Peripheral Inhibitor of AChE, Neostigmine, Prevents the Inflammatory Dependent Suppression of GnRH/LH Secretion during the Follicular Phase of the Estrous Cycle.

The study was designed to test the hypothesis that the inhibition of acetylcholinesterase (AChE) activity at the periphery by Neostigmine (0.5 mg/animal) will be sufficient to prevent inflammatory dependent suppression of the gonadotropin-releasing hormone (GnRH)/luteinising hormone (LH) secretion in ewes in the follicular phase of the estrous cycle, and this effect will be comparable with the systemic AChE inhibitor, Donepezil (2.5 mg/animal). An immune/inflammatory challenge was induced by peripheral administration of lipopolysaccharide (LPS; 400 ng/kg). Peripheral treatment with Donepezil and Neostigmine prevented the LPS-induced decrease (P < 0.05) in LHß gene expression in the anterior pituitary gland (AP) and in LH release. Moreover, Donepezil completely abolished (P < 0.05) the suppressory effect of inflammation on GnRH synthesis in the preoptic area, when pretreatment with Neostigmine reduced (P < 0.05) the decrease in GnRH content in this hypothalamic structure. Moreover, administration of both AChE inhibitors diminished (P < 0.05) the inhibitory effect of LPS treatment on the expression of GnRH receptor in the AP. Our study shows that inflammatory dependent changes in the GnRH/LH secretion may be eliminated or reduced by AChE inhibitors suppressing inflammatory reaction only at the periphery such as Neostigmine, without the need for interfering in the central nervous system.

Endotoxin-induced inflammation disturbs melatonin secretion in ewe.

OBJECTIVE: The study examined the effect of intravenous administration of bacterial endotoxin-lipopolysaccharide (LPS) -on the nocturnal secretion of melatonin and on the expression of enzymes of the melatonin biosynthetic pathway in the pineal gland of ewes, taking into account two different photoperiodic conditions: short-night (SN; n = 12) and long-night (LN; n = 12). METHODS: In both experiments, animals (n = 12) were randomly divided into two groups: control (n = 6) and LPS-treated (n = 6) one. Two hours after sunset, animals received an injection of LPS or saline. Blood samples were collected starting one hour after sunset and continuing for 3 hours after the treatment. The ewes were euthanized 3 hours after LPS/saline treatment. The concentration of hormones in plasma was assayed by radioimmunoassay. In the pineal gland, the content of serotonin and its metabolite was determined by HPLC; whereas the expression of examined genes and protein was assayed using real-time polymerase chain reaction and Western Blot, respectively. RESULTS: Endotoxin administration lowered (p<0.05) levels of circulating melatonin in animals from LN photoperiod only during the first hour after treatment, while in ewes from SN photoperiod only in the third hour after the injection. Inflammation more substantially suppressed biosynthesis of melatonin in ewes from SN photoperiod, which were also characterised by lower (p<0.05) cortisol concentrations after LPS treatment compared with animals from LN photoperiod. In the pineal gland of ewes subjected to SN photoperiod, LPS reduced (p<0.05) serotonin content and the expression of melatonin biosynthetic pathway enzymes, such as tryptophan hydroxylase and arylalkylamine-N-acetyltransferase. Pineal activity may be disturbed by circulating LPS and proinflammatory cytokines because the expression of mRNAs encoding their corresponding receptors was determined in this gland. CONCLUSION: The present study showed that peripheral inflammation reduces the secretion of melatonin, but this effect may be influenced by the photoperiod.

Maternal High-Fat Diet during Pregnancy and Lactation Influences Obestatin and Ghrelin Concentrations in Milk and Plasma of Wistar Rat Dams and Their Offspring.

The study aims to establish the effect of a maternal high-fat diet on obestatin concentration, total ghrelin, and ghrelin/obestatin ratio during pregnancy and lactation of Wistar rats and their offspring in the first 21 days of life. On the mating day, females were randomly allocated and fed either a high-fat diet (30% of fat; HF) or breeding diet (5% fat; BD) till the 21st day of lactation. Hormones were analyzed in the blood plasma and milk of rat dams as well as in the blood plasma of their offspring. HF resulted in a significant decrease in obestatin level on the 14th day of lactation and elevation on the 21st day. Plasma obestatin in HFD offspring was significantly higher than in BD ones. HF diet did not significantly affect dam plasma ghrelin until the 21st day of lactation. The ghrelin concentrations in milk after both diets were significantly lower than in blood plasma. Milk ghrelin in HF dams was significantly higher than in the BD ones. Plasma ghrelin from HF offspring was significantly higher than that from BD dams. Our results demonstrate that a maternal HF diet during pregnancy and lactation influences ghrelin and obestatin level in both dams and their offspring.

Leptin and ghrelin levels in colostrum, milk and blood plasma of sows and pig neonates during the first week of lactation.

Radioimmunology was used to determine leptin and ghrelin levels in sow colostrum and milk in relation to those in sow and neonatal pig blood plasma and to the body weight of piglets during the first week of lactation. The highest concentration of leptin was found in colostrum on the second day of lactation (69.3 ± 6.3 ng/mL). Leptin concentrations in sow plasma were significantly lower than in colostrum/milk (2.19 ± 0.9 ng/mL, P = 0.7692) and were stable in the first 7 days of lactation. Total and active ghrelin concentrations in colostrum/milk were stable in the measured time points (6734 ± 261 pg/mL, P = 0.3397; 831 ± 242 pg/mL, P = 0.3988, respectively). Total ghrelin concentrations in sow plasma were lower than in colostrum/milk. These results indicate that pigs follow a unique species-specific pattern of leptin and ghrelin synthesis, release and existence, and that the mammary gland is an important source of leptin and ghrelin contained in colostrum/milk.

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.

The influence of salsolinol on dopaminergic system activity within the mediobasal hypothalamus of anestrous sheep: a model for studies on the salsolinol-dopamine relationship.

Salsolinol with its derivatives has been considered as a potential neurotoxin for the dopaminergic system in the human and rat brain. Investigating a sheep model for studies on the action of salsolinol within the central nervous system we examined whether this compound is able to affect the hypothalamic neuroendocrine dopaminergic (NEDA) system during its high seasonal activity, when sheep entered to anestrus under the long day conditions. Therefore, salsolinol was infused into the third ventricle of the brain in combination with the in vivo push-pull perfusion of the mediobasal hypothalamus/median eminence (MBH/ME). The effects of this drug on either perfusate noradrenaline (NA) or plasma prolactin concentration were also studied. The infusion of salsolinol resulted in rapid and permanent diminution in dopamine (DA) release into the extracellular spaces of the MBH/ME up to an undetectable level and in the 57% decrease in DA metabolite 3,4-dihydroxyphenylacetic acid concentration, compared to the control. This effect of salsolinol was accompanied by the significant enhancement of the pituitary prolactin release into circulation. The concentration of other DA metabolite, homovanillic acid, as well as NA in the MBH/ME was not affected. Thus, our results in the anestrous sheep underline the role played by salsolinol as a neuromodulator for the hypothalamic NEDA system and as a signal transmitter for the pituitary prolactin release. We suggest that the hypothalamic NEDA system of anestrous sheep during its high secretory activity may be set as a model for studies on the salsolinol-dopamine relationship.

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.

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.

Effects of salsolinol and its antagonistic analogue, 1-MeDIQ, on growth hormone release in nursing sheep.

Suckling induces a GH surge simultaneously to that of prolactin, so we tested whether salsolinol, a dopamine derivative (1-methyl-6,7-dihydroxy-1,2,3,4-tetrahydroisoquinoline), participates in the regulatory process of GH secretion in lactating sheep. A series of intracerebroventricular (i.c.v.) infusions of salsolinol, in two doses, was performed in nursing sheep, without suckling, during the fifth week of lactation. In other suckling sheep, we infused i.c.v. a structural analogue of salsolinol-1-methyl-3,4-dihydroisoqinoline (1-MeDIQ), which is able to antagonize salsolinol's action. Intracerebroventricular treatment of nursing sheep with a lower dose of salsolinol (total 50 ng) significantly increased plasma GH concentration, as compared with the concentrations noted before the infusion and in nursing controls. A higher dose of salsolinol (total 5 micrograms) did not affect GH release significantly. Intracerebroventricular treatment with 1-MeDIQ (total 300 micrograms) significantly reduced basal GH release, not affecting a pattern of GH surge in response to suckling. In conclusion, salsolinol may affect the regulatory process of GH secretion in lactating sheep, but its role seems not to be major.

Effects of a structural analogue of salsolinol, 1-MeDIQ, on pituitary prolactin release and dopaminergic activity in the mediobasal hypothalamus in nursing sheep.

The prolactin release caused by salsolinol (a derivative of dopamine, DA) in rats could be prevented by its structural analogue 1-methyl-3,4-dihydroisoqinoline (1-MeDIQ). To study the participation of salsolinol in the neural stimulatory mechanism of prolactin release in lactating sheep, we tested whether 1-MeDIQ, acting at the central nervous system (CNS) level, would diminish basal prolactin release and reduce prolactin surge induced by suckling. A series of intracerebroventricular (icv) infusions of 1-MeDIQ (5 x 60 microg/60 microl/30 min, at 30-min intervals) were performed in nursing ewes (n=8) during the fifth week of lactation. Additionally, by combining these infusions with push-pull perfusion, we studied the concentration of dopaminergic components, i.e., salsolinol, DA and 3,4-dihydroxyphenylacetic acid (DOPAC) within the infundibular nucleus/median eminence (IN/ME) in four of the ewes. Treatment with 1-MeDIQ significantly (P<0.001) reduced either the basal prolactin release during the non-suckling period or the suckling-induced prolactin surge. Specifically, the suppressive effect occurred gradually, affecting both the duration and amplitude of the prolactin surge. In the control ewes, the perfusate salsolinol concentration increased significantly (P<0.001) during suckling, while in the ewes treated with 1-MeDIQ only vestigial amounts of this compound were found during the non-suckling period. No DA was detected in the perfusates collected from the IN/ME of control and 1-MeDIQ-treated groups and no significant differences were found in the DOPAC concentrations between these groups. In conclusion, 1-MeDIQ is able to inhibit prolactin secretion in lactating sheep, acting at the CNS level. In addition, one of the way of 1-MeDIQ action may be directed to the local salsolinol release within the mediobasal hypothalamus.

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.

Identification of salsolinol in the mediobasal hypothalamus of lactating ewes and its relation to suckling-induced prolactin and GH release.

The push-pull perfusions of the infundibular nucleus-median eminence (IN/ME) were made in lactating ewes (n=7) twice, to identify dopamine (DA)-derived salsolinol and the changes in its extracellular concentration in response to suckling. The perfusate collecting period in every ewe consisted of control non-suckling period, 1000-1230 h (five perfusates), and suckling period, 1230-1500 h (next five perfusates). Simultaneously, blood samples were collected from 1000 to 1500 h at 10-min intervals. The perfusate concentrations of salsolinol and DA were measured by HPLC, and plasma prolactin and GH concentrations were assayed by the RIA. Mean concentrations of salsolinol in perfusates collected from the anterior and posterior parts of the IN/ME (according to post-mortem localization of a perfusion site) increased significantly (P<0.05 and P<0.001 respectively) during the suckling period, when compared with those noted during the non-suckling period. While no DA was found in the anterior part, only vestigial amounts of DA were found in a few perfusates collected from the posterior part. Salsolinol was not detected in the IN/ME of ewes 10 weeks after weaning (seasonal anoestrus). Mean plasma prolactin and GH concentrations during suckling were significantly (P<0.001) higher than those noted during the non-suckling period. In conclusion, our current study reveals that salsolinol is present in the IN/ME of lactating ewes and that its extracellular concentration increases during suckling. Moreover, it supports the role of salsolinol as a neurotransmitter involved in the regulatory process of prolactin secretion at least during lactation.

Differential endocrine response in rams to intracerebroventricular infusion of genistein.

The intracerebroventricular infusions of genistein (total 40 mug) were made in male sheep (November) to test its influence on melatonin, growth hormone (GH) and luteinizing hormone (LH) secretion. The analysis of the results encompassed 3 similar periods: before the infusion (afternoon hours) the first (evening hours) and the second (night hours) halves of the treatment. The night plasma concentration of melatonin in genistein-infused rams was significantly lower than that noted during the respective period in vehicle-infused rams. Plasma GH concentration increased significantly in both vehicle- and genistein-infused rams during the night hours, as compared with the concentrations noted during the afternoon and evening, however, genistein significantly stimulated the amplitude of GH pulses in these latter. The LH concentration was significantly lower during the second part of genistein treatment, than in vehicle-infused rams. The frequency and amplitude of LH pulses clearly tended to decrease following genistein infusion. In conclusion, genistein, acting at the central nervous system level in sexually active rams is able to reduce the secretion of melatonin and LH and has also a slight stimulatory effect on the amplitude of GH pulses.

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.

Genistein-induced pituitary prolactin gene expression and prolactin release in ovariectomized ewes following a series of intracerebroventricular infusions.

The aim of the study was to evaluate whether genistein, a phytoestrogen commonly present in feed plants, affects prolactin release and its gene expression in the pituitary gland. In the experimental model, genistein was infused into the third ventricle (IIIv) of the brain in ewes during the short-daylight period (November-December), when the physiological plasma level of prolactin is low. Animals were ovariectomized six weeks before the experiment, to remove the main source of endogenous estrogens, and three weeks later a stainless steel guide cannula was implanted into IIIv. Genistein (10 ng/100 microl/h, n=5) or vehicle (control, n=5) were infused in a series of four one-hour infusions at 30-min intervals (from 16:30 to 22:00). Plasma samples were collected at 15-min intervals from 14:00 to 22:00 through a catheter inserted into the jugular vein and after the experiment ewes were slaughtered. Northern blot analysis revealed that pituitary prolactin mRNA content increased significantly in response to genistein, compared to the vehicle-infused ewes (p<0.05). Prolactin concentration in plasma rose significantly during the periods of genistein infusion, as compared to the values found before infusion (p<0.05-p<0.01) as well as to the values of the concomitant periods in vehicle-infused ewes (p<0.001). Our results show an effective estrogenic action of genistein on prolactin synthesis and release in ovariectomized ewes that might in part be exerted at the central nervous system level.

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.

In vitro evidence that leptin suppresses melatonin secretion during long days and stimulates its secretion during short days in seasonal breeding ewes.

Recent observations in seasonal-breeding mammals indicate that the hypothalamus is programmed to become leptin resistant during long days (LD) and leptin sensitive during short days (SD). These observations support the possibility that photoperiod mediates at least part of its effects on melatonin secretion through changes in leptin sensitivity. Herein we examined the interaction of season and recombinant ovine leptin (oleptin) on melatonin secretion by pineal explants in short-term culture. Glands were collected after sunset from eight ewes during LD (March, April, May, June) and from an additional eight ewes during SD (September, October, November, December). Glands were transected saggitally and coronally into quarters, with each equilibrated in 2.5ml of DMEM for 120min, followed by a 3h incubation in medium containing either 0 or 50ng/ml of oleptin. Treatment with oleptin reduced (P<0.001) melatonin secretion compared to controls during LD by approximately 22% at 2, 2.5 and 3h of culture. However, in cultures from glands collected during SD, oleptin stimulated (P<0.078) melatonin secretion approximately 50% compared to control. These effects were consistent throughout each respective season. We conclude that the secretion of melatonin from the ovine pineal gland is negatively responsive to leptin during LD, whereas leptin may stimulate melatonin secretion during SD.

Differential effects of soy-containing diets on the reproductive tissues growth and reproductive hormone secretion in male rats.

The effects of feeding a breeding diet containing soy products to pregnant and lactating females on reproductive tissues and secretion of the reproductive hormones in their male progeny, immediately after weaning (postnatal day - PND 22) and after reaching puberty (PND 60) were studied. Similarly, the response of adult males to a soy maintenance diet over shorter (PND 160) and longer (PND 280) periods of time was examined. The relative weights (standardized by body weight) of the testes, epididymis and prostate, and the concentrations of luteinizing hormone (LH), testosterone and prolactin (PRL) were used as the examined endpoints. In rats on PND 22, no significant differences in the relative organs weights and the plasma hormones concentrations were found between the experimental and control groups. In rats on PND 60 which continued consuming a soy breeding diet, the relative tissue weights did not differ significantly, while the mean plasma LH and PRL concentrations were higher (p<0.01-0.001) compared to the controls. In rats on PND 160 fed soy maintenance diet, the higher relative testes (p<0.01) and epididymis (p<0.05) weights as well as plasma testosterone (p<0.001) concentration were recorded compared to the controls. In rats on PND 280 fed a soy maintenance diet, the relative weights of all reproductive tissues were similar to those of controls, however, the weight of the body and the real weights of the reproductive tissues were lower (p<0.05) than in controls. The mean plasma concentrations of the reproductive hormones did not differ significantly between the two groups. In conclusion, a supplement of soy in the rat diet may affect growth and/or development of the reproductive tissues in male rats and also affect concentrations of reproductive hormones. The effects depend on the period of life when the soy diet is applied.

Does prolactin influence the hypothalamo-pituitary GnRH-LH system in preovulatory-phase ewes?

The effects of prolonged infusions of prolactin (PRL) into the third ventricle of the brain of cycling ewes on the secretory activity of hypothalamic GnRH neurons and pituitary LH cells in the pars distalis during the proestrous day were studied. Mature Blackhead ewes were infused with vehicle (control, n=5) or with prolactin (200 mug/day, n=5) during 4 consecutive days prior to the next spontaneous ovulation. The dose of PRL was infused each day in 4 series of 50 mug/100 mul/h at 30-min. intervals, from 8.30 to 14.00 h. The animals were slaughtered on the 16th (proestrous) day of the estrous cycle immediately after the last infusion and their brains were fixed in situ. Plasma samples were collected for 6 h at 10 min. intervals, on days 12 (before the infusions) and 16 of the cycle. The distribution pattern, number and morphology of GnRH neurons in vehicle- and PRL-infused ewes were found to be similar and typical for the proestrous phase of the cycle. The immunoreactive (ir) GnRH stores in the median eminence were high and similar in both groups. There were no differences between control and PRL-treated ewes in the number or features of irLH cells. The area fraction and optical density for irLH cells and mRNA LHbeta-expressing cells did not differ between control and experimental groups. Irrespective of the kind of infusion, changes in LH secretion during the estrous cycle were similar in control and PRL-infused ewes. Mean plasma LH concentrations were higher (p<0.001) on day 16 compared to day 12 of the cycle. There were no differences in plasma LH concentrations or in the parameters of pulsatile LH secretion between groups. In conclusion, repeated, several-hour-long infusions of PRL into the CNS prior to the next spontaneous ovulation in ewes has no direct effect on the secretory activity of GnRH neurons, and/or the synthesis, accumulation, or tonic release of LH from the pituitary gonadotrophs.