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.

Influence of testicular hormones on the somatostatin-GH system during the growth promoted transition to puberty in sheep.

The aim of the present study was to investigate whether the growth promoted transition to puberty in lambs involved changes in the effects of testicular hormones on somatostatin in hypothalamic neurons and GH secretion. The study was performed in infants (9-week-old) testis-intact (TEI) and orchidectomized (ORCHX) at the sixth week of age, and pubertal lambs (16-week-old) TEI and ORCHX at the 12th week of age (n = 20). In TEI lambs, the changes included a pubertal increase in immunoreactive somatostatin in the periventricular nucleus and median eminence with simultaneous neuropeptide depletion in the median eminence, and a decrease in the percentage of the hypophyseal area (PA) occupied by GH-immunoreactive cells (P < 0.05). The mean concentration of GH in the peripheral blood plasma was greater (P < 0.001) in early infancy (5 wk), because of the greater (P < 0.0001) pulse amplitude, and then uniformly low until puberty. The postnatal increase in the body weight (BW) was prominent (P < 0.01) in middle-late infancy (9-12 wk) because of the large daily live-weight gain. After orchidectomy somatostatin was abundant. This effect on nerve terminals in the median eminence was greater (P < 0.01) in infancy and lesser (P < 0.05) in puberty. Conversely, the PA occupied by GH cells was lower in the ORCHX pubertal lambs compared to TEI lambs (P < 0.05). The GH concentration and pulse characteristics were less (P < 0.05) in the infantile and pubertal ORCHX lambs compared to the TEI lambs. However, this effect was weak (P < 0.05) until middle infancy because of no influence on the GH basal concentration, and strong (P < 0.001) after late infancy. The BW did not differ (P > 0.05) between TEI and ORCHX lambs. Findings suggest activation of GH negative autofeedback loop in middle infancy. Testicular factors may play an inhibitory role in regulating somatostatin accumulation and a stimulatory role in GH secretion until puberty. The start of puberty is related to an attenuation in the stimulatory role of gonadal factors in regulating somatostatin depletion in nerve terminals associated with an intensification of the stimulatory role of gonadal factors in regulating GH secretion. From a somatic perspective of growth rate, these mechanisms do not seem to be important. Thus, testicular factors modulate mechanisms within the somatostatin-GH system to integrate somatotropic and gonadotropic functions at the time of growth-promoted sexual maturation in sheep.

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.

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.

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.

Prepubertal changes in the synthesis, storage and release of the somatostatin in the hypothalamus of female lambs: a morphofunctional study.

It is assumed that hypothalamic somatostatin plays a role in the preovulatory phase of the oestrous cycle in sheep. The aim of the study was to investigate the processes of synthesis, storage and release of somatostatin in hypothalamic neurons, in immature female lambs, in the period approaching to puberty. Experiments were carried out on 10 prepubertal (17 weeks old) and 10 peripubertal (32 weeks old) ovary-intact lambs. Morphofunctional changes in the somatostatin neurons were assayed with immunohistochemistry and hybridisation in situ. Computer image analysis was used to determine the density of both reactions and the percentage of the area exhibiting immunohistochemical staining. These parameters express the content of immunoreactive (ir) somatostatin and expression of mRNA for pre-pro-somatostatin (PPS). Two populations of ir somatostatin perikarya were localized in the hypothalamus: a very large number of perikarya in the periventricular (PEV) nucleus, and single cell bodies in the arcuate (ARC) nucleus. Only ir somatostatin fibres, but no perikarya were seen in the ventromedial (VM) nucleus and preoptic area. The analysis of mRNA PPS showed perikarya filled with silver grains localized in the PEV, ARC and VM. There were differences in the content of ir somatostatin and the intensity of the PPS mRNA signal between the two periods investigated. In the median eminence, the content of ir somatostatin in the terminals decreased in the peripubertal compared to the prepubertal group (P<0.001). In the PEV, the content of ir somatostatin in the perikarya and the expression of PPS mRNA decreased in the peripubertal compared to the prepubertal group (P<0.001). In the ARC, the content of ir somatostatin in the perikarya increased (P<0.001), but expression of PPS mRNA decreased (P<0.001) in the peripubertal compared to the prepubertal group. There were no differences in the expression of PPS mRNA in the VM. We concluded, that the different secretory activity of the two hypothalamic populations of somatostatin neurons can be related to their different physiological functions in the prepubertal period of female lambs.

The neuroendocrine events during the ovine growth-promoted maturation: the developmental importance of hypophysiotrophic action of somatostatin in ewes.

The comparison of hypothalamic somatostatin (SRIH)-neuronal systems, hypophyseal somatotroph populations and growth hormone (GH) blood plasma patterns among developmental stages, from infancy until puberty, may help to describe the nature of the hypothalamo-hypophyseal mechanisms underlying the changes in GH on the systemic level leading to the somatic, that is growth and sexual maturity in sheep. The aim of this study was to elucidate (i) developmental importance of hypophysiotrophic action of SRIH, (ii) precise time of maturation of this action and (iii) photoperiodic regulation of the postnatal ontogeny in ewes. The central and peripheral activity of the SRIH-GH axis is described through a sequence of histomorphological and functional changes in Merino ewes born after the summer solstice. The actual time of puberty of these animals was delayed until the following breeding season, when the sheep were 14-month old. Histomorphometric examinations have been made in 21 infantile (preweanling, 12-week old), prepubertal (15- and 22-week old), peripubertal (30- and 52-week old) and pubertal (63-week old) ovary-intact sheep. Functional examinations of the GH plasma levels were determined every 1-2 weeks during the period from the 12th to 63rd week of age. The highest GH level was observed at the 13th week of age, on the beginning of the breeding season. The fluctuations in the GH level just after the winter and summer solstice were detected as the one and only deviation from a rule of uniformly low GH concentrations observed until puberty. The age of the fall in serum GH levels corresponded with the postweaning period and the beginning of the phase of the lower daily live-weight gains (growth rate). Thus, the development of GH secretion was finished before the 15th week of age, that is together with the ending of the transitional infantile/prepubertal period, whereas the maturational processing within the hypothalamo-hypophyseal unit prolonged after the 15th week of age until 22 weeks of age and concerned the role of SRIH as the hypophysiotrophic factor regulating somatic maturation, i.e. attenuating growth. Altogether, the pattern of GH secretion during weaning is important for the shift between infancy and prepuberty depended upon an intensive growth and defined as growth maturation. The maturation of the SRIH-GH axis is finished by 22 weeks of age, independently of photoperiodic influences, whereas the neuroendocrine mechanisms to integrate somatic, that is growth and sexual maturation, are seasonal in nature in the ewe. Our observations confirm the hypothesis of the inherent endogenous rhythm controlling somatic maturation in the sheep.

Prepubertal changes in the synthesis, storage and release of growth hormone and luteinising hormone and in the immunoreactivity of oestrogen receptor-alpha in lamb pituitary cells. A morphofunctional study.

The present study was designed to determine the changes in the synthesis, storage and release of luteinising hormone (LH) and growth hormone (GH) in the hypophyseal cells by investigating the presence of oestrogen receptor-alpha (ERalpha) in developing prepubertal female lambs. The experiment was carried out on 14 prepubertal (17-week-old) and 14 peripubertal (32-week-old) ovary-intact lambs. Morphofunctional changes in the cells of the adenohypophyseal population were assayed with immunohistochemistry (IH), in situ hybridisation (ISH), Real-time PCR and radioimmunoassay (RIA). Blood samples (n=14) were taken every 2 weeks from 17 to 32 weeks of age for estimation of GH and LH by RIA. Computer image analysis was used to determine the percent of cells exhibiting IH and/or ISH reaction. The percentage of cells stained for LHbeta and GH increased for both LH- and GH-producing cells and were higher (P<0.001) in the peripubertal than prepubertal group. The percentage of mRNA LHbeta-expressing cells decreased and were lower for the peripubertal (P<0.001) than prepubertal group. The GH mRNA in pituitaries of prepubertal lambs was higher in comparison to peripubertal ones (P<0.001). The percentage of ERalpha positive cells increased significantly (P<0.001) in peripubertal compared to prepubertal lambs and this increase was significant (P<0.001) in both LH- and GH-producing cells. Plasma LH concentrations increased from 27 weeks of age, while GH concentrations gradually decreased from 17 weeks of age (P<0.05). The histomorphological changes in the LH- and GH-producing cells reflect the increasing pattern of the regulation of secretory processes of these hormones and an escalating regulatory role of oestrogen in the physiology of these cells during the prepubertal period. These results support the involvement of both hormones in the events leading up to puberty.

Central estrogen-like effect of genistein on growth hormone secretion in the ewe.

The present study tested a hypothesis, whether plant-derived genistein influences the secretion of growth hormone (GH) in ewes, acting directly within the central nervous system (CNS). Starting six weeks after ovariectomy, ewes were infused intracerebroventricularly with genistein (n = 5) or 17beta-estradiol (n = 5), both in a total dose of 40 microg/400 microl/4 h, or with a vehicle (control, n = 5). All infusions were performed from 10:00 AM to 2:00 PM and blood samples were collected from 8:00 AM to 8:00 PM at 10-min intervals. Five genistein- and three vehicle-infused ewes were slaughtered the following morning. The plasma GH concentration was assayed by the radioimmunoassay method, and immunoreactivity of GH in the adenohypophysis was determined by immunohistochemistry. In genistein-infused ewes, mean plasma GH concentration was significantly higher during the whole period of infusion than the concomitant concentration in vehicle-infused ewes. However, examining data within group, GH secretion rose gradually, reaching a significant value during the second phase of genistein infusion. In 17beta-estradiol-infused animals, a significant increase in GH concentration was noted during the first two hours of the infusion, in comparison with vehicle-infused and also in comparison with genistein-infused ewes. Although a gradual increase in basic GH secretion continued in all treated groups during the afternoon and evening, mean plasma GH concentrations in genistein- and 17beta-estradiol-infused ewes were still significantly higher than in the vehicle-infused. The percentage of GH-positive cells in the adenohypophysis and the density of immunoreactive material in these cells decreased significantly in genistein-infused ewes, compared to the control, indicating diminished hormone storage. In conclusion, genistein as 17beta-estradiol, is an effective stimulator of GH secretion in ewes and may exert its effect at the level of the CNS.

Aspects of central and peripheral regulation of reproduction in mammals.

To increase our knowledge concerning the central and peripheral regulation of reproduction in mammals a series of studies were performed. In the first experiment, we found that exogenous leptin altered the activity of the hypothalmo-pituitary-gonadotropic axis in sheep during insufficient feeding. The action of leptin appears to be mediated by changes in GnRH and LH secretion as well as NPY immunoreactivity. The aim of the second experiment was to investigate the role of the adipoinsular axis hormones during pregnancy in rats. The elevated levels of plasma leptin as wells as the increased mRNAs expression of the leptin receptors in placenta indicate the significant role of the hormone in fetal growth and development. On the other hand, a decrease in leptin receptors mRNA content within hypothalamus and pituitary together with unchanged plasma insulin level may suggest that during rat pregnancy leptin resistance was developed in the hypothalamus, pituitary and pancreatic islets. The third experiment was carried out to establish the role of opioids and glucocorticoids in the regulation of the hypothalmo-pituitary-gonadal axis in ewes during natural or synchronized estrous cycle. Prolonged treatment with progesterone resulted in significant changes in plasma levels of Met-enkephalin, cortisol and steroids and altered the expression of proenkephalin mRNA in the hypothalamus, pituitary, ovary and adrenals. Injections of Met-enkephalin or naltrexone (blocker of opioid receptors) modulated the progesterone influence in tested tissues. The data clearly suggest that opioids are involved in the regulation of the estrous cycle at the hypothalamo-pituitary-gonadal/adrenal axes.

The effect of intracerebroventricular infusions of leptin on the immunoreactivity of neuropeptide Y and gonadotrophin releasing hormone neurons in the hypothalamus of prepubertal sheep in conditions of short fasting.

In the study we evaluated the effects of infusion of exogenous leptin to the third ventricle of the brain on the expression of immunoreactive (ir) neuropeptide Y (NPY) neurons in the hypothalamus and ir gonadotrophin releasing hormone (GnRH) nerve terminals in the median eminence of prepubertal lambs in the conditions of short fasting. Merino female sheep (n=16) were randomly divided into four groups, two fed with standard feeds and two fasted for 72 h. One standard and one fasted groups were infused with Ringer saline (controls), remaining standard and fasted groups with leptin (25 microg/120 microl/h), for 4 h during three consecutive days, and then slaughtered. Ir NPY and ir GnRH were localized by immunohistochemistry using specific polyclonal antibodies. Detection of both hormones was followed by the image analysis and expressed as the percent area stained and integral density of immunostaining. In the hypothalami from all groups the ir NPY perikarya and varicose nerve fibers were localized in three distinct sub-areas, in the arcuate (ARC), paraventricular and periventricular nuclei. In fasted sheep the percent area and integral density for immunoreactivity of NPY increased significantly (P<0.001) in three sub-areas compared to the standard-fed animals. Leptin infusion lowered the both parameters (P<0.001) but solely in the ARC NPY population of fasted sheep. The percent area and integral density of immunostaining for ir GnRH in fasted sheep revealed the augmentation (P<0.001) compared to standard-fed sheep. Leptin infusions diminished (P<0.001) both parameters in fasted, without effects in standard-fed lambs. In conclusion, the enhanced by fasting immunoreactivity of the ARC NPY perikarya and varicose nerve fibers and restrained immunoreaction of GnRH terminals in the median eminence were reversed by exogenous leptin. It is suggested that leptin can affect GnRH release via ARC NPY neurons in conditions of deficit of nutrients in prepubertal, female lambs.