Obestatin may affect the GnRH/KNDy gene network in sheep hypothalamus.

The effects of obestatin on gonadotrophic axis activity in ruminants have not yet been determined. The aim of this study was to investigate the effect of intracerebroventricular infusions of obestatin on the gonadotrophin-releasing hormone (GnRH) mRNA and protein expressions as well as on KNDy mRNA and kisspeptin (Kiss) peptide expressions in peripubertal female sheep. Animals were randomly divided into two groups: the control group received intracerebroventricular infusions of the vehicle, and the obestatin group was infused with obestatin (25 µg/120 µL h-1). The series of four 1-h infusions per day during three consecutive days were performed. After the end of the experiment parts of sheep brains were fixed in situ for immunohistochemical analysis, while the remaining brains were frozen for Real Time qPCR analysis. Substantial changes in the activity of the GnRH and KNDy gene network were observed in obestatin-infused sheep. In those animals an increase of GnRH mRNA expression in the preoptic area, a decrease of GnRH mRNA expression in the median eminence and an increase of GnRH immunoreactivity in the median eminence were found. Moreover, changes in the KNDy mRNA expression in mediobasal hypothalamus as well as decrease Kiss expression in arcuate nucleus and median eminence were observed. It was revealed that obestatin affects the GnRH and KNDy gene network as well as Kiss at the level of mRNA and protein expression. Thereby, it can be concluded that obestatin participates in the mechanism modulating gonadotrophic axis activity at the central level in peripubertal female sheep.

Expression of kisspeptin protein in hypothalamus and LH profile of growing female lambs.

Kisspeptin (kp) is considered to be one of the major regulators of the induction of pubertal events via the activation of the gonadotrophin-releasing hormone-LH system. The aim of the present study was to analyse expression of immunoreactive (ir) kp in the hypothalamic neurons of female lambs from the neonatal to the peripubertal period (5 days to 32 weeks) in relation to the plasma LH pattern using immunohistochemistry and image analysis. Hypothalami were collected from female lambs (n=33) from the infantile, juvenile, prepubertal and peripubertal periods. The population of kp-ir perikarya was detected mainly in the arcuate nucleus and their number increased gradually from 5 to 16 weeks of age and was maintained at a high level up to the peripubertal stage. This was reflected by the significant (P<0.05) gradual increase in the percentage of hypothalamic area occupied by kp-ir neurons and increase in the number of kp-ir perikarya within the arcuate nucleus. The same pattern of kp immunoreactivity was observed in the median eminence. Plasma LH concentration increased from Week 5 to Weeks 12-16 and further increased at Week 32. LH pulse frequency increased from Week 5 to 32 (P<0.05). Thus, changes in kp expression reflected changes in the LH pattern during lamb growth. The data obtained provide evidence about the participation of kp in the mechanisms of ontogenic development of ovine reproductive processes.

Obestatin stimulates the somatotrophic axis activity in sheep.

The effects of obestatin (an anorexigenic peripheral peptide) on somatotrophic axis activity in ruminants have not yet been determined. The aim of this study was to investigate the consequence of intracerebroventricular infusions of obestatin on the activity of the somatotrophic axis in peripubertal female sheep. Animals were randomly divided into two groups: control group received intracerebroventricular infusions of the vehicle, and the obestatin group was infused with obestatin (25 µg/120 µL h-1). The series of four hourly infusions on three consecutive days were performed. The blood samples were collected on day 0 and on day 3. Immediately after the end of experiment sheep were slaughtered. Parts of the brains were fixed in situ for further immunohistochemical analysis, while the remaining brains were frozen for Real Time RT-qPCR analysis. Substantial changes in the activity of the somatotrophic axis were observed in obestatin-infused sheep. In those animals obestatin evoked an increase in growth hormone-releasing hormone (GHRH) mRNA expression and a decrease in somatostatin mRNA expression in the anterior hypothalamic area. Moreover, a decrease in somatostatin immunoreactivity in the periventricular nucleus and an increase in somatostatin immunopositive fibers in the median eminence were noted. Changes in the GHRH and somatostatin activity are associated with an increase in growth hormone (GH) gene expression and in the amount of GH immunoreactive material stored in the somatotrophic pituitary cells. Consequently, an increase in GH concentration in the peripheral blood, due to an increase in the number of pulses was observed. It was revealed that obestatin affects the somatostatin/GHRH/GH system at the level of protein synthesis, accumulation and release. It is suggested that obestatin participates in the mechanism modulating somatotrophic axis activity at the central level by stimulating GH release through suppression of somatostatin output. Thereby, it can be concluded that obestatin may be involved in the modulation of growth processes in sheep.

Effects of intracerebroventricular infusions of ghrelin on secretion of follicle-stimulating hormone in peripubertal female sheep.

Reproduction depends on mechanisms responsible for the regulation of energy homeostasis and puberty is a developmental period when reproductive and somatic maturity are achieved. Ghrelin affects the activity of the hypothalamo-pituitary-gonadal axis under conditions of energy insufficiency. An in vivo model based on intracerebroventricular (i.c.v.) infusions was used to determine whether centrally administered acyl ghrelin affects transcriptional and translational activity of FSH in peripubertal lambs and whether ghrelin administration mimics the effects of short-term fasting. Standard-fed lambs received either Ringer-Lock (R-L) solution (120µL h-1) or ghrelin (120µL h-1, 100µg day-1). Animals experiencing a short-term (72h) fast were treated only with R-L solution. In each experimental group, i.c.v. infusions occurred for 3 consecutive days. Immunohistochemistry, in situ hybridisation and real-time reverse transcription quantitative polymerase chain reaction analyses revealed that short-term fasting, as well as exogenous acyl ghrelin administration to standard-fed peripubertal lambs, augmented FSHß mRNA expression and immunoreactive FSH accumulation. In addition to the effects of ghrelin on FSH synthesis in standard-fed animals, effects on gonadotrophin release were also observed. Acyl ghrelin increased the pulse amplitude for gonadotrophin release, which resulted in an elevation in mean serum FSH concentrations. In conclusion, the present data suggest that ghrelin participates in an endocrine network that modulates gonadotrophic activity in peripubertal female sheep.

The effect of short fasting on the hypothalamic neuronal system of kisspeptin in peripubertal female lambs.

Changes in the metabolic state induced by feed restrictions have a negative effect on the reproduction in mammals and result in the delayed puberty onset. Kisspeptin (kp) has been demonstrated as a pivotal regulator of GnRH/LH secretion during puberty. To elucidate the involvement of kp in the hypothalamic secretory function in altered metabolic state, the expression of kp protein was investigated in peripubertal female lambs after short fasting. The experiment was conducted on immature 32-weeks old Merino lambs fed standard diet (n=5) or fasted for 72h (n=5). The localization and expression of kp was evaluated using immunohistochemistry. Serum LH concentration was determined using radioimmunology. In the hypothalami of fasted sheep, the number of kp perikarya and the percent of density of neuronal kp network in the caudal part of the nucleus arcuatus were significantly less (P<0.001) than in standard fed lambs. The decrease of kp axons throughout areas extending from area preoptica to medial basal hypothalamus and in the median eminence in fasted lambs compared to standard fed ones was observed. Plasma LH concentrations and amplitude of pulses decreased (P<0.05) after 3 days of fasting compared to standard fed group. The decrease of the kp expression is likely due to diminished kp protein synthesis, and its storage in the neurons. In summary, the data are the first to demonstrate interactions between metabolic status and kp neuronal system in lambs before puberty, and suggest that kp neurons may represent a link between metabolic signals and central control of reproduction.

The effect of intracerebroventricular infusions of ghrelin or short fasting on the gene expression and immunoreactivity of neuropeptide Y in the hypothalamic neurons in prepubertal female lambs: a morphofunctional study.

The role of exogenous ghrelin in the regulation of neuropeptide Y (NPY) neuronal system in the hypothalamus of intact lambs has not been yet determined. The aim of present study was to investigate the effects of intracerebroventricular infusion of ghrelin or short fasting on the secretory activity of the NPY neurons in the hypothalamus of prepubertal female sheep. Animals (n=30) were randomly divided into three groups, two groups were fed standard diet and one group was fasted for 72h. One group fed standard diet and fasted group were infused to the 3rd ventricle of the brain with vehicle, while the remaining group fed standard diet was infused with ghrelin (25µg/120µl/h) for 6h during three consecutive days. Immediately after the treatment, tissues were collected. Parts of the brains were fixed in situ for further immunohistochemical analysis, and remaining parts were frozen for RT-PCR analysis. Both, fasting and ghrelin infusion elicited the same kind of changes in the mRNA and intra-neuronal levels of the NPY hypothalamic neurons. Namely, the expression of NPY mRNA in the medial basal hypothalamus and immunoreactivity of NPY in the arcuate and periventricular nuclei increased in fasted and standard fed with ghrelin's infusion groups compared to standard fed sheep (P<0.05). These data demonstrate that ghrelin takes part in the mechanisms linking the nutritional status with an activity of the hypothalamic NPY at the level of the central nervous system by stimulating NPY secretion in sheep.

The influence of ovarian factors on the somatostatin-growth hormone system during the postnatal growth and sexual development in lambs.

The aim of the study was to elucidate the effects of ovarian hormones on somatostatin in the hypothalamic neurons and growth hormone (GH) secretion during the postnatal growth and development of sheep. The study was performed on 9-week-old (infantile) lambs that were ovary-intact (OVI) or ovariectomized (OVX) at 39 days of age, and on 16-week-old (juvenile) lambs that were OVI or OVX at 88 days of age. Hormones in neurons and somatotropic cells were assayed with immunohistochemistry and radioimmunoassay. Following ovariectomy, immunoreactive somatostatin was more abundant (p<0.05) in the hypothalamus of infantile lambs, whereas in juvenile lambs it was more abundant (p<0.05) in the periventricular nucleus but reduced (p<0.01) in the median eminence. In contrast to somatostatin in the hypothalamus, the content of immunoreactive GH in the hypophysis was less in OVX infantile lambs, but greater in OVX juvenile lambs (p<0.05). Basal blood serum concentrations of GH were greater (p<0.05) in OVX infantile lambs, whereas in OVX juvenile lambs, mean and basal concentrations of GH and amplitude of GH pulses were less than in OVI lambs (p<0.05). The postnatal increase in body weight was greatest in middle-late infancy (p<0.01). The body weight did not differ (p>0.05) between OVI and OVX lambs. In conclusion, ovarian factors may inhibit the GH secretion in infantile lambs but enhance the GH secretion in juvenile lambs. Transition to puberty, as related to the growth rate, appears to be due mainly to change in gonadal influence on the somatostatin neurosecretion. A stimulation of somatostatin output in the median eminence by gonadal factors in infancy is followed by a stimulation of somatostatin accumulation after infancy. Thus, ovarian factors modulate mechanisms within the somatotropic system of lambs to synchronize the somatic growth with sexual development.

Isolated ß-turn model systems investigated by combined IR/UV spectroscopy.

The functionality of bioactive molecules sensitively depends on their structure. For the investigation of intrinsic structural properties, molecular beam experiments combined with laser spectroscopy have proven to be a suitable tool. Herein we present an analysis of the two isolated tripeptide model systems Ac-Phe-Tyr(Me)-NHMe and Boc-Phe-Tyr(Me)-NHMe. For this purpose, mass-selective combined IR/UV spectroscopy is applied to both substances in a molecular beam experiment. The comparison of the experimental data with DFT calculations, including different functionals as well as dispersion corrections, allows an assignment of both tripeptide models to ß-turns formed independently from the protection groups and supported by the interaction of the two aromatic chromophores.

The effect of intracerebroventricular infusions of ghrelin and/or short fasting on the gene expression and immunoreactivity of somatostatin in the hypothalamic neurons and on pituitary growth hormone in prepubertal female lambs. Morphological arguments.

The effect of exogenous ghrelin on somatostatin distribution in the ruminant's hypothalamus has not been yet determined. The aim of the present study was to investigate the consequence of central infusion of ghrelin and/or short fasting on the secretory activity of the somatostatin/GH system in prepubertal female sheep. Animals were randomly divided into three groups, two standard fed and one fasted for 72 h. One standard group and one fasted group were infused icv with vehicle, while the remaining standard group was infused with ghrelin (25 µl/120 µl/h). Infusions were performed for 6 h during three consecutive days; blood samples were collected during the "day 0" (before the infusion) and "day 3" Immediately after the experiment the sheep were slaughtered. Parts of the brains were fixed in situ for further immunohistochemical analysis The remaining brains were frozen for RT-PCR analysis. Fasting and ghrelin infusion elicited the same kind of changes in the secretory activity of the somatostatin/GH system compared to standard fed sheep. The expression of somatostatin mRNA and ir somatostatin in the PEV nucleus and ir stores in the median eminence increased in both these groups compared to standard fed sheep (P<0.001). The population of ir GH pituitary cells decreased (P<0.001), the mean GH plasma concentrations increased in all fasted and ghrelin infused animals between day 0 and day 3 of infusions (P<0.05) compared to the standard fed group. It can be suggested that ghrelin takes part in the mechanisms linking the nutritional status of an organism with an activity of the somatotrophic axis on the level of the CNS by stimulating GH release through suppression of the somatostatin output.

Influence of gonadal hormones on endocrine activity of gonadotroph cells in the adenohypophysis of male lambs during the postnatal transition to puberty.

Using histomorphological and functional criteria we describe the feedback mechanisms which could play a role in the regulation of the gonadotrophic axis during the postnatal transition to puberty in male lambs. The working hypothesis was that the testicular factors change the peripheral levels of luteinizing hormone (LH) and follicle-stimulating hormone (FSH) by influencing the synthesis rate and storage of LH and FSH in adenohypophyseal gonadotroph cells of weanling and weaned pubertal lambs. The examination was made in (i) 9-week-old infantiles, suckling lambs undergoing weaning, testis-intact (TEI) and orchidectomised (ORCHX) at the 6th week of age, and (ii) 16-week-old pubertal lambs TEI and ORCHX at the 12th week of age (n=5 per group). Changes in gonadotrophs were assayed with hybridohistochemistry, immunohistochemistry and radioimmunoassay. The percentage of the adenohypophyseal area (PA) occupied by cells containing LHß-mRNA and FSHß-mRNA and peripheral levels of both gonadotrophins were lower (P<0.01) in the 16-week-old TEI lambs in comparison with the 9-week-old ones. The PA occupied by cells immunoreactive for LHß was lower (P<0.01), whereas in the case of FSH was greater (P<0.001) in the 16-week-old lambs. After orchidectomy the PA occupied by gonadotrophs stained for LHß-mRNA was greater (P<0.01) in 16-week-old lambs. The PA occupied by LHß-labelled cells was lower (P<0.05) in the 9-week-old ORCHX lambs, whereas in 16-week-old ones was higher (P<0.05) in comparison with the TEI lambs. The circulating LH was greater (P<0.01) in the ORCHX 9- and 16-week-old lambs compared to the TEI ones. The PA occupied by cells containing FSHß-mRNA and the plasma FSH concentration were greater (P<0.001) after orchidectomy in lambs from both age stages. The PA occupied by FSHß-labelled cells was greater (P<0.01) in the 9-week-old ORCHX lambs, whereas in 16-week-old ones was lower (P<0.05) compared to the lambs from TEI groups. In conclusion, in infantile lambs testicular factors may play inhibitory role in regulating FSH synthesis rate, storage and release in contrast to the stimulatory role in regulating LH storage reflected by the inhibitory role in regulating LH release. In lambs at the beginning of puberty, testicular factors may play inhibitory role in regulating LH synthesis rate, storage and release in contrast to the stimulatory role in regulating FSH storage reflected by the inhibitory role in regulating FSH synthesis rate and release. The effects of testicular hormones on the gonadotrophin storage, i.e. releasable pools in adenohypophyseal cells, are specific for both LH and FSH in lambs during the postnatal transition to puberty. Thus, the initiation of puberty in male sheep is a function of change of the inhibitory role of gonadal factors in regulating FSH storage to the stimulatory one and the stimulatory role of gonadal factors in regulating LH storage to the inhibitory one.

Effects of maternal deprivation on the somatotrophic axis and neuropeptide Y in the hypothalamus and pituitary in female lambs. The histomorphometric study.

The effects of maternal deprivation on the somatotrophic axis and neuropeptide Y (NPY) neuronal system in the hypothalamus of female lambs were evaluated. Twelve-week-old lambs were divided into two groups: the control (lambs stayed with mothers) and maternally deprived (MD; lambs separated for 3 days from mothers). The expression of immunoreactive (ir) somatostatin in the neurons of the periventricular nucleus (PEV) and in nerve terminals of the median eminence (ME), growth hormone (GH) in the adenohypophyseal cells and NPY in the neurons of the PEV and arcuate (ARC) nuclei of the hypothalamus using immunohistochemistry followed by the image analysis were estimated. Concentrations of GH in the blood plasma were determined by radioimmunoassay. The expression of ir somatostatin in the PEV and ME, ir NPY in the ARC and PEV, ir GH in adenohypophyseal cells, and blood plasma GH concentrations were greater (p<0.05) in MD than in the control lambs. In conclusion, MD affects the somatotrophic axis by enhancement of GH secretion via restraining of somatostatin output. The simultaneous increase of expression of hypothalamic ir NPY suggests NPY involvement in the regulation of psychoemotional stress through the somatotrophic axis in the female lambs.

Hypothalamic arcuate neuropeptide Y-neurons decrease periventricular somatostatin-neuronal activity before puberty in the female lamb: morphological arguments.

It is assumed that hypothalamic somatostatin plays a dominant role in the regulation of growth of developing lambs. On the other side, neuropeptide Y (NPY) neurons of the arcuate (ARC) nucleus are potentially involved in the control of gonadotrophins in prepubertal lambs and also of growth hormone (GH) secretion in adults. This study therefore investigated whether the transition from the prepubertal to the peripubertal period is accompanied by changes in NPY-ir and NPY mRNA content in neurons of the ARC nucleus and their putative projections to somatostatin neurons in both the ARC and periventricular (PEV) nuclei. The hypothalami of prepubertal (17-week-old) and peripubertal (32-week-old) female lambs were compared using single and double-labelling immunohistochemistry, and hybridisation in situ for NPY. Single-labelling for NPY mRNA and NPY-ir was quantified by image analysis using a light microscope and expressed as the percent area stained and/or the integral density of the reaction. Double-labelling for NPY-somatostatin relationships was analysed by confocal microscopy. Our data suggest that there are no detectable changes in NPY-ir in the PEV nucleus in the period leading up to puberty, whereas both the distributional area and intensity of NPY-labelling in the ARC are significantly higher in peripubertal compared to prepubertal sheep. In contrast, NPY mRNA levels are higher in prepubertal than in peripubertal ewes in the ARC nucleus. Confocal microscopy suggests the existence of NPY-somatostatin axo-somatic contacts in both PEV and ARC nuclei. In the PEV nucleus, the number of close appositions between NPY-ir fibres and somatostatin-ir perikarya is higher in prepubertal than in peripubertal ewes, but in the ARC no such difference was observed. In conclusion, our observations suggest that there is decreased activity of the NPY neurons of the ARC nucleus closely related to somatostatin neurons in the PEV nucleus at the onset of puberty. The withdrawal of this NPY effect may allow a higher release of somatostatin, which consequently inhibits GH secretion and stops growth. Both peptides are involved in the transmission of signals leading to stop growth at puberty.

Influence of ovarian hormones on endocrine activity of gonadotroph cells in the adenohypophysis of lambs during the postnatal transition to prepuberty.

There is juvenile hiatus during maturation of larger mammals with relatively long life spans. Using histomorphological and functional criteria we describe the feedback mechanisms which could play a role in the regulation of the gonadotrophic axis during the postnatal transition to the quiescent prepubertal period in sheep. The aim of this study was to determine the influence of ovarian factors on the endocrine activity of gonadotroph cells, the site of synthesis, storage and release of luteinizing hormone (LH) and follicle-stimulating hormone (FSH), in adenohypophyses of weanling and weaned prepubertal lambs. The examination was made in (i) 9-week-old infantiles, suckling lambs undergoing weaning, ovary-intact (OVI) and ovariectomised (OVX) at the 6th week of age, and (ii) 16-week-old juveniles OVI and OVX at the 12th week of age (n=5 per group). Changes in gonadotrophs were assayed with hybridohistochemistry, immunohistochemistry and radioimmunoassay. The percentage of the adenohypophyseal area (PA) occupied by gonadotrophs containing LHbeta-mRNA and immunoreactive for LHbeta was lower (P<0.05), whereas the PA occupied by cells containing FSHbeta-mRNA and immunoreactive for FSHbeta was higher (P<0.05) in the 16-week-old OVI lambs in comparison with the 9-week-old ones. The mean concentration and basal level of LH in the peripheral blood plasma were greater (P<0.05) in the 16-week-old OVI lambs in comparison with the 9-week-old group, whereas the circulating FSH was not different. In the OVX 9-week-old lambs, the PA occupied by gonadotrophs containing LHbeta-mRNA and the plasma LH concentration, basal level, pulse frequency and amplitude were greater (P<0.05), whereas the PA occupied by cells immunoreactive for LHbeta was lower (P<0.05) in comparison with the OVI group. In the OVX 16-week-old lambs, the PA occupied by gonadotrophs containing LHbeta-mRNA and immunoreactive for LHbeta, the LH plasma concentration, basal level and pulse frequency were (P<0.05) greater in comparison with the OVI group. The PA occupied by gonadotrophs containing FSHbeta-mRNA and the plasma FSH concentration were greater (P<0.05) in the OVX 9- and 16-week-old lambs in comparison with the OVI ones. The ovariectomy had no effect on the PA occupied by cells immunoreactive for FSHbeta in both age stages. In conclusion, ovarian factors may play inhibitory role in regulating both LH and FSH synthesis rate and release and stimulatory role in regulating LH storage in adenohypophyseal gonadotrophs in infantile lambs. In lambs at the beginning of the juvenile period, ovarian factors may play only inhibitory role in regulating both LH and FSH synthesis and release and LH storage. The effects of ovarian hormones on the gonadotrophin storage, i.e. releasable pools in adenohypophyseal cells, are specific for LH, no such effects are apparent on FSH in lambs during the postnatal transition to prepuberty. Thus, the initiation of the juvenile period in female sheep is a function of change of the stimulatory role of ovarian hormones in regulating LH storage to the inhibitory one.

Effect of maternal deprivation on the gonadotrophin-releasing hormone (GnRH) and GnRH-associated peptide neurobiology in lambs during the transition from infancy to prepuberty.

Using morphological criteria we describe the effect of maternal deprivation on the gonadotrophin-releasing hormone (GnRH) and GnRH-associated peptide (GAP) of the GnRH prohormone (proGnRH) in the preoptic area (POA)-hypothalamus during the weaning period. The immunohistochemical GnRH- and GAP-neuroanatomy was investigated in female 12-week-old weanling and maternally deprived lambs and 15-week-old weaned lambs. The GnRH-immunoreactive (ir) nerve elements in the POA were more numerous in weanling and weaned lambs in comparison with maternally deprived lambs, whereas the nerve elements ir for GAP were numerous in weanlings and scarce in remaining lambs. In the hypothalamus, GnRH-ir fibers were more numerous in weaned lambs in comparison with others. Immunoreactive GnRH in the median eminence was scarce in weanlings and comparable greater in maternally deprived and weaned lambs. In contrast to ir GnRH, the GAP-ir fibers and nerve terminals in the hypothalamus and median eminence were numerous in weanlings and maternally deprived lambs and scarce in weaned lambs. In conclusion, maternal deprivation affects the intraneuronal locations involved in the maturation of GnRH from proGnRH in the POA-hypothalamus of weanlings. The described effect involves the increase in the GnRH posttranslational processing and terminal accumulation in the median eminence, which reflects the maturational increase from the low infantile terminal storage to the high prepubertal one.

The pituitary endocrine mechanisms involved in mammalian maturation: maternal and photoperiodic influences.

This review is designed to describe some pituitary mechanisms indispensable for growth and sexual maturation during the neuroendocrine adaptation of the female mammal to the extrauterine environment. We define the phases of postnatal development on the basis of secretory patterns of hormones. The infantile period is characterized by accelerated growth, and elevated secretion of growth hormone (GH) and follicle-stimulating hormone (FSH) in contrast to the diminished secretion of luteinizing hormone (LH). The transition from infancy to prepuberty generates the attenuation of somatic growth in non-primate mammals and the beginning of sexual maturation. The mechanisms of this transition involve the effects of weaning, which is associated with a rupture of the young-mother bond and, if abrupt, results in the stress of maternal deprivation. Maternal deprivation involves the stress-like endocrine response of pituitary and influences the mechanisms underlying the secretion of GH and FSH. An acute decrease in the secretion of GH and FSH at the initiation of prepuberty and an increase in the storage and pulsatile release of LH according to progressive prepubertal stages are pituitary endocrine features of post-infantile maturation. There are two factors important for timing of puberty, the maturity of gonadotroph population manifested by the adequate size of LH-containing cell subpopulation and the circumstances of an external environment optimal for reproductive functions in adults. Thus, the intrapituitary endocrine mechanisms of maturation have a psychosomatic nature during weaning and histomorphological nature during the postinfantile transition to puberty. In seasonal breeders, the endocrine timing of puberty has a circumannual seasonal nature.

Host-guest interactions between molecular clips and multistate systems based on flavylium salts.

Flavylium salts contain the basic structure and show a pH-dependent sequence of reactions identical to natural anthocyanins, which are responsible for most of the red and blue colors of flowers and fruits. In this work we investigated the effect of the water-soluble molecular clips C1 and C2 substituted by hydrogen phosphate or sulfate groups on the stability and reactions of the flavylium salts 1-4 by the use of UV-vis absorption, fluorescence, and NMR spectroscopy as well as of the time-resolved pH jump and flash photolysis methods. Clip C1 forms highly stable host-guest complexes with the flavylium salts 1 and 2 and the quinoidal base 3A in methanol. The binding constants were determined by fluorometric titration to be log K = 4.1, 4.7, and 5.6, respectively. Large complexation-induced (1)H NMR shifts of guest signals, Delta delta(max), indicate that in the case of the flavylium salts 1 and 2 the pyrylium ring and in the case of the quinoidal base 3A the o-hydroxyquinone ring are preferentially bound inside the clip cavity. Due to the poor solubility of these host-guest complexes in water, the association constants could be only determined in highly diluted aqueous solution by UV-vis titration experiments for the complex formation of clip C1 with the flavylium salt 3AH(+) at pH = 2 and the quinoidal base 3A at pH = 5.3 to be log K = 4.9 for both complexes. Similar results were obtained for the formation of the complexes of the sulfate-substituted clip C2 with flavylium salt 4AH(+) and its quinoidal base 4A which are slightly better soluble in water (log K = 4.3 and 4.0, respectively). According to the kinetic analysis (performed by using the methods mentioned above) the thermally induced trans-cis chalcone isomerization (4Ct --> 4Cc) and the H(2)O addition to flavylium cation 4AH(+) followed by H(+) elimination leading to hemiketal 4B are both retarded in the presence of clip C2, whereas the photochemically induced trans-cis isomerization (4Ct --> 4Cc) is not affected by clip C2. The results presented here are explained with dominating hydrophobic interactions between the molecular clips and the flavylium guest molecules. The other potential interactions (ion-ion, cation-pi, pi-pi, and CH-pi), which certainly determine the structures of these host-guest complexes to a large extent, seem to be of minor importance for their stability.

Gonadotrophin-releasing hormone and GnRH-associated peptide neurobiology from the rearing period until puberty in the female sheep.

Using morphological criteria we describe the postnatal ontogeny of gonadotrophin-releasing hormone (GnRH) and GnRH-associated peptide (GAP) of the GnRH prohormone in the ovine preoptic area (POA)-hypothalamus. The immunohistochemical GnRH- and GAP-neuroanatomy was investigated in infantile (12-week-old weanlings), prepubertal (15-, 22-week-old juveniles), peripubertal (30-, 52-week-old during maturational quiescence) and pubertal (63-week-old) females born after the summer solstice, thus undergoing delayed puberty. The changes observed in the medial POA included a postinfantile increase in the number of multipolar GnRH-immunoreactive (ir) perikarya until late prepuberty (p < 0.05). Whereas in peripubertal lambs were observed both an increase in the number of GnRH-ir and GAP-ir cell clusters in the medial POA and an increase in the mediobasal hypothalamus (MBH) subpopulation of GnRH-ir and GAP-ir neurons (p < 0.05). The GnRH-ir perikarya, nerve fibers and terminals showed a progressive increase in numbers until peripuberty. Conversely, the acute postinfantile alterations of decrease and then increase in the population of GAP-ir nerve elements were observed in the POA-hypothalamus until late prepuberty (p < 0.05). The preoptico-hypothalamic ir GAP and GnRH were of comparable abundance in both peripubertal groups and scarce during puberty. In conclusion, the infantile/prepubertal transition period changes GnRH posttranslational biosynthesis and GnRH-GAP-neuroanatomy from the infantile to the pubertal pattern, which allows the inauguration of the breeding season. The postinfantile MBH distribution of GnRH-containing neurons and increase in the POA population of GnRH-GAP neurons and their somatic appositions seem important for maturation. The nature of GnRH-GAP neurobiology during inauguration of puberty seems morphotic and in female sheep is seasonal.

A mechanism of efficient G6PD inhibition by a molecular clip.

Triple duty: A synthetic molecular clip traps nicotinamide adenine dinucleotide phosphate (NADP(+); see picture) as well as occupying both the cofactor- and the substrate-binding site in glucose-6-phosphate (G6P) dehydrogenase. This combination of two inhibition mechanisms makes the clip highly effective and selective for this enzyme over other dehydrogenases.

Effect of intracerebroventricular infusion of leptin on the secretory activity of the GnRH/LH axis in fasted prepubertal lambs.

Leptin is believed to link metabolic status to reproductive processes. The aim of the present study was to investigate the effect of exogenous leptin on the secretory activity of GnRH/LH system in acutely undernourished prepubertal, female lambs. Merino lambs were randomly divided into four groups, two standard-fed and two fasted for 72 h. One standard and one fasted groups were infused intracerebroventricularly (i.c.v.) with the vehicle; the remaining standard and fasted groups were infused with leptin (25 microg/120 microl/h). Leptin was administered in series of four 1-h infusions at 30-min intervals for 3 consecutive days from 08:30 to 14:00 h. Blood samples were collected on day 0 (before infusions) and on day 3 every 10 min over a 6-h period. Immediately after the experiment, the sheep were slaughtered and brains fixed in situ. Hypothalamic and pituitary tissues were prepared for further immunohistochemical and hybridization in situ analysis. In fasted sheep, increased GnRH levels in the median eminence (P<0.001) and LH beta levels in the pituitary cells (P<0.001) plus decreased LH beta mRNA and LH pulsatility in blood plasma were observed (P<0.05). In leptin-infused fasted sheep, GnRH levels in the median eminence decreased (P<0.001), LH beta mRNA hybridization signal increased, LH beta levels decreased in the pituitary cells (P<0.001) and LH pulsatility increased (P<0.05) in the blood plasma. These results indicate that, in prepubertal sheep, the GnRH/LH axis is sensitive to the fasting signal, that influence of which can be reversed by leptin. Leptin cancels out the suppressing effect of fasting on LH secretion by augmentation of GnRH.