Anna Wójcik-Gładysz

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.

Intracerebroventricular infusion of neuropeptide Y up-regulates synthesis and accumulation of luteinizing hormone but not follicle stimulating hormone in the pituitary cells of prepubertal female lambs

Neuropeptide Y (NPY) is a putative neuroregulator of the reproductive axis in the central nervous system. In this study we evaluated the effects of central infusion of exogenous NPY on the secretory activity of pituitary gonadotrophic cells in prepubertal lambs. Immature female Merino sheep (n=12) were infused of Ringer solution (control) or 50 microg of NPY to the third ventricle for 5 min and then slaughtered 3 h later. Immunoreactive luteinizing hormone (LH) and follicle stimulating hormone (FSH) cells were localised by immunohistochemistry using antibody raised against LHbeta and FSHbeta. Messenger RNA analyses were performed by in situ hybridisation using sense and antisense riboprobes produced from beta subunits of LH and FSH cDNA clones. The results were generated by computer image analysis to determine the area fraction occupied by immunoreactive and/or hybridising cells and optical density for immunostaining and hybridisation signal. LH in the blood plasma was determined by radioimmunoassay. It was found, that in the lambs infused with NPY the area fraction and optical density for immunoreactive LH cells and mRNA LHbeta-expressing cells increased significantly (P<0.001), compared to the vehicle-infused animals. The concentration of LH in the blood plasma did not differ between control and treated groups. The NPY infusions had no effect on the immunoreactivity of FSH cells or on expression of mRNA for FSHbeta. In conclusion we suggest that NPY may be an important component of mechanisms stimulating the synthesis and storage but not the release of LH in the pituitary gonadotrophs from prepubertal female sheep. In addition, this effect is specific for LH, no such effect was apparent on FSH.

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.

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 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 LHβ-mRNA and FSHβ-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 LHβ 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 LHβ-mRNA was greater (P<0.01) in 16-week-old lambs. The PA occupied by LHβ-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 FSHβ-mRNA and the plasma FSH concentration were greater (P<0.001) after orchidectomy in lambs from both age stages. The PA occupied by FSHβ-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.

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.

Prepubertal changes in the synthesis, storage and release of growth hormone and luteinising hormone and in the immunoreactivity of oestrogen receptor-α 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.

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 ghrelins 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.

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 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.