, and the sheep in obestatin group were infused with obestatin (diluted in the Ringer-Locke solution) at 25 µg per 120 µl/hr. The series of four 1-hr infusions on 3 consecutive days were performed, and immediately after the experiment, the sheep were decapitated. Selected brain regions were fixed in situ for further immunohistochemical analysis, while the remaining ones were frozen for real-time RT-qPCR analysis. Obestatin infusion elicited changes in the neuropeptide Y (NPY) neuronal network in the hypothalamus. The results obtained show that exogenous obestatin evoked an increase in npy and agrpmRNA expression in the mediobasal hypothalamus (MBH), while the immunoreactivity for NPY was decreased in the arcuate and periventricular nuclei. The increase in cart and pomcmRNA expression in the MBH was also observed. Moreover, increased levels of gpr39 receptor and npy receptor 1 mRNA expression were evident in obestatin-infused sheep. Based on these results, it can be concluded that obestatin plays a role in the modulation of appetite-regulating network at the central level in sheep. The results obtained suggest that the underlying mechanism may involve the modification of the activity of NPY/AgRP and CART/α-MSH neurons in the arcuate nucleus.

Unraveling the function of paralogs of the aldehyde dehydrogenase super family from Sulfolobus solfataricus.

Aldehyde dehydrogenases (ALDHs) have been well established in all three domains of life and were shown to play essential roles, e.g., in intermediary metabolism and detoxification. In the genome of Sulfolobus solfataricus, five paralogs of the aldehyde dehydrogenases superfamily were identified, however, so far only the non-phosphorylating glyceraldehyde-3-phosphate dehydrogenase (GAPN) and α-ketoglutaric semialdehyde dehydrogenase (α-KGSADH) have been characterized. Detailed biochemical analyses of the remaining three ALDHs revealed the presence of two succinic semialdehyde dehydrogenase (SSADH) isoenzymes catalyzing the NAD(P)(+)-dependent oxidation of succinic semialdehyde. Whereas SSO1629 (SSADH-I) is specific for NAD(+), SSO1842 (SSADH-II) exhibits dual cosubstrate specificity (NAD(P)(+)). Physiological significant activity for both SSO-SSADHs was only detected with succinic semialdehyde and α-ketoglutarate semialdehyde. Bioinformatic reconstructions suggest a major function of both enzymes in γ-aminobutyrate, polyamine as well as nitrogen metabolism and they might additionally also function in pentose metabolism. Phylogenetic studies indicated a close relationship of SSO-SSALDHs to GAPNs and also a convergent evolution with the SSADHs from E. coli. Furthermore, for SSO1218, methylmalonate semialdehyde dehydrogenase (MSDH) activity was demonstrated. The enzyme catalyzes the NAD(+)- and CoA-dependent oxidation of methylmalonate semialdehyde, malonate semialdehyde as well as propionaldehyde (PA). For MSDH, a major function in the degradation of branched chain amino acids is proposed which is supported by the high sequence homology with characterized MSDHs from bacteria. This is the first report of MSDH as well as SSADH isoenzymes in Archaea.

Gemini-like molecular clips and tweezers: the influence of structure and guest binding on interfacial tension.

In a series of experiments, we studied the interfacial activity of aromatic aliphatic molecules with rigid gemini-like structures at the interface between toluene and water. These molecules, called clips and tweezers, have rigid central benzene or naphthalene spacer-units, each substituted with two polar groups as well as two rigid aromatic side walls. They can serve as host molecules and selectively bind a variety of electron-deficient aromatic and aliphatic guest molecules. In different experiments, we compared the interfacial tensions with the calculated hydrophilic-lipid-balance (HLB) values of these molecules. The measured interfacial tensions depend as much on the HLB values as on the geometric structure of the water insoluble molecules. The concentration dependence of the surface tension gave evidence for the formation of inverse micellar aggregates, which were formed in the oil phase above a well-defined value of the bulk concentration. The presence of aggregates in the organic liquid could also be investigated by dynamic light scattering measurements. We observed typical diameters of the inverse micellar aggregates in the order of 5.6 nm, and the critical micelle concentrations (cmc's) coincided well with the results of interfacial tension measurements. From the surface excess in the vicinity of the cmc, we calculated the space occupied by a single clip molecule on the self-assembled monolayer. The observed molecular surface area was in agreement with the effective molecular diameters of the molecules. In additional experiments, we could also show that complexes with aromatic guest molecules such as 1,2-4,5-tetracyanobenzene (TCNB) led to a reduction of the amphiphilic clip properties.

The intrapituitary endocrine events during maturation and timing of puberty in the female sheep.

The aim of this study was to determine the maturational activity of gonadotroph cells, the site of synthesis, storage and release of luteinizing hormone (LH) and follicle-stimulating hormone (FSH) in Polish Merino female sheep born after the summer solstice. The actual time of puberty of these lambs was delayed until the following breeding season, when they were 14 months old. Changes were examined in 12 peripubertal (30-, 52-week-old) and pubertal (Days 15 and 17 of the second ovarian cycle) females. Histomorphological and functional changes in the gonadotroph population were assayed with hybridohistochemistry, immunohistochemistry, computer-assisted image analysis and radioimmunoassay. The percentage of the adenohypophyseal area (PAA) occupied by gonadotrophs containing LHbeta-mRNA was higher and the LH plasma concentration and pulse frequency were lower in the 52-week-old sheep in comparison with the 30-week-old sheep (P<0.05). The PAA occupied by immunoreactive (ir)-LHbeta-cells remained stable at the 30th and 52nd weeks of age and then increased at the pubertal follicular phase. The PAA occupied by ir-FSHbeta-cells was higher in the 52-week-old sheep compared with the 30-week-old sheep and then lower at the pubertal follicular phase (P<0.05). The PAA occupied by gonadotrophs containing LHbeta-mRNA or FSHbeta-mRNA was lower at the pubertal follicular phase in comparison with the 52nd week of age (P<0.05). In pubertal sheep, the PAA occupied by gonadotrophs containing LHbeta-mRNA or FSHbeta-mRNA was higher and the PAA occupied by ir-LHbeta or ir-FSHbeta-cells was lower at the preovulatory phase in comparison with the follicular phase of the cycle (P<0.05). In conclusion, the photoperiodic suspension of gonadotroph population's maturational functions has been observed at the level of LH storage and release but not at the level of LH synthesis during the expected time of puberty in female sheep of an aseasonal breed such as Merino. The findings show the heterogeneity in the patterns of LH and FSH post-transcriptional processing during the period of peripubertal/pubertal transition, explained by the different intrapituitary regulation at the level of post-transcriptional synthesis and storage rather, than at the level of release. Altogether, intrapituitary mechanisms of ovine maturation could have the histomorphological feature. Our observations prompt the hypothesis that the female lamb may be able to transduce changes in day length into the appropriate endocrine cues for sexual maturation after attainment by the pituitary gonadotroph population the full peripubertal efficiency, manifested by the sufficient storage of LH.

Langmuir and Langmuir-Blodgett monolayers of molecular tweezers and clips.

Molecular clips and tweezers are able to selectively bind electron-deficient aromatic and aliphatic substrates. By means of pressure-area isotherms and Brewster angle microscopy (BAM), the self-association process and phase behavior of dimethylene-bridged molecular clips and tetramethylene-bridged molecular tweezers each substituted with two acetoxy groups as polar head groups were investigated. In a series of experiments, we observed that the molecular surface area of the clips and tweezers only depended on the skeletal structure and not on the polar groups. The measured areas agreed with the effective molecular diameters of the molecules if the aromatic side walls of the clips or tweezers were assumed to be aligned perpendicularly to the water surface. We compared the phase behavior of the pure molecular clips and tweezers with that of the host-guest complexes of these molecules, which were formed with 1,2,4,5-tetracyanobenzene (TCNB) as the guest molecule. For the clips with a central benzene (I) and naphthalene spacer unit (II), the complex formation with TCNB had no measurable influence on the phase diagrams of the films. We observed, however, a dramatic difference in the BAM images and pi-A isotherms between the pure molecular tweezers III and its complex with TCNB (TCNB@III). In addition to the pi-A isotherms, we used the surface potential (V)-area (A) isotherms to compare the pure tweezers III with the corresponding complex (TCNB@III). There was a strong difference in the maximum surface potential value for the pure tweezers (450 mV) and that for the complex (300 mV). In additional experiments, we prepared LB layers of such molecules, which were investigated by fluorescence spectroscopy. In comparison to the pure tweezers III, a luminescence emission of charge-transfer (CT) origin was observed for the host-guest complex (TCNB@III) fixed on the solid substrate. It turned out that the spectra were in good agreement with the results observed in chloroform solution.

The effect of dietary protein restriction on the secretion of LH and FSH in pre-pubertal female lambs.

The effect of restricted dietary protein on the synthesis, storage and release of LH and FSH was studied in pre-pubertal female lambs. The experiment started when the lambs were aged 12 weeks and weighed 26.0+/-1.6 kg. It was conducted for 25 weeks. The lambs were fed isocaloric diets containing either a restricted level of crude protein (8% CP; n=6; treatment R) or an elevated one (18% CP; n=4; treatment E). At 37 weeks of age and before the first oestrous cycle, blood samples were collected over 6 h at 10 min intervals for LH assay. The lambs were slaughtered and their brains recovered and fixed in situ. Immuno-reactive (IR) LH and FSH cells were localised by immunohistochemistry techniques. Messenger RNA analyses used by non-isotope in situ hybridisation with sense and anti-sense riboprobes from beta subunits of LH and FSH cDNA clones. Data were generated using computer analysis to measure the proportion of IR and/or hybridising cells and their optical density for immuno-staining and hybridisation signal. Plasma LH was measured by RIA. The daily live-weight gains were 56.5+/-13.1 g and 97.8+/-14.3 g for R and E lambs, respectively (P<0.05), so that final weights at slaughter were 36.1+/-1.97 kg and 39.1+/-3.44 kg, respectively (P<0.05). The number of cells expressing LH beta mRNA and the optical density of this hybridisation signal was significantly (P<0.001) lower in the R lambs but the number of IR LH positive cells was higher (P<0.001) than for the E lambs. The concentration of LH in the plasma of R sheep was lower (P<0.05) than the E group and this response was associated with a decrease (P<0.05) in LH pulse frequency and amplitude. Dietary protein concentration appeared to have no effect on the IR in FSH cells or on the expression of FSH beta mRNA. In summary, the low protein diet influenced the body weight and weight gain of growing lambs and exerted an inhibitory effect on the synthesis and the release of LH in the pituitary gonadotrophs. No such effect was observed for FSH. It was concluded that the protein concentration of the diet consumed during the growth of female lambs may be an important modulator of processes leading to the pre-pubertal rise in LH.

Effect of food manipulation on the neuropeptide Y neuronal system in the diencephalon of ewes.

The expression of neuropeptide Y (NPY), which is involved in the neuromodulatory function associated with animal nutrition, growth and reproduction, depends on the nutritional status. However, the roles of individual components of food are not fully recognised. In this study, we evaluated the effects of dietary protein levels on the diencephalic population of NPY neurones. Female lambs were fed two diets equilibrated energetically but containing 8% protein in restricted diet or 18% protein in elevated diet, for 15 weeks starting with 6 months of age, during the first breeding period. Then, brain tissues were collected, fixed and used for the immunohistochemical localisation of NPY. Detection of NPY in diencephalon sections was followed by the image analysis and expressed as the percent area stained and optical density of immunostaining. Two distinct populations of the immunoreactive NPY perikarya were found, one in the infundibular nucleus; and the other in areas of diencephalon adjacent to the rostral hypothalamus. Long-term feeding the protein restricted diet caused a prominent expression of the immunoreactive material solely in the hypothalamic NPY neurones, particularly in those located in the entire periventricular area and in the infundibular nucleus. Both, percent area exhibiting positive staining and the density of immunoreactive NPY measured in the periventricular subdivision of the paraventricular nucleus were significantly higher (P<0.05) in sheep fed restricted diet, than in sheep fed on elevated diet. This study describes the distribution of NPY neurones in the sheep diencephalon, and shows the relationships between the expression of hypothalamic population of NPY neurones and the level of protein feeding.

Effects of central infusions of neuropeptide Y on the somatotropic axis in sheep fed on two levels of protein.

Effects of infusions of neuropeptide Y (NPY) into 3rd ventricle of growing sheep fed on diets containing restricted (R) or elevated (E) levels of protein on the immunoreactive (ir) somatostatin neurones, ir somatotrophs, growth hormone (GH) concentration in the blood plasma were studied. The long-term restriction of protein in the diet elicited: enhancing irSS content in periventricular perikarya; diminishing irSS stores in the median eminence and elevating the number ir somatotrophs and content of irGH. NPY infusions enhanced the content of irSS in perikarya in sheep fed on E diet and diminished the number of ir somatotrophs and content of irGH of sheep fed on R diet. The R diet as well as NPY infusions caused an increase in GH mean concentrations in the blood plasma. Obtained results suggest that stimulatory effect of restricted feeding and/or NPY action on GH secretion can be due to attenuated SS output. Since dietary restrictions and exogenous NPY have similar influence on the activation of GH secretion, we suggest that NPY could be a neuromodulatory link between nutritional cues and somatotropic axis in sheep.

The effect of corticotropin-releasing factor (CRF) on the gonadotropin hormone releasing hormone (GnRH) hypothalamic neuronal system during preovulatory period in the ewe.

Effects of infusions of corticotropin releasing factor (CRF) into the 3rd ventricle of the brain of ewes during the proestrus on the immunoreactive (ir) gonadotropin hormone releasing hormone (GnRH) neuronal system, pituitary luteinizing hormone(LH) producing cells and LH concentrations in the blood plasma were studied. None of the CRF-treated sheep displayed the estrous activity nor ovulated on the day of estrus (17th day of the cycle), and two days later when they were slaughtered. The GnRH center of CRF treated ewes situated in the preoptico-septal area was well organized, but irGnRH stores in the median eminence were low in comparison to the controls (sheep from the late follicular phase of the estrous cycle). The feature and the number of LH-cells in CRF-treated ewes were typical for the preovulatory phase of the cycle but the plasma concentrations of LH did not exceed basal levels. These results suggest that CRF induced decrease of irGnRH stores in the nerve terminals of the ME can be responsible for the blockade of the preovulatory surge of GnRH/LH in the sheep.

The long-term effect of low protein diet on the somatostatin hypothalamic neuronal system and the pituitary growth hormone cells in growing ewe.

Three-month old Polish Lowland female lambs were fed isocaloric diets containing 14.2% (standard) or 8.1% of proteins for twenty weeks. Changes in body weight were characteristic for normal growth performance in all animals, but daily body gain calculated for the whole experimental period was significantly lower in lambs fed a low protein diet (87.9 +/- 13.5 and 158 +/- 13.8 g/day, respectively). Two series of blood collections (4 hrs with 15 min interval) were performed at age of 21 and 26 weeks in order to analyze the growth hormone (GH) concentration in the peripheral blood. The results obtained by radioimmunoassay showed that at both ages the mean concentration of GH was significantly higher in the group fed a low protein diet (4.84 +/- 2.23 and 3.68 +/- 1.86 vs 1.46 +/- 0.72 and 1.48 +/- 0.44 ng/ml, respectively) and this difference was associated with significant elevation of the pulse amplitude (3.83 +/- 4.23 and 4.54 +/- 3.06 vs 1.48 +/- 1.11 and 1.31 +/- 0.68 ng/ml, respectively). Using immunocytochemistry, the somatostatin in the hypothalamus and the GH in the pituitary cells were analyzed in all animals slaughtered at age of 8 months. Lowering the content of dietary proteins diminished markedly the content of immunoreactive somatostatin in the median eminence (ME) and augmented the concentrations of the immunoproduct in the somatostatin perikarya. In the pituitary gland, a marked increase of the number of GH-producing cells was observed. The results obtained indicate that chronic restriction of dietary proteins, irrespective of sufficient energy supply, augment the secretion of GH via a decrease in the hypothalamic somatostatin output due to the suppression of its axonal transport.

Stress and nutritional influences on GnRH and somatostatin neuronal systems in the ewe.

The effect of stressful stimulation and protein malnutrition on the gonadotrophic and somatotrophic axis of sheep is discussed with special references to the relationship between these stimuli and the GnRH and somatostatin neuronal systems in the hypothalamus. Generally, long-term stimulation and chronic underfeeding reduce gonadal functions in the sheep. There is evidence for the GnRH-dependent pathway for the mechanism of these phenomena in female sheep. GnRH neurons respond to long-term stress in diminishing of neuropeptide release from the nerve terminals due to the depression of its axonal transport. Chronic restriction of dietary proteins in lambs reduces the plasma LH concentrations but does not impair the development of GnRH neurons nor the synthesis and processing of GnRH. It is suggested that malnutrition delays the first ovulation probably due to the neural mechanism responsible for the preovulatory GnRH/LH output. Stress has rather unclear effect on growth hormone (GH) secretion in the sheep. Prolonged, but not short stressful stimulation provokes the rapid release of somatostatin, which is sustained during long-term stimulation. These results suggest that effect of stress on somatotrophic axis depends on the period of stressful stimulation. Chronic malnutrition enhances secretion of GH by an increase in amplitude of GH pulses and reduces the secretory activity of somatostatin neurons. It is postulated that nutrients can influence GH secretion in the sheep by mechanism dependent on the hypothalamic somatostatin.

Development of the gonadotrophic and somatotrophic axes of sheep.

The hypothalamo-pituitary-gonadotrophic axis develops in the sheep fetus from midgestation to late gestation. The GnRH neuronal centres seem to be fully developed in the fetus and their localization complies with the adult pattern. Pituitary gonadotrophs are responsive to exogenous GnRH and release LH and FSH in a pulsatile fashion; the highest concentrations in plasma are found during late gestation. In sheep, maturational changes of this axis continue through to the prepubertal period. The GnRH neuronal system is established at about 12 weeks of age. The pattern of LH and FSH release is characteristic for each gonadotrophin depending on age and sex. The responsiveness of the gonadotrophs to GnRH increases up to 3 weeks of age. It is concluded that the changes in morphology and physiology of the hypothalamo-pituitary-gonadotrophic axis reflect the progressive maturation of the central mechanisms involved in the control of gonadotrophin secretion throughout fetal and prepubertal growth in sheep. Development of the hypothalamo-pituitary-somatotrophic axis begins in the fetus around mid-gestation. The central regulation of growth hormone (GH) in the fetus probably has a dual character, although the growth hormone releasing hormone (GHRH) neuronal system has not yet been observed in sheep. The somatostatin neuronal system develops in diverse neuronal centres in the fetus. The somatostatin centre involved in hypophysiotrophic functions does not develop fully before birth and is established over the first 10 weeks after birth. Plasma GH concentrations are very high in the fetus and fall suddenly in the perinatal period, and after a temporary increase they decline with age.(ABSTRACT TRUNCATED AT 250 WORDS)

Effect of protein deficiency on luteinizing hormone releasing hormone (LHRH), gonadotropin releasing hormone associated peptide (GAP) and luteinizing hormone (LH) immunocytochemistry in the hypothalamus and pituitary gland of prepubertal ewes.

Growing female lambs were fed diets containing 14.2% (standard) or 8.1% (protein restricted) of proteins to determine their effects on puberty and luteinizing hormone releasing hormone (LHRH), gonadotropin hormone associated peptide (GAP), luteinizing hormone (LH) hormonal system. At the end of the experiment (30-34 weeks of age), hypothalamic LHRH, GAP and pituitary LH were analysed by immunocytochemical methods using specific antibodies. Plasma LH were determined by radioimmunoassay at 21 weeks of age. It was found that lowering of the dietary proteins content decreased the concentration of basal plasma LH significantly in lambs of 21 weeks of age. None of the sheep of this group reached sexual maturity at the same time as the animals of the standard group. However, immunoreactive (ir) LHRH neuronal system of protein restricted lambs was normally developed: Numerous irLHRH perikarya, dense network of axons and abundant material stored in the nerve terminals were well visualized in the typical sites of the preoptico-septal area, hypothalamus and the median eminence (ME). Gonadotropin associated peptide (GAP) of the LHRH precursor was present in the same populations of neurons that contained LHRH in the sheep brain. The proportion of pituitary LH-cells was three fold higher in pituitaries of the nutritionally restricted group. They displayed hypertrophy and very strong immunoreaction. These results show that protein deficiency in diets of growing female sheep delays their puberty but does not impair the synthesis and processing of LHRH in the brain neurons and synthesis of LH in pituitary cells.(ABSTRACT TRUNCATED AT 250 WORDS)

Bihormonal cells producing gonadotropins and prolactin in a rat pituitary tumor cell line (RC-4B/C).

Using single hormone-staining immunocytochemical methods, we have recently characterized a novel cell line, RC-4B/C, established from an aged male rat pituitary adenoma. This cell line contained all known anterior pituitary cell types including gonadotropes. Gonadotropin-releasing hormone receptors were also present. A recent re-examination of the cell types using single hormone stains showed that the cell line underwent an alteration in the percentage of different cell types as compared to the data obtained 2 years ago. The proportion of follicle-stimulating hormone-beta (FSH beta), luteinizing hormone-beta (LH beta), prolactin (PRL), and adrenocorticotropic hormone cells increased significantly (p less than or equal to 0.001), while the proportion of growth hormone (GH), and thyrotropin-beta cells did not change. Dual staining of the monolayers showed that the cell line contained many bihormonal cells producing FSH beta + LH beta, FSH beta + PRL and LH beta + PRL. The proportion of bihormonal FSH beta + LH beta and FSH beta + PRL cells was preponderant over monohormonal cells, while the proportion of bihormonal LH beta + PRL cells was in the same range as that of monohormonal cells. Preliminary data with dual labeling also revealed the presence of GH and PRL in the same cell, but complete absence of a combination such as FSH beta + GH. No search for the presence of other bihormonal or multihormonal cells was performed. In short, our data show that the majority of the cells in the cell line RC-4B/C contain FSH beta, LH beta and PRL and that among these cells many bihormonal cells are present.

Gonadotropes in a novel rat pituitary tumor cell line, RC-4B/C. Establishment and partial characterization of the cell line.

An epithelial cell line (RC-4B/C) was established from a pituitary adenoma obtained from a 3-yr-old (ACI/fMai X F344/fMai)F1 male rat. Before Year 5 in vitro, RC-4B/C cells could not be viably recovered from cryogenic storage. Recovery of viable cells from cryogenic storage in Year 5 was associated with a more transformed phenotype, including the appearance of endogenous C-type rat retroviral particles. The ultrastructural appearance of the cells was similar to that of differentiated anterior pituitary cells; the cultured cells contained numerous, electron dense, secretory granules, Golgi complexes, and extended arrays of rough endoplasmic reticulum. Immunocytochemical study showed that all cell types present in the rat anterior pituitary gland were present in the cell line. The percentage of luteinizing hormone beta (LH beta) cells in the cell line was higher (19.9%) and that of growth hormone cells was lower (12.2%) than in normal male rat pituitary, whereas the cell line contained a comparable percentage of follicle stimulating hormone beta (FSH beta), prolactin (PRL), ACTH, and thyrotropin beta cells. Radioimmunoassay data demonstrated the PRL content of the cells was comparable to that of normal male rat pituitary gland, whereas the content of LH and FSH was 70- and 800-fold lower, respectively. Assay of specific receptor sites for gonadotropin releasing hormone (GnRH) using Scatchard plots of the data established the RC-4B/C cells contained GnRH receptor sites of the same affinity as in the pituitary gland, but of twofold lower capacity. These data suggest the RC-4B/C cell line warrants further study as a model for the induction and maintenance of the gonadotropic function of the pituitary gland.

[Characterization of gonadotropic cells in a new pituitary tumor cell line]. / Caractérisation de cellules gonadotropes dans une nouvelle lignée tumorale hypophysaire.

The pituitary tumor cell line RC-4B/C was established in The Jackson Laboratory from an aged rat pituitary adenoma. Immunocytochemical studies of this cell line showed that all pituitary cell types were present. Approximately 20% reacted with antisera (AS) to ovine (o) LH beta, 8.6% with AS to oFSH beta, 15% with AS to rat PRL, 12% with AS to equine GH, 9% with AS to porcine TSH beta and 8.6% with AS to ACTH1-24. Using NIDDK rat kits, RIA showed about 0.38, 0.08 and 607.50 ng per 10(6) cells of LH, FSH and PRL, respectively, vs 33.9, 75.6 and 573 ng in freshly dispersed rat pituitary cells. The GnRH receptor content of the cell line was about a half that of normal rat pituitary cells but the receptor affinity was the same. A chronic treatment of the cells for about 5 months with a "sub-physiological" concentration (3.7 pM) of a GnRH agonist had 3 major effects: 1) as compared to the controls, a 3-fold increase in the cell number in the log phase; 2) an increase of the percentage of FSH beta cells from 8.6 to 21.9% whereas LH beta cells and the cell content of LH and FSH remained stationary; 3) a decrease of the percentage of PRL cells from 15 to 6.5% and an almost 250-fold decrease of PRL cell content. Incorporation studies with [35S] Met demonstrated that the alpha subunit in the cell line was only partly glycosylated. Pretreatment of the cells with 5 nM estradiol restored, at least partly, glycosylation of alpha.(ABSTRACT TRUNCATED AT 250 WORDS)

Effect of protein deficiency on some hypothalamic and pituitary hormones in growing male lambs. An immunohistochemical study.

Growing male lambs were fed with diets containing 14.0, 10.8 and 7.6% protein for 3 months to determine their effects on the content of hypothalamic LHRH and SRIH and pituitary LH and GH, using immunohistochemical methods. Lowering the concentration of dietary proteins caused marked changes in the immunoreactivity of these hormones. The immunoreactive (IR) content of LHRH stored in the median eminence was enhanced, and the proportion of LH cells and their IR content increased. Opposite effects were observed in the SRIH/GH system; IR SRIH content stored in the median eminence markedly diminished, and, although hyperplasia of GH cells was observed, their IR content was diminished. This study indicates that prolonged restrictions of protein in the diet of growing male sheep affects the immunoreactive content of the investigated hormones. It seems that they suppress LHRH/LH release and augment GH release, possibly via suppression of hypothalamic somatostatin.

The effect of prolonged stress on the hypothalamic luteinizing hormone-releasing hormone (LHRH) in the anoestrous ewe.

A study was performed to examine the effect of the intermittent long-term (20 min/h daily during 3 consecutive days) electric stress (footshocks) on the hypothalamic LHRH in anoestrous ewe. On the third day, immediately after the last stimulation, the animals were decapitated and the hypothalami and pituitaries were taken for analysis of LHRH and LH by immunocytochemistry; LHRH was also assayed by radioimmunoassay. In the stressed animals the concentration of immunoreactive LHRH (IR-LHRH) and LHRH analysed by radioimmunoassay (RI-LHRH) increased significantly in the median eminence (ME) and the medial preoptic area (MPOA) when compared with the controls. Additionally, IR-LHRH perikarya appeared in the MPOA of stressed animals, whereas they were not seen in the controls. RI-LHRH in the medial basal hypothalamus (MBH) of stressed ewes was markedly lower than in control ones. No significant differences in RI-LHRH concentration were found in the septum between stressed and control ewes. The present study clearly indicates that prolonged stressful stimuli modify LHRH concentration in the discrete areas of the hypothalamus of the anoestrous ewe. It is suggested that these changes are attributed mainly to an inhibition of LHRH release from the ME and a suppression of LHRH transport along neural fibres within the hypothalamus.

Immunocytochemical changes in hypothalamic and pituitary hormones after acute and prolonged stressful stimuli in the anestrous ewe.

We studied the effect of short (acute (20 min/h, for 4 h) and intermittent, long-term (20 min/h for 9 h on 3 consecutive days) electric foot shocks on the immunocytochemical localization of CRH and SRIH in the hypothalamus and of ACTH, beta-endorphin, GH and PRL in the pituitary of the anestrous ewe. Acute stress greatly reduced immunoreactive (ir) CRH in the median eminence and cellular irACTH, beta-endorphin and PRL, as well as the proportion of these cell types in the pituitary. A slight reduction of irSRIH in the median eminence was also observed. After long-term stress, reduction of irCRH in the median eminence was still observed. However, ACTH/beta-endorphin cells in the pituitary gland displayed increased secretory activity, manifested by hypertrophy and hyperplasia. A marked depletion of irSRIH in the nerve terminals of the median eminence was observed. The proportion of PRL cells but not their ir content returned to control levels. No effects were observed on the features of the GH cells. This study indicates that there are differences in the effect of short- and long-term stressful stimuli on the activity of hormonal systems in the anestrous ewe. Short-term stress immediately activates the CRH/ACTH/beta-endorphin axis. Prolonged stress appears to augment the activation of the SRIH hypothalamic system and probably has a restraining effect on ACTH/beta-endorphin release.

Maturation of luteinizing hormone-releasing hormone (LHRH) and somatostatin (SRIF) neuronal systems in the hypothalamus of growing ewe lambs.

Using the immunoperoxidase method, neurons containing luteinizing hormone-releasing hormone (LHRH) and somatostatin (SRIF) were identified in the hypothalamus of growing ewe lambs between 3 days and 16 weeks of age. Both peptidergic neuronal systems were well developed in the early postnatal period. The centres producing LHRH were found to be situated in the anterior region of the hypothalamus and preoptic area. Immunoreactive (ir) LHRH perikarya in growing lambs were readily stained by the various anti-LHRH antibodies used and they were generally more numerous in young animals than in foetuses and adults. The concentration of irLHRH material stored in the median eminence (ME) increased with lamb age, reaching a maximum around 12 weeks of age. The centre producing somatostatin, situated in the anterior periventricular area of the hypothalamus, did not develop before the postpartum period. The irSRIF material in the ME was greatly depleted during the first neonatal days but regained its former level by 8 weeks of age. From 10 to 16 weeks of age, these stores in the ME were again depleted. The results show that the two neuronal systems investigated developed in a different way in the growing lamb. The secretory activity of these systems changed during subsequent periods of growth. The importance of these events in the processes of body growth and reproductive system maturation are still to be determined.