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We have shown that cortisol infusion reduced the luteinizing hormone (LH) response to fixed hourly GnRH injections in ovariectomized ewes treated with estradiol during the non-breeding season (pituitary-clamp model). In contrast, cortisol did not affect the response to 2 hourly invariant GnRH injections in hypothalamo-pituitary disconnected ovariectomized ewes during the breeding season. To understand the differing results in these animal models and to determine if cortisol can act directly at the pituitary to suppress responsiveness to GnRH, we investigated the importance of the frequency of GnRH stimulus, the presence of estradiol and stage of the circannual breeding season. In experiment 1, during the non-breeding season, ovariectomized ewes were treated with estradiol, and pulsatile LH secretion was restored with i.v. GnRH injections either hourly or 2 hourly in the presence or absence of exogenous cortisol. Experiments 2 and 3 were conducted in hypothalamo-pituitary disconnected ovariectomized ewes in which GnRH was injected i.v. every 2 h. Experiment 2 was conducted during the non-breeding season and saline or cortisol was infused for 30 h in a cross-over design. Experiment 3 was conducted during the non-breeding and breeding seasons and saline or cortisol was infused for 30 h in the absence and presence of estradiol using a cross-over design. Samples were taken from all animals to measure plasma LH. LH pulse amplitude was reduced by cortisol in the pituitary clamp model with no difference between the hourly and 2-hourly GnRH pulse mode. In the absence of estradiol, there was no effect of cortisol on LH pulse amplitude in GnRH-replaced ovariectomized hypothalamo-pituitary disconnected ewes in either season. The LH pulse amplitude was reduced in both seasons in experiment 3 when cortisol was infused during estradiol treatment. We conclude that the ability of cortisol to reduce LH secretion does not depend upon the frequency of GnRH stimulus and that estradiol enables cortisol to act directly on the pituitary of ovariectomized hypothalamo-pituitary disconnected ewes to suppress the responsiveness to GnRH; this effect occurs in the breeding and non-breeding seasons.

The present study investigated photorefractoriness in the prolactin (PRL) axis in hypothalamopituitary-disconnected (HPD) sheep exposed to prolonged long days. In experiment 1, HPD Soay rams transferred from short (8L:16D) to long (16L:8D) days for 48 wk to induce a cycle of activation, decline (photorefractoriness), and reactivation in PRL secretion were treated chronically with bromocriptine (dopamine-receptor agonist) or vehicle from the onset of photorefractoriness. Bromocriptine (0.01-0.04 mg kg-1 day-1; 12-24 wk of long days) blocked PRL release and caused a rebound response after the treatment, but it had no effect on the long-term PRL cycle (posttreatment PRL minimum, mean +/- SEM, 35.3 +/- 0.6 and 37.0 +/- 0.4 wk for bromocriptine and control groups, respectively; not significant). In experiment 2, HPD rams were treated with sulpiride (dopamine-receptor antagonist) during photorefractoriness. Sulpiride (0.6 mg/kg twice daily; 22-30 wk of long days) induced a marginal increase in blood PRL concentrations, but again, it had no effect on the long-term PRL cycle (PRL minimum, 37.9 +/- 0.4 and 37.6 +/- 0.9 wk for sulpiride and control groups, respectively; not significant). The 24-h blood melatonin profile consistently reflected the long-day photoperiod throughout, and blood FSH concentrations were minimal, confirming the effectiveness of the HPD surgery. The results support the conclusion that photorefractoriness is regulated at the level of the pituitary gland independently of the PRL output signal.

The gonadotrope is a complex cell that expresses receptors for gonadotropin releasing hormone (GnRH) and estrogen. It has synthetic machinery for the production of 3 gonadotropin subunits which are assembled into two gonadotropins, luteinising hormone (LH) and follicle stimulating hormone (FSH). The production and secretion of LH and FSH are differentially regulated by GnRH and estrogen. Patterns of secretion of LH are dictated by the pulsatile release of GnRH from the median eminence as well as the feedback effects of estrogen. The means by which estrogen plays such an important role in the regulation of LH and FSH is reviewed in this chapter, with emphasis on work that has been done in the sheep. Estrogen regulates the second messenger systems in the gonadotrope as well as the number of GnRH receptors and the function of ion channels in the plasma membrane. Estrogen also regulates gene expression in these cells. Additionally, GnRH appears to regulate the level of estrogen receptor in the ovine gonadotrope, so there is substantial cross-talk between the signalling pathways for GnRH and estrogen. No clear picture has emerged as to how estrogen exerts a positive feedback effect on the gonadotrope and it is suggested that this might be forthcoming from more definitive studies on the way that estrogen regulates the second messenger systems and the trafficking of secretory vesicles.

During the oestrous cycle, there is an alteration in gonadotrope responsiveness to gonadotropin releasing hormone (GnRH). One cellular mechanism that may be involved in these changes at the pituitary level is the hormonal regulation of oestrogen receptor (ER) expression. Using double-label immunohistochemistry, we examined the proportion of gonadotropes, lactotropes and somatotropes with immunoreactive (ir) oestrogen receptor alpha (ERalpha) in pituitary sections from ewes at three stages of the ovine oestrous cycle (n = 8 per group). The percentage of ERalpha positive cells that also stained positive for luteinizing hormone (LH) increased in the transition from the luteal phase to the follicular phase (n = 8), with no further increase at the time of oestrus (n = 8). In the pituitaries from the luteal phase sheep, only a small number (15%) of lactotropes and 4% of somatotropes were found to contain ir-ERalpha and there were no alterations across the oestrous cycle. When we examined pituitaries from ovariectomized (OVX) ewes treated (i.m.) with either oestradiol benzoate (50 microg) or oil vehicle for 2, 4, 6 or 16 h (n = 4 per group), there was no effect of treatment. In fact, the percentage of gonadotropes that were ERalpha-positive in OVX ewes was similar to that observed in the pituitaries from the follicular phase ewes, both of which display a high frequency of pulsatile GnRH secretion. We conclude that the number of gonadotropes that contain ir-ERalpha increases in the follicular phase of the oestrous cycle and this may enhance the responsiveness of these cells to oestrogen and GnRH. We suggest that this may be due to increased pulsatile GnRH input rather than rising oestrogen levels.

This experiment determined if the degree of stimulation of the pituitary gland by GnRH affects the suppressive actions of inhibin and testosterone on gonadotropin secretion in rams. Two groups (n = 5) of castrated adult rams underwent hypothalamopituitary disconnection and were given two i.v. injections of vehicle or 0.64 microg/kg of recombinant human inhibin A (rh-inhibin) 6 h apart when treated with i.m. injections of oil and testosterone propionate every 12 h for at least 7 days. Each treatment was administered when the rams were infused i.v. with 125 ng of GnRH every 4 h (i.e., slow-pulse frequency) and 125 ng of GnRH every hour (i.e., fast-pulse frequency). The FSH concentrations and LH pulse amplitude were lower and the LH concentrations higher during the fast GnRH pulse frequency. The GnRH pulse frequency did not influence the ability of rh-inhibin and testosterone to suppress FSH secretion. Testosterone did not affect LH secretion. Following rh-inhibin treatment, LH pulse amplitude decreased at the slow, but not at the fast, GnRH pulse frequency, and LH concentrations decreased at both GnRH pulse frequencies. We conclude that the degree of stimulation of the pituitary by GnRH does not influence the ability of inhibin or testosterone to suppress FSH secretion in rams. Inhibin may be capable of suppressing LH secretion under conditions of low GnRH.

Influx of Ca2+ via Ca2+ channels is the major step triggering exocytosis of pituitary somatotropes to release growth hormone (GH). Voltage-gated Ca2+ and K+ channels, the primary determinants of the influx of Ca2+, are regulated by GH-releasing hormone (GHRH) through G-protein-coupled intracellular signalling systems. Using whole-cell patch-clamp techniques, the changes of the Ca2+ and K+ currents in primary cultured ovine and human somatotropes were recorded. Growth hormone-releasing hormone (10 nmol/L) increased both L- and T-type voltage-gated Ca2+ currents. Inhibition of the cAMP/protein kinase A (PKA) pathway by either Rp-cAMP or H89 blocked this increase in both L- and T-type Ca2+ currents. Growth hormone-releasing hormone also decreased voltage-gated transient (IA) and delayed rectified (IK) K+ currents. Protein kinase C (PKC) inhibitors, such as calphostin C, chelerythrine or downregulation of PKC, blocked the effect of GHRH on K+ currents, whereas an acute activation of PKC by phorbol 12, 13-dibutyrate (1 micromol/L) mimicked the effect of GHRH. Intracellular dialysis of a specific PKC inhibitor (PKC19-36) also prevented the reduction in K+ currents by GHRH. It is therefore concluded that GHRH increases voltage-gated Ca2+ currents via cAMP/PKA, but decreases voltage-gated K+ currents via the PKC signalling system. The GHRH-induced alteration of Ca2+ and K+ currents augments the influx of Ca2+, leading to an increase in [Ca2+]i and the GH secretion.

The electrophysiological properties of anterior pituitary somatotropes integrally involve the function of voltage-gated K+ currents. In this study, we have used GH4C1 cell lines to investigate the effect of human GHRH on voltage-gated K+ currents. Because of a clear 'rundown' of the K+ current with classic whole cell recording (WCR) without ATP in pipette solution, nystatin-perforated WCR was the major recording configuration used. Using a low Ca2+ (0.5 mM) bath solution containing Co2+ (1 mM) and TTX (1 microM), GH4 cells predominantly exhibited an outward delayed rectifier K+ current (IK). Local application of growth hormone releasing hormone (GHRH) (100 nM) reversibly reduced the amplitude of the K+ currents (to 83% of control). There was no effect of GHRH on the activation curve of the K+ current and no difference observed using 2.5 mM Ca2+ or low Ca2+ (0.5 mM Ca2++1 mM Co2+) bath solutions. Under the condition of low Ca2+ bath solution, the application of apamin (1 microM) or charybdotoxin (1 microM), two specific blockers of the Ca2+-activated K+ current, did not alter the K+ current or the response to GHRH. This reduction in the K+ current by GHRH was also observed with classic WCR with a pipette solution containing ATP (2 mM). The GHRH-induced reduction in the K+ current was completely abolished by the presence of GDP-beta-s (500 microM) in the pipette solution or by addition of PKC inhibitors, calphostin C (100 nM) and chelerythrine (1 microM), in bath solution. Inhibitor for cAMP-PKA system (Rp-cAMP and H89) did not affect the K+ current response to GHRH. These results suggest that GHRH reduces the voltage-gated K+ current in GH4C1 cells, a response that is mediated by G-proteins and PKC system but not by cAMP-PKA system. The reduction in the K+ current may partially contribute to the GHRH-stimulated growth hormone (GH) secretion.

Hypothalamo-pituitary disconnected Soay rams were exposed to two photoperiodic treatments: 1) constant long days (16L:8D) for 48 wk after pretreatment under short days (LD group), and 2) constant short days (8L:16D) for 48 wk after pretreatment under long days (SD group). In the LD group, plasma prolactin (PRL) concentrations increased from 0 to 8 wk (maximum: 143.3 +/- 8.4 microg/l; 8.8 +/- 1. 2 wk), decreased from 9 to 34 wk (minimum: 15.6 +/- 1.6 microg/l; 34. 5 +/- 1.5 wk), and finally increased again under the constant conditions, with a similar cyclical pattern for all individuals. In the SD group, PRL concentrations showed an inverse pattern (minimum: 8.6 +/- 2.6 microg/l; 17.1 +/- 2.0 wk; maximum: 46.4 +/- 5.5 microg/l; 30.2 +/- 3.2 wk), with more variability. Plasma concentrations of FSH were basal in both groups. The duration of the daily nocturnal melatonin peak (measured at 10, 24, and 44 wk) remained close to 8 h under long days (high-fidelity melatonin signal) but decreased significantly (13.8 h to 9.3 h) under short days (low-fidelity melatonin signal). The results support the conclusion that the melatonin signal encoding photoperiod acts within the pituitary gland to induce both acute (inductive) and chronic (refractory) effects photoperiod on PRL secretion.

There is a sex difference in the hypothalamo-pituitary-adrenal (HPA) axis of many species, although there are sparse data on the sheep. In the present study we have compared the HPA axes of intact and gonadectomised adult male and female sheep at the level of the median eminence, pituitary and adrenal glands using a variety of in vitro approaches. The concentration of arginine vasopressin (AVP) was higher (P<0.01) in the median eminence of male than female sheep, and was also elevated by gonadectomy of either sex (P<0.01). The concentration of corticotrophin-releasing factor (CRF) in the median eminence did not differ between the sexes, but was also elevated in both sexes following gonadectomy (P<0.01). Anterior pituitary pro-opiomelanocortin mRNA concentrations were higher (P<0.05) in intact male sheep than in intact females, with the levels in gonadectomised animals of both sexes being intermediate. In contrast to this finding, basal ACTH secretion from anterior pituitary cells was higher (P<0.05) in cultures derived from female sheep than those from males, but gonadectomy was without effect. There was no effect of sex or gonadectomy on in vitro ACTH secretion in response to AVP, CRF or the combination of AVP and CRF, and in all cases the combination of AVP and CRF generated greater (P<0.0001) ACTH secretion than AVP alone. AVP alone was more effective (P<0.01) than CRF alone as an ACTH secretagogue. The adrenal glands were larger (P<0.05) in female than male sheep, with no effect of gonadectomy. Basal cortisol production was greatest (P<0.05) in cultures of adrenal cells from intact male sheep, though ACTH- and 8BrcAMP-induced cortisol production was greater in the cultures of cells from females (P=0.05); there were no effects of gonadectomy. Cultures of adrenocortical cells from male sheep had greater (P<0.05) basal cAMP production, but ACTH-stimulated cAMP production did not differ between any of the groups of animals. These findings show a range of differences in the HPA axis of male and female sheep. Furthermore, they suggest that the heightened activity of the axis in the female occurs primarily due to differences at the level of the adrenal gland, and that greater adrenal responsiveness of female animals is due to differences in the latter stages of steroidogenesis, rather than an effect on ACTH signal transduction at its receptor.

Treatment of gonadotropes with estrogen (E) changes the electrophysiological response to gonadotropin-releasing hormone (GnRH) such that the cells are hyperpolarised immediately after stimulation with GnRH and then generate action potentials more frequently than non-E-treated cells. We investigated the role of K+ current in this altered response to GnRH using cultures of ewe pituitary cells enriched for gonadotropes. K+ current density was measured using nystatin-perforated whole-cell recordings in the voltage clamp mode. Treatment of cells with E for 16-20 h significantly (p < 0.01) increased the unit K+ current to 180% of that in vehicle-treated cells. Outward current in these cells flows predominantly through voltage-dependent, delayed rectifier K+ channels (IK), and E alters the magnitude of this current. The effect of E to increase the K+ current was dose- and time-dependent and was maximal after 16-20 h. The unit K+ current values returned to pre-treatment levels after 36 h of E treatment. Several cells were studied both before and after E treatment and the average effect of E on these cells was to increase the unit K+ current by 90%. The time-course of the effect of E on K+ current density is the same as the effect of E to increase LH release in vitro and in vivo. We conclude that the increase in K+ current may be an important part of the mechanism whereby E acts on gonadotropes to facilitate the LH surge which triggers ovulation.

In this study we used an isolation/restraint stress to test the hypothesis that stress will affect the secretion of LH differently in gonadectomised rams and ewes treated with different combinations of sex steroids. Romney Marsh sheep were gonadectomised two weeks prior to these experiments. In the first experiment male and female sheep were treated with vehicle or different sex steroids for 7 days prior to the application of the isolation/restraint stress. Male sheep received either i.m. oil (control rams) or 6 mg testosterone propionate injections every 12 h. Female sheep were given empty s.c. implants (control ewes), or 2x1 cm s.c. implants containing oestradiol, or an intravaginal controlled internal drug release device containing 0.3 g progesterone, or the combination of oestradiol and progesterone. There were four animals in each group. On the day of application of the isolation/restraint stress, blood samples were collected every 10 min for 16 h for the subsequent measurement of plasma LH and cortisol concentrations. After 8 h the stress was applied for 4 h. Two weeks later, blood samples were collected for a further 16 h from the control rams and ewes, but on this day no stress was imposed. In the second experiment, separate control gonadectomised rams and ewes (n=4/group) were studied for 7 h on 3 consecutive days, when separate treatments were applied. On day 1, the animals received no treatment; on day 2, isolation/restraint stress was applied after 3 h; and on day 3, an i. v. injection of 2 microg/kg ACTH1-24 was given after 3 h. On each day, blood samples were collected every 10 min and the LH response to the i.v. injection of 500 ng GnRH administered after 5 h of sampling was measured. In Experiment 1, the secretion of LH was suppressed during isolation/restraint in all groups but the parameters of LH secretion (LH pulse frequency and amplitude) that were affected varied between groups. In control rams, LH pulse amplitude, and not frequency, was decreased during isolation/restraint whereas in rams treated with testosterone propionate the stressor reduced pulse frequency and not amplitude. In control ewes, isolation/restraint decreased LH pulse frequency but not amplitude. Isolation/restraint reduced both LH pulse frequency and amplitude in ewes treated with oestradiol, LH pulse frequency in ewes treated with progesterone and only LH pulse amplitude in ewes treated with both oestradiol and progesterone. There was no change in LH secretion during the day of no stress. Plasma concentrations of cortisol were higher during isolation/restraint than on the day of no stress. On the day of isolation/restraint maximal concentrations of cortisol were observed during the application of the stressor but there were no differences between groups in the magnitude of this response. In Experiment 2, isolation/restraint reduced the LH response to GnRH in rams but not ewes and ACTH reduced the LH response to GnRH both in rams and ewes. Our results show that the mechanism(s) by which isolation/restraint stress suppresses LH secretion in sheep is influenced by sex steroids. The predominance of particular sex steroids in the circulation may affect the extent to which stress inhibits the secretion of GnRH from the hypothalamus and/or the responsiveness of the pituitary gland to the actions of GnRH. There are also differences between the sexes in the effects of stress on LH secretion that are independent of the sex steroids.

Three experiments were conducted with castrated Romney Marsh rams (wethers) to investigate the ability of testosterone and inhibin to suppress the secretion of LH and FSH during the breeding and the non-breeding seasons. In Experiment 1, wethers (n=5/group) were treated every 12 h for 7 days with oil or 16 mg testosterone propionate (i.m.) and were then given two i.v. injections either of vehicle or of 0.64 microg/kg human recombinant inhibin A (hr-inhibin) 6 h apart. Blood samples were collected for 4 h before inhibin or vehicle treatment and for 6 h afterwards for the assay of LH and FSH. In Experiments 2 and 3 wethers underwent hypothalamo-pituitary disconnection (HPD) and were given 125 ng GnRH i.v. every 2 h. In Experiment 2, HPD wethers (n=3/group) were injected (i.m.) every 12 h with oil or testosterone and blood samples were collected over 9 h before treatment and 7 days after treatment. In Experiment 3, HPD (n=5/group) wethers were treated with vehicle or hr-inhibin, as in Experiment 1, after treatment with oil, or 4, 8 or 16 mg testosterone twice daily for 7 days. Blood samples were collected over 4 h before treatment with vehicle or hr-inhibin and for 6 h afterwards. Treatment of wethers with testosterone (Experiment 1) resulted in a significant decrease in the plasma concentrations of LH and number of LH pulses per hour but the magnitude of these reductions did not differ between seasons. Testosterone treatment had no effect on LH secretion in GnRH-pulsed HPD wethers in either season and treatment with hr-inhibin did not affect LH secretion in wethers or HPD wethers in any instance. Plasma concentrations of FSH were significantly (P<0.05) reduced following treatment with testosterone alone during the breeding season but not during the non-breeding season. FSH levels were reduced to a greater extent by treatment with hr-inhibin but this effect was not influenced by season. During the non-breeding season, the effect of hr-inhibin to suppress FSH secretion was enhanced in the presence of testosterone. These experiments demonstrate that the negative feedback actions of testosterone on the secretion of LH in this breed of rams occurs at the hypothalamic level and is not influenced by season. In contrast, both testosterone and inhibin act on the pituitary gland to suppress the secretion of FSH and these responses are affected by season. Testosterone and inhibin synergize at the pituitary to regulate FSH secretion during the non-breeding season but not during the breeding season.

We have studied the direct effect of leptin on the secretion of GH from the anterior pituitary gland of the sheep. Using primary cultures of ovine pituitary cells, leptin (10(-9)-10(-7) M) treatment for 30 min did not affect basal or GHRH (10(-7) M)-stimulated GH secretion. Following treatment for 24 h, a dose of 10(-7) M leptin stimulated basal GH secretion. In contrast, doses of 10(-7) and 10(-8) M leptin inhibited GHRH-stimulated GH secretion after 24 h of treatment. These results suggest that leptin can have long-term effects on the somatotropes, but no acute effect. Furthermore, leptin appears to have opposite effects on basal and GHRH-stimulated GH secretion.

The number of pituitary gonadotropin-releasing hormone receptors (GnRH-R) varies across the estrous cycle. We report that there is variable expression of the differently-sized GnRH-R transcripts in cyclic ewes and in an experimental model. During the follicular phase of the cycle, and compared to the luteal phase, there was increased expression of the 1.5, 2.3 and 3.7 kilobase (kb) transcripts with no change in the levels of the 5.6 or the 1.2 kb transcripts. Steady state levels of mRNA for luteinising hormone beta and common alpha subunit were also increased in the follicular phase of the cycle. In hypothalamo-pituitary disconnected ovariectomised ewes given pulsatile GnRH replacement, injection of estrogen increased the 1.5, 2.3 and 3.7 kb, while the levels of the 5.6 and 1.2 kb transcripts were not altered. We conclude that the differential regulation of GnRH-R mRNA occurs through a direct effect of E on the pituitary.

Effect of growth hormone-releasing peptide-2 (GHRP-2) on ovine somatotrophs is abolished by a growth hormone-releasing factor (GRF) receptor antagonist, which raises the possibility that GHRP-2 may act on GRF receptors. In the present study, we used rat pituitary GC cells with or without stable transfection of cDNA coding for the human GRF receptor (GC/R+ or GC/R-) to determine whether or not GHRP-2 acts via the GRF receptor. Northern blot analysis indicated that GRF receptor mRNA was undetectable in GC/R-cells, whereas a high level of expression occurred in GC/R+ cells that were transfected by GRF receptor cDNA. In GC/R- cells, incubation with up to 10(-7)M of either hGRF or GHRP-2 did not alter the intracellular cAMP, [Ca2+]i, or GH secretion. In GC/R+ cells, hGRF (10(-11)-10(-7)M) increased cAMP levels in a concentration-dependent manner up to 20-fold. This increase in cAMP levels was blocked by a GRF receptor antagonist, [Ac-Tyr1, D-Arg2]-GRF 1-29, but not by a Ca2+ channel blocker, NiCl2 (0.5 mM). GH secretion and [Ca2+]i were, however, not increased by hGRF. Incubation of the transfected cells with 10(-1)-10(-8)MGH RP-2 did not modify intracellular cAMP levels. This result suggests that GHRP-2 does not act through the GRF receptor.

Hypothalamo-pituitary disconnected Soay rams (HPD rams) were treated chronically with a pulsatile infusion of GnRH (250 ng/2 h) for 10 weeks to reactivate the pituitary-gonadal axis under long days (16L:8D; LD group n=8) and short days (8L:16D, SD group n=8). The aim was to establish whether photoperiod modulates the secretion of gonadotrophins (presumably through the daily melatonin signal) by an action at the level of the pituitary gland. The treatment of the HPD rams with GnRH induced a marked, long-term increase in the peripheral blood concentrations of FSH, LH and testosterone, growth of the testes and reappearance of the androgen-dependent sexual skin colouration. There were parallel changes in the short-term, pulsatile fluctuations in the blood concentrations of LH and FSH, the pituitary responsiveness to GnRH and the testicular responsiveness to a standardized injection of oLH, as observed previously in normal intact rams during reactivation of the reproductive axis induced by a switch from long to short days. There were no significant differences in any of the reproductive parameters between the LD and SD HPD groups, although the blood concentrations of prolactin were significantly higher in the LD group, and GnRH provoked a chronic increase in the blood concentrations of prolactin, particularly in the SD group. Overall, the results support the conclusion that (i) melatonin does not act within the pituitary gland to mediate effects of photoperiod on GnRH-induced gonadotrophin secretion; and (ii) changes in prolactin secretion which result from the local action of melatonin in the pituitary gland do not affect gonadotrophin secretion in the ram.

In Soay rams in which the pituitary gland has been surgically separated from the hypothalamus, blood prolactin concentrations vary in response to changes in photoperiod and the administration of melatonin, as in intact animals, providing evidence that melatonin acts within the pituitary gland to control prolactin secretion. In this study the presence of potentially functional melatonin receptors in the pars tuberalis and zona tuberalis (PT/ZT) of hypothalamo-pituitary disconnected (HPD) Soay rams is confirmed using both in vitro autoradiography with the ligand 2-(125I)-iodomelatonin and in situ hybridization for the melatonin receptor. There was no effect of the HPD operation on the pattern and quantity of 2-(125I)iodomelatonin binding in the brain demonstrating that this binding is independent of hypothalamic regulation. The possibility that melatonin may control prolactin secretion directly via specific receptors on lactotrophs was investigated using dual in situ hybridization with a (35S) labelled probe for the ovine melatonin receptor (Mel 1a(b)) and a Digoxigenin labelled probe for ovine prolactin. Melatonin receptor gene expression was observed in the PT/ZT in both intact and HPD rams, however, there was no colocalization with prolactin gene expression; only in the ZT was there a close association between cells expressing the melatonin receptor and lactotrophs. The results provide strong support for the view that melatonin acts via the PT/ZT to mediate the effects of photoperiod on the seasonal cycle in prolactin secretion.

The involvement of protein kinase C (PKC) in the action of GH-releasing factor (GRF) and synthetic GH-releasing peptides (GHRP-2 and GHRP-6) was investigated in ovine somatotrophs in primary culture. In partially purified sheep somatotrophs, GRF and GHRP-2 caused translocation of PKC activity from the cytosol to the cell membranes and caused GH release in a dose- and time-dependent manner. GHRP-6 did not cause PKC translocation. The PKC inhibitors, calphostin C, staurosporine and chelerythrine, partially reduced GH release in response to GRF and GHRP-2 at doses which selectively inhibit PKC activity. These inhibitors totally abolished GH release caused by phorbol 12-myristate 13-acetate (PMA). Down-regulation of PKC by the treatment of cells with phorbol 12,13-dibutyrate for 16 h caused a significant (P < 0.001) reduction in total PKC activity and totally abolished PKC translocation in response to a challenge with GRF, GHRP-2 or PMA. In addition, down-regulation abolished GH release in response to GRF, GHRP-2 or GHRP-6. Treatment of cells with H89, a selective PKA inhibitor, totally blocked GH release caused by either GRF or GHRP-2 and partially reduced PMA-induced GH release. H89 had no effect on PKC translocation caused by GRF, GHRP-2 or PMA and did not affect GH release caused by GHRP-6. These data suggest that GHRP-2 and GRF activate PKC in addition to stimulating adenylyl cyclase activity. Although the cAMP-protein kinase A (PKA) pathway is the major signalling pathway employed by GRF and GHRP-2, the activation of PKC may potentiate signalling via the cAMP-PKA pathway in ovine GH secretion. Importantly, the effect of PMA in increasing the secretion of GH from ovine somatotrophs is effected, in part, by up-regulation of the cAMP-PKA pathway. We conclude that there is cross-talk between the PKC pathway and the cAMP-PKA pathway in ovine somatotrophs during the action of GRF or GHRP.

Hypothalamo-pituitary disconnected (HPD) and control Soay rams were treated chronically for 48 wk with s.c., continuous-release implants of melatonin while under long days (16L:8D). The implants produced continuously elevated blood concentrations of melatonin 2-3 times higher than the normal nocturnal maximum. The long-term treatment induced a biphasic effect on prolactin secretion in both the HPD and control rams, with a marked decrease in the blood prolactin concentrations for 10 wk followed by a gradual increase. The introduction of a second melatonin implant after 20 wk failed to affect prolactin secretion. The treatment with melatonin also caused a dynamic effect on FSH secretion, but this occurred in the control rams only. Blood concentrations of FSH in the HPD rams were very low throughout, but there were minor changes in testicular diameter that were correlated with variations in prolactin. Overall, the results support the conclusion that 1) melatonin acts primarily in the pituitary gland to affect prolactin secretion, and partial refractoriness develops at this level for control of prolactin; and 2) melatonin acts most probably in the hypothalamus to affect gonadotropin secretion, and refractoriness develops at the level of the neural tissue regulating GnRH release for control of gonadotropins.

The pituitary intermediate lobe (IL) contains a single population of cells and has recently been shown to express endothelin (ET)-like peptides. The IL thus provides an excellent in vivo model to study regulation, function and processing of ET in an endocrine cell. The primary aims of the present study were to locate and characterize the precise molecular forms of ET in the ovine IL and determine if levels and/or processing of ET is under dopaminergic or other influences. We have developed a radioimmunoassay (RIA) that detects each form of ET and, when combined with reverse phase-HPLC (RP-HPLC), shows the ovine IL to contain predominantly the ET-1 isoform. In addition, using a specific anti-endothelin antiserum for immunohistochemistry (IHC), we localized ET-1 with alpha-melanocyte stimulating hormone (alpha-MSH) within the melanotroph. The effects of dopamine agonists, antagonists and hypothalamo-pituitary disconnection (HPD) on both tissue levels and processing of ET in the ovine IL were also examined. Normal sheep were treated chronically with haloperidol or bromocriptine to investigate the possibility of dopaminergic regulation of ET in the IL. In the haloperidol-treated group, plasma prolactin levels did not vary significantly from day 0 to day 8, but the bromocriptine treatment reduced prolactin levels (t = 9.4 P < 0.01). Neither bromocriptine nor haloperidol, however, affected tissue ET peptide levels or forms. After HPD, the HPLC profile of pooled IL showed that ET-1 levels in the IL are slightly increased with no change in molecular forms.

The effects of growth hormone-releasing peptide-6 (GHRP-6) on peripheral plasma concentrations of growth hormone (GH) and hypophysial portal plasma concentrations of growth hormone-releasing hormone (GHRH) and somatostatin (SRIF) were investigated in conscious ewes. Paired blood samples were collected from the hypophysial portal vessels and from the jugular vein of nine ewes for at least 2 hr. The sheep were then given a bolus injection of 10 micrograms of GHRP-6 per kg followed by a 2-hr infusion of GHRP-6 (0.1 microgram/kg.hr). Blood sampling continued throughout the infusion and for 2 hr afterwards. An increase in plasma GH concentration was observed in the jugular samples of six of the nine ewes (1.4 +/- 0.3 vs 7.4 +/- 2.0 ng/ml, P < 0.05) 5-10 min after the GHRP-6 bolus injection, but in no case did we observe a significant coincident release of GHRH. During the infusion period, mean plasma GHRH levels were not significantly increased but there was a 50% increase (P < 0.05) in GHRH pulse frequency; GHRH pulse amplitude was not changed. Mean SRIF concentration, pulse frequency, and pulse amplitude were unchanged by GHRP-6 treatment. These data indicate that GHRP-6 causes a small, but significant effect on the pulsatile secretion of GHRH, indicating action at the hypothalamus or higher centers of the brain. The large initial GH secretory response to GHRP-6 injection does not appear to be the result of GHRP-6 action on GHRH or SRIF secretion.

1. Somatotroph-enriched cells (up to 85%) were obtained from ovine pituitary glands by means of collagenase dissociation and Percoll-gradient centrifugation. Further identification was based on the reduction in Ca2+ currents by 10 nM somatostatin (SRIF). 2. The whole-cell configuration of the patch-clamp technique was employed to study the membrane Ca2+ currents with K+ ions replaced by Cs+ and the addition of K+ and Na+ channel blockers in bath and pipette solutions. 3. A significant reduction in Ca2+ currents was obtained in response to local application of SRIF (10 nM) but vehicle application had no effect. 4. Intracellular dialysis of antibodies to alpha(o), alpha(i)-1-2, or alpha(i)-3 subunits of G proteins into the cells via patch-clamp pipettes was confirmed by immunofluorescent staining of the antibodies. Antibody dialysis did not modify resting voltage-gated Ca2+ currents across the cell membrane. 5. Dialysis of anti-alpha(o) antibodies significantly attenuated the reduction in Ca2+ currents that was obtained upon application of 10 or 100 nM SRIF. Dialysis of neither anti-alpha(i)-1-2 nor anti-alpha(i)-3 antibodies diminished the effect of SRIF on Ca2+ currents. 6. Intracellular dialysis of antisense oligonucleotides directed against the alpha(o) subunit mRNA (alpha(o) ASm, for alpha(o) common) or against the alpha(i)-3 subunit mRNA (alpha(i)-3 AS) blocked expression of alpha(o) or alpha(i)-3 subunits in the cells, respectively, as assessed by fluorescent staining with anti-alpha(o) or anti-alpha(i)-3 antibodies 48 h after dialysis. 7. Dialysis of alpha(o) ASm, but not alpha(i)-3 AS, significantly diminished the inhibitory effect of SRIF on Ca2+ currents. This effect of alpha(o) ASm dialysis occurred within 12 h after dialysis and reached a maximum at 48 h; partial recovery was seen at 72 h. 8. Antisense oligonucleotides specific for alpha(o)-1 (alpha(o)-1 AS) or alpha(o)-2 (alpha(o)-2 AS) were dialysed into somatotrophs and only alpha(o)-2 AS significantly attenuated the inhibition of Ca2+ currents by SRIF. 9. We conclude that the G(o)-2 protein mediates the effect of SRIF on Ca2+ currents in ovine somatotrophs in primary culture.

Episodic release of luteinizing hormone (LH) by the pituitary gland is controlled by hypothalamic gonadotropin-releasing hormone (GnRH). In the period leading up to the preovulatory surge of LH, estrogen increases the number of pituitary receptors for GnRH and sensitises the gonadotropes to GnRH. The postreceptor events that are responsible for the increase in responsiveness to GnRH are not clearly delineated, but LH release is known to be Ca2+ dependent. The present study addressed the question as to whether or not estrogen may act to modify voltage-dependent Ca2+ entry in normal gonadotropes. Primary cultures enriched in gonadotropes or somatotropes were produced from anestrous female sheep. Conventional whole-cell patch-clamp recording was used to measure inward membrane current in the absence of GnRH treatment, with and without 10 nM estradiol-17beta (E2) treatment for 0 to 36 h. Nystatin-perforated whole-cell patch-clamp recording was used to record membrane voltage responses to GnRH. Ca2+ current density (ICa, pA/pF) began to increase after 2 h exposure to E2, and reached peak values of about 200% of control by 16-20 h (p < 0.005), then declined. If E2 was withdrawn at 24 h, ICa returned towards control values by 36 h. If E2 treatment was continued beyond 24 h, however, ICa fell to about 75% of control by 36 h (p < 0.005). Actinomycin D prevented the enhancement of ICa. E2 was without effect on Na+ current density in gonadotropes, or on ICa in somatotropes. The proportion of ICa carried by L-type and N-type channels in gondadotropes was not changed by E2. Ovine gonadotropes respond to GnRH with membrane potential fluctuations driven by periodic activation of Ca2+-dependent K+ channels, and synchronised action potential generation. This response was found to be sensitive to E2. Responses were categorised according to the pattern of activity evoked by 10 nM GnRH. Without E2 treatment, 11/14 cells responded with oscillations and 3/14 cells responded with spiking (hyperpolarizations following single action potentials). After 20 h 10 nM E2, just 1/14 cells responded with hyperpolarizing oscillations while 13/14 cells showed spiking activity. The predominance of the spiking pattern in E2-treated cells is consistent with the increased Ca2+ flux, and with enhanced LH release. We conclude that E2 has a transient effect on gonadotropes to enhance voltage-gated Ca2+ channel function. The time-course and biphasic nature of the influence of E2 on ICa may be physiologically appropriate to the preovulatory LH surge. Enhanced Ca2+ influx may participate in increased Ca2+-dependent hormone release, while the delayed inhibitory action of E2 on ICa may serve to limit the duration of the surge.

A previous study provided evidence that melatonin acts in the pituitary gland to mediate the effects of daylength on the secretion of prolactin in sheep. This was based on the observation that hypothalamo-pituitary disconnected (HPD) Soay rams showed normal patterns in the changes in the peripheral blood concentrations of prolactin in response to alterations in photoperiod (10-fold higher concentrations under long than short days), and in response to exogenous melatonin (rapid decline following the administration of a constant-release implant of melatonin). The purpose of this study was to establish whether dopamine (DA) might be involved in mediating the effects of melatonin on the secretion of prolactin. Groups of HPD (n = 7) and control Soay rams (n = 8) were treated with vehicle (control, 2.0 ml 0.1 M tartaric acid/saline sc), bromocriptine (DA agonist, 0.06 mg/kg sc) or sulpiride (DA antagonist, 0.6 mg/kg sc), and the acute prolactin responses were measured over the next 4 h. Treatments were carried out under short days (8L: 16D, low prolactin), long days (16L: 8D), high prolactin), and under long days in the presence of a constant-release implant of melatonin (low prolactin). The prolactin response to TRH (1.25 micrograms/kg iv) was also measured. Bromocriptine caused a decrease in the plasma concentrations of prolactin in both HPD and control rams under short and long days. Sulpiride had no effect in the HPD rams on any occasion, but caused a very marked increase in the plasma concentrations of prolactin in the control rams under short days, long days, and under long days + melatonin. TRH caused an acute increase in the plasma concentrations of prolactin in the HPD rams under both long and short days although the responses were notably reduced compared with the controls especially under long days + melatonin. Overall, the inhibitory response to the DA agonist in HPD rams indicates the presence of DA D2 receptors linked to functional lactotrophs in the isolated pituitary gland. However, the total lack of a response to the DA antagonist indicates the absence of endogenous DA mechanisms regulating the secretion of prolactin in the HPD rams. The conclusion is that melatonin acts directly on the pituitary gland to mediate effects of photoperiod through a DA-independent mechanism.

We have used conventional whole-cell patch-clamp to investigate the membrane currents of ovine anterior pituitary gonadotropes, and nystatin-perforated whole-cell patch-clamp to record the membrane potential changes elicited by the natural hypothalamic secretagogue, gonadotropin-releasing hormone (GnRH). A large basal inward current found by voltage clamp was blocked by tetrodotoxin (TTX) (ED50 < 10 nM), identifying it as a Na+ current. Slowly inactivating inward current, activated at potentials more positive than -30 mV, remained in Na(+)-free medium or in the presence of 1 microM TTX. This current was abolished by ionic Ca2+ channel blockade. In the presence of nifedipine about 70% of this high voltage-activated Ca2+ current was abolished, leaving a slowly inactivating component. No transient Ca2+ current was found. The nifedipine-insensitive slowly inactivating inward current was eliminated by 1 microM omega-conotoxin GVIA (CGTX), consistent with the presence of N-type channels. Outward K+ currents sensitive to membrane voltage and intracellular Ca2+ concentration ([Ca2+]i) were present. The resting membrane potential lay between -20 and -75 mV (mean = -43 +/- 1.5) with spontaneous TTX-sensitive action potentials occurring in 34% of cells. GnRH had concentration-dependent effects on gonadotrope membrane potential. Application of 100 nM GnRH resulted in a rapid hyperpolarization, followed by a gradual depolarization during which action potentials returned briefly. This was followed by protracted electrical quiescence. Application of 1 or 10 nM GnRH led to hyperpolarization, followed by gradual depolarization, upon which rhythmic hyperpolarizations were superimposed, giving membrane potential oscillations. During the depolarising stage of each oscillation, a burst of action potentials occurred. Action potentials, then oscillations, ceased after 5-15 min. Depolarization was then maintained (at -20 to -35 mV) for up to 1 h. Apamin, the SK-type Ca(2+)-dependent K+ channel blocker, prevented the hyperpolarizing oscillations and produced membrane depolarisation, but Ca2+ channel blockade did not. Microfluorimetric detection of [Ca2+]i showed that 10 nM GnRH induced [Ca2+]i oscillations. We conclude that Ca2+ derived from intracellular pools is involved in producing the membrane potential oscillations. The [Ca2+]i fluctuations may activate the apamin-sensitive, Ca(2+)-dependent SK-type K+ channel, and entrain TTX-sensitive action potentials to a bursting pattern of generation following GnRH stimulation. In the absence of T-type currents, the Na+ current spikes may be crucial for activation of the nifedipine- and CGTX-sensitive high-voltage-activated Ca2+ channels.

Experiments were performed to test the hypothesis that there is a negative feedback 'clamp' of ovarian hormones on the hypothalamus and pituitary gland during the follicular phase of the oestrous cycle that limits the secretion of GnRH and LH. GnRH secretion was monitored by sampling the hypophysial portal blood of ewes during the luteal phase of the oestrous cycle and either 24 h or 48 h after the induction of luteolysis by the injection of cloprostenol, a prostaglandin analogue. There was an increase in GnRH pulse frequency in the transition from the luteal to the follicular phase of the cycle. A reduction in the amplitude of GnRH pulses did not occur until 48 h after cloprostenol, suggestive of negative feedback at the level of the hypothalamus that is more profound in the latter part of the follicular phase. The responsivity of the pituitary gland to GnRH was monitored in ewes during the luteal phase of the oestrous cycle and 24 h or 48 h after cloprostenol. Injections of 250 ng or 1000 ng GnRH were given (i.v.) to ewes that had been anaesthetised to suppress endogenous secretion of GnRH and LH. Using the lower dose, the responses 48 h after cloprostenol were not significantly different from those in the luteal phase. With the higher dose of GnRH, a significant (P < 0.05) increase in mean responsivity was seen 48 h after cloprostenol. There was, however, a marked variation in response, with some ewes showing profound increases in LH secretion in response to GnRH and others showing responses that were similar to those obtained during the luteal phase of the cycle. These data are interpreted to mean that the secretion of LH is 'clamped' during the follicular phase of the oestrous cycle and the 'clamp' is only released near the time of the preovulatory LH surge. To test whether or not a rise in GnRH input to the pituitary gland could over-ride the 'clamp' on the pituitary secretion of LH in the late follicular phase of the cycle, sheep were treated 40 h after cloprostenol with either a bolus injection of 500 ng GnRH or four pulses of 125 ng GnRH given at 10-min intervals. These treatments caused small elevations in LH secretion but did not always cause preovulatory LH surges. In some cases, a small rise in LH secretion was induced by GnRH treatments and levels of LH in plasma returned to baseline with the preovulatory LH surge occurring a few hours later.(ABSTRACT TRUNCATED AT 400 WORDS)

The putative negative feedback effects of IGF-I and IGF-II on GH secretion were tested by intracerebroventricular (icv) and intrapituitary administration to sheep. Over two consecutive days, serial jugular blood samples were taken at 10 min intervals for 6 h from ewes (n = 3/group) fitted with indwelling stainless steel cannulae into the lateral or third cerebral ventricles. The sheep were injected (icv) with either vehicle or purified ovine IGF-I (2, 4 or 8 micrograms). IGF-I injection had no effect on plasma GH secretion. Serial blood samples were taken from a second group of nine ewes in which ovine or recombinant human (rh) IGF-I was infused (2.5 micrograms/h for 2 h) into the third ventricle; once again, IGF-I failed to affect the episodic pattern of GH secretion. Three ewes fitted with indwelling stainless steel cannulae placed in the anterior pituitary gland were consecutively infused with either ovine or rhIGF-I (2.5 micrograms/h for 2 h) or vehicle. Plasma GH concentrations were suppressed in 3/3 sheep from 1-1.5 h after the commencement of infusion and GH levels remained low for the remainder of the sampling period. In another group of five ewes synergistic effects of IGF-I and IGF-II on GH secretion were tested by icv infusion of rhIGF-I, rhIGF-II, or rhIGF-I+rhIGF-II (5 micrograms/h for 2 h) or vehicle (sterile 10 mM HCl/saline). Each sheep received each treatment in a randomised design. Infusion (icv) of IGF-I and IGF-II alone or in combination failed to alter GH secretion.(ABSTRACT TRUNCATED AT 250 WORDS)

Voltage-gated Ca2+ currents were recorded using the nystatin-perforated whole cell recording configuration on the ovine somatotrophs. With the use of Ca(2+)-tetraethylammonium chloride bath solution and Cs+ electrode solution, two types of Ca2+ currents were obtained with a predominant long-lasting (L) current blocked by nifedipine. A transient (T) current was isolated in the presence of nifedipine (3 microM) and was not blocked by omega-conotoxin (5 microM), but diminished to 47 +/- 5% of control by Ni2+ (0.3 mM) or to 52 +/- 10% of control by amiloride (0.5 mM). The nifedipine-blockable L-type current was not affected by omega-conotoxin (5 microM); it was, however, attenuated to 80 +/- 4% of control by Ni2+ (0.3 mM) and to 48 +/- 6% of control by amiloride (0.5 nM). Cd2+ (1 mM) totally prevented both T and L currents. Application of growth hormone-releasing factor (GRF, 10 nM) reversibly increased the amplitude of both Ca2+ currents without modifying their kinetic properties. The effect of GRF was observed approximately 30 s after application, peaked (142 +/- 11% of control, n = 5) rapidly, and lasted > 10 min if GRF treatment was continuous. Intracellular Ca2+ concentration ([Ca2+]i) was increased by GRF (10 nM) within seconds, reaching a peak within 30 s and lasting > 250 s. Blockade of Ca2+ channels (Cd2+, 1 mM) or the use of Ca(2+)-free solution reduced basal [Ca2+]i and significantly (P < 0.05) diminished the effect of GRF on [Ca2+]i.(ABSTRACT TRUNCATED AT 250 WORDS)

The roles of testicular hormones in the negative feedback regulation of the secretion and actions of GnRH in male domestic ruminants are reviewed, concentrating mainly on research conducted with rams. Testicular steroids have major feedback actions directly at the hypothalamus to inhibit the secretion of GnRH, although it is apparent that, under certain circumstances, the steroids also have actions directly at the pituitary gland. Further research is necessary to delineate these actions and to determine the contribution of testosterone and its primary metabolites, dihydrotestosterone and oestradiol, to negative feedback on the hypothalamo-pituitary unit. Since GnRH neurones do not possess receptors for steroids, testicular steroids must evoke other neuronal pathways to influence GnRH producing neurones. While the opioids may be important in this regard, it is necessary to determine which other neuronal pathways may also be involved to understand fully the mechanism of action of testicular steroids. It appears that the feedback regulation of the secretion of LH can be accounted for by testicular steroids, whereas the secretion of FSH is influenced by inhibin and steroids, and possibly the recently isolated proteins follistatin and activin. The actions of inhibin to suppress the secretion of FSH occur at the pituitary gland and not on the synthesis or secretion of GnRH. There is a complex interaction between testosterone and inhibin in the control of FSH secretion that results in synergistic effects during the non-breeding season but not during the breeding season. Activin has been shown to have FSH-stimulating properties and follistatin has been shown to have FSH-inhibiting properties, but it is unknown if these proteins play a physiological role in the feedback regulation of FSH in male domestic ruminants.

The effect of hypothalamopituitary disconnection (HPD) on growth hormone (GH) secretion was studied in sheep. Plasma GH levels were measured in serial blood samples (10 min x 6-8 h) taken from 12 Romney wethers and 5 ewes which had undergone HPD 40-506 days earlier and from 4 wethers (10 min x 7 h) to serve as controls. Five wethers and 5 ewes were taken approximately 300 days after HPD and injected with vehicle or 10 micrograms/kg of the synthetic hexapeptide growth hormone-releasing peptide 6 (GHRP-6); GH responses were monitored. In a second series of sheep, 4 wethers and 6 HPD wethers were given saline, 0.5 micrograms/kg of synthetic growth hormone-releasing factor (GRF) or 5 micrograms/kg GHRP-6 and were blood-sampled to measure the plasma GH response. A further group of 4 HPD wethers and 3 control wethers were killed and the anterior pituitary glands collected for the quantification of GH and LH beta mRNA by Northern analysis. Three HPD wethers and 1 HPD ewe and 1 control ewe were killed and their brains were perfused; the hypothalami were sectioned and immunostained for the presence of GRF-containing fibres in the median eminence. Normal episodic GH secretion was abolished by HPD in both wethers and ewes but plasma values did not fall below the assay detection limit, indicating constitutive secretion. Northern blot analyses showed that the GH mRNA was detectable in HPD wethers at a lower (p < 0.05) level than in control animals but mRNA for LH beta was undetectable in the HPD wethers. Immunohistochemistry revealed GRF-positive staining in the median eminence of the controls but GRF-positive staining was not seen below the operation site in the median eminence of the HPD animals. In the first series, 3/5 wethers and 3/5 of the ewes responded to GHRP-6 challenge; the magnitude of the response was similar in both sexes. In the second series, responses to GRF were lower (48%) (p < 0.03) in HPD wethers than in control wethers, and responses to GHRP-6 were much lower (p < 0.01) than those to GRF in both controls and HPD wethers. These studies show that HPD removes GRF inputs to the pituitary gland and abolishes pulsatile GH secretion in most cases but constitutive non-pulsatile secretion continues. The HPD wether pituitary glands had lower GH mRNA levels than controls. Accordingly, HPD sheep were able to respond to a single injection of GRF although the response was half that seen in control animals.(ABSTRACT TRUNCATED AT 400 WORDS)

Long term changes in the secretion of prolactin were monitored in groups of hypothalamo-pituitary disconnected rams (HPD rams, n = 8) and control rams (HPD sham-operated and unoperated, n = 8) while exposed to an artificial lighting regimen of alternating 16-weekly periods of long days (16L:8D) and short days (8L:16D) for 72 weeks, and during a treatment with subcutaneous constant-release implants of melatonin under long days. The HPD rams showed all the clinical characteristics of complete pituitary disconnection (diabetes insipidus, gonadal regression and slight obesity), and were unresponsive to a range of provocation tests (exposure to a barking sheep dog, cannulation of the jugular vein, injection of serotonin and NMDA) which caused acute changes in the blood plasma concentrations of prolactin in the controls. Nevertheless, there was a clearly defined cycle in the blood concentrations of prolactin in the HPD rams related to the imposed lighting regimen with values 10-fold higher under long days compared to short days (HPD mean +/- SEM: 90.1 +/- 24.7 vs 9.4 +/- 2.0 micrograms/l, long vs short day respectively, P < 0.001). The temporal pattern was very similar to that observed in the controls, although the concentrations of prolactin were higher in the HPD rams and more variable (control mean +/- SEM: 55.6 +/- 3.6 vs 3.0 +/- 0.5 micrograms/l, long vs short day, P < 0.001). There was a corresponding cycle in the growth and moulting of the wool in the HPD rams consistent with a biological response to the photoperiodically-induced changes in the secretion of prolactin. The diurnal rhythm in the blood concentrations of prolactin was absent in the HPD rams, but there was a normal rhythm in the secretion of melatonin. The treatment of the animals with constant-release implants of melatonin under long days caused a marked decrease in the blood concentrations of prolactin in both the HPD and control rams. The overall conclusion is that the endogenously generated daily melatonin signal which encodes daylength acts directly in the pituitary gland to mediate the effects of photo-period on the secretion of prolactin. The photo-period transduction pathway thus by-passes the hypothalamus.

This study examined the effect of thyroidectomy (TX) on the GH axis in sheep. The secretion of GH was monitored 10 and 77 days after TX or sham-TX when the effects on plasma GH and prolactin levels of the injection of 0.5 micrograms GH-releasing factor (GRF)/kg and 1 microgram thyrotrophin-releasing hormone (TRH)/kg were also assessed. There were no significant differences in GH pulse amplitude, pulse frequency, inter-pulse interval and GH secreted/h between sham-TX and TX animals at 10 or 77 days after TX. There was no difference in the GH response to GRF injection in sham-TX sheep at any time but in TX sheep the GH response was significantly (P < 0.05) attenuated 10 days after TX. After 77 days the GH response was similar to the response before TX. There was no measurable GH response to injection of TRH in sham-operated or TX sheep at any time. The prolactin response to TRH was not affected by TX or sham-TX. These results suggest that TX in sheep does not affect GH secretion but paradoxically the response to GRF is attenuated in hypothyroid sheep in the short term. TRH causes release of prolactin but not GH in sheep.

A newly synthesised GH-releasing peptide, KP 102 (also named GHRP-2), was studied in an in vitro perifusion system of primary cultured ovine anterior pituitary cells. Application of KP 102 to the perifusion medium caused a dose-dependent increase in GH secretion. Dose-response relationships indicated that KP 102 had similar potency to GRF and was 10-fold more potent than earlier generations of GH-releasing peptide (GHRP-6 and GHRP-1) tested in same system. The response to a second application of KP 102 given within 1 h of initial application was significantly lower than the response to the first application. When KP 102 (or GRF) was applied first and then GRF (or KP 102) given 1 h later, the second response was not attenuated. When GRF and KP 102 were coadministered, an additive effect on release of GH was obtained. The effect of maximal dose of KP 102 (100 nM) on GH release was totally abolished by [Ac-Tyr1,D-Arg2] GRF 1-29 (1 microM) which is believed to be a specific antagonist for the GRF receptor. Blockade of Ca2+ channels by Cd2+ (2 mM) diminished the basal GH secretion and abolished the increase in GH release in response to KP 102 (100 nM). These data suggest that the action of KP 102 is blocked by a GRF receptor antagonist and therefore acts through a different receptor to that employed by earlier generations of GH-releasing peptides. GH release in response to KP 102 involves an increase in Ca2+ influx and there is no cross-desensitization between KP 102 and GRF responses.

We have measured the relative levels of proopiomelanocortin (POMC), prolactin (PRL) and growth hormone (GH) messenger ribonucleic acid (mRNA) in the anterior and neurointermediate lobes of the fetal pituitary during the last 2-3 weeks of gestation. The mean POMC mRNA/18S RNA ratio in the fetal anterior pituitary was significantly greater (p < 0.02) at 130-136 days (0.90 +/- 0.08; N = 9) than at 141-143 days of gestation (0.67 +/- 0.07; N = 6). In contrast, the mean PRL mRNA/18S RNA ratio increased significantly (p < 0.02) between 130 and 136 days (0.31 +/- 0.05; N = 9) when compared with 141-143 days of gestation (0.58 +/- 0.10; N = 6). There was no significant difference, however, between the mean GH mRNA/18S RNA ratio in fetal anterior pituitaries at 130-136 days (0.95 +/- 0.04; N = 9) when compared with 141-143 days of gestation (1.08 +/- 0.14; N = 6). The POMC mRNA/18S RNA ratio in the neurointermediate lobes was seven-, five- and tenfold higher than anterior pituitaries at 130-134, 135-136 and 141-143 days of gestation, respectively. We hypothesize that elevated circulating cortisol levels after 140 days of gestation act in the slow time domain (i.e. over days) to suppress POMC gene expression and that the increase in fetal pituitary PRL mRNA levels may be a consequence of oestrogen stimulation in late gestation.

The roles of inhibin and testosterone in the negative feedback control of the secretion of FSH were explored in experiments using castrated rams administered human recombinant inhibin A (hr-inhibin) and testosterone propionate (TP). Two experiments were conducted in the non-breeding season. In experiment 1, two groups of long-term castrated rams (wethers) were treated with an i.v. injection of either vehicle or hr-inhibin in two doses (25 and 50 micrograms) given 2 weeks apart. Plasma concentrations of FSH, measured by radioimmunoassay, were suppressed significantly (P < 0.01) and equally by both doses of hr-inhibin with a mean (+/- S.E.M.) maximal suppression of FSH of 19.9 +/- 2.60% occurring 6-10 h after injection. In experiment 2, hypothalamo-pituitary disconnected (HPD) wethers given 125 ng gonadotrophin-releasing hormone (GnRH) every 2 h, were treated with vehicle or 25 or 50 micrograms hr-inhibin before or after treatment (32 mg/day) with TP. A cross-over design was used so that each wether was treated with vehicle and hr-inhibin. Treatment with TP significantly (P < 0.001) suppressed plasma concentrations of FSH by 56%. Both doses of hr-inhibin were similarly effective in significantly (P < 0.05) suppressing plasma concentrations of FSH causing a mean suppression of 31.1 +/- 5.60% 6-10 h after injection. The suppressive effect of hr-inhibin was significantly (P < 0.05) increased when the wethers were treated with TP to a mean suppression of 50.7 +/- 5.6% 6-10 h after injection.(ABSTRACT TRUNCATED AT 250 WORDS)

To study the mechanism underlying the effect of dopamine withdrawal on prolactin release, continuous perfusion experiments were performed on rat lactotroph-enriched primary cultures. Removal of dopamine (10(-7) M) after a short-term application (15 min) produced a rebound of prolactin secretion, which was enhanced by pretreatment of the cell culture with 17 beta-estradiol (10(-8) M for 48 h). Ca2+ channel blockade by Co2+ (1 mM) abolished the rebound in prolactin release. An increase in intracellular adenosine 3',5'-cyclic monophosphate by either forskolin (5 microM) or 3-isobutyl-1-methylxanthine (100 microM) enhanced the prolactin rebound after dopamine withdrawal. Application of thyrotropin-releasing hormone (10(-7) M) increased the prolactin rebound after dopamine withdrawal with a maximum effect obtained by commencing treatment immediately after removal of dopamine. Pretreatment of cell cultures with pertussis toxin (100 ng/ml, for 10 h) totally abolished the effects of dopamine on prolactin secretion. The dopamine agonist bromocriptine (10(-9) M) significantly decreased prolactin secretion, but no rebound effect was observed after its removal. We conclude that the rebound of prolactin release after dopamine treatment involves the influx of Ca2+.

Studies were conducted in vitro and in vivo to determine whether or not inhibin affects the transcription rate of the gene for the beta subunit of follicle stimulating hormone (FSH beta). Pituitary cells in primary culture were incubated with 0-3000 milli-units/ml inhibin; a dose-related decrement in mRNA was obtained but a parallel effect was not observed for the transcription rate of the FSH beta gene in a nuclear run-on experiment. To determine effects in vivo, ovariectomized ewes were treated with saline (group 1), 75 micrograms inhibin 6 h before slaughter (group 2), inhibin 6 h and 12 h before slaughter (group 3) or inhibin 12 h before slaughter (group 4). In samples taken each 2 h, plasma FSH levels were seen to be maximally reduced 6 h after a single injection of inhibin; at this time mRNA levels were reduced up to 100% whereas FSH beta gene transcription rate was reduced by 50%. A second injection at 6 h (group 3) caused a further reduction in plasma FSH levels with no additional effect on transcription rate. In those sheep killed 12 h after a single inhibin injection, transcription rate for the FSH beta gene, cytoplasmic mRNA levels and plasma FSH concentrations had recovered. These studies show that the rapid effect of inhibin on FSH beta mRNA levels may be due, in part, to an effect on transcription rate of the FSH beta gene. An additional mechanism is required, however, to fully explain the inhibin effect on FSH beta mRNA levels.(ABSTRACT TRUNCATED AT 250 WORDS)

Since it has become possible to sample hypophysial portal blood from sheep without totally compromising pituitary function, several important features of the secretion of hypothalamic hormones have been elucidated. The secretion of gonadotropin-releasing hormone (GnRH) has been detailed most thoroughly with the important observation that each pulsatile discharge of luteinizing hormone (LH) is the direct result of a large secretory episode of GnRH from the hypothalamus. There is high fidelity in the GnRH relationship in terms of frequency and amplitude. During the LH surge, additional factors such as an alteration in the degree of enzymic degradation of GnRH may be important physiological mechanisms. The secretion of factors that control the release of growth hormone (GH), prolactin and adrenocorticotropic hormone (ACTH) have also been studied. Hypothalamic factors controlling GH and ACTH release do not bear such an explicit relationship to the secretory episodes of pituitary hormone as seen with the GnRH/LH axis. The factor involved in the acute stress-induced release of prolactin has not yet been identified in sheep.

These studies were undertaken to test the hypothesis that stimulation of the central noradrenergic and adrenergic pathways activates the hypothalamic-pituitary-adrenal axis in vivo in the conscious sheep. Blood samples were taken at 10-min intervals over 4 h to establish the baseline state, and then each animal received an intracerebroventricular (icv) injection of NaCl (control animals) or catecholamine [norepinephrine (NE) or epinephrine (EPI)]. A more frequent rate of venous sampling was used for the 30-min period after the icv injection, after which time the 10-min rate of blood sampling was continued for another 3.5 h. NaCl (n = 4) caused no change in pituitary-adrenal secretion. In contrast, 10 micrograms NE (n = 4) caused acute 1.9- and 3.2-fold increases in mean plasma ACTH and cortisol levels over the 1 h period post injection, and 1.6- and 2.3-fold increments in their concentrations over the 4 h postinjection period. Although 10 micrograms EPI (n = 4) did not elevate mean plasma ACTH, it produced significant 1.7- and 1.5-fold increases in plasma cortisol during the 1- and 4-h periods post injection. However, when 100 micrograms EPI was injected (n = 4), acute 9.5- and 5.5-fold increases in plasma ACTH and cortisol were seen over the 1 h period post injection, and 6.1- and 4.2-fold increments in their plasma concentration were noted during the entire post-injection period. To determine the predominant site of action of the catecholamines, we also examined the ability of NE and EPI to release ACTH from cultured ovine anterior pituitary cells. NE and EPI (10(-9)-10(-6) M) stimulated the release of ACTH in a dose-dependent manner, but with maximal increments only 1.5-fold greater than the basal secretion. NE and EPI also increased the maximal ACTH response to CRF, but did not alter the maximal ACTH release induced by arginine vasopressin.(ABSTRACT TRUNCATED AT 400 WORDS)

To test whether the F gene-specific differences in the plasma concentrations of FSH and LH are due to differences in the pituitary responsiveness to exogenous GnRH, ovariectomized Booroola ewes with hypothalamic-pituitary disconnection (HPD-ovx) were treated with GnRH (250 ng i.v.) once every 2 h for up to 5 weeks. In Exp. 1, jugular venous blood was collected once weekly from 13 FF and 14 ++ HPD-ovx ewes for 6 weeks before GnRH treatment and every 2nd, 3rd or 6th day for 5 weeks during treatment. In Exp. 2, jugular venous blood was collected from another 8 FF and 7 ++ HPD-ovx ewes at 5- or 10-min intervals over 4 GnRH pulses (250 ng i.v. once every 2 h) on 3 separate occasions after the animals had been subjected to the GnRH pulse regimen for approximately 7 days beforehand. Also in Exp. 2, the animals were extensively sampled around a larger (10 micrograms) i.v. injection of GnRH and the pituitary FSH and LH contents assessed after the animals had been re-exposed to the once every 2 h GnRH (250 ng i.v.) pulse regimen for several days following the larger GnRH bolus. In Exp. 3 the distributions of mean plasma concentrations of FSH and LH in individual GnRH-treated HPD-ovx ewes were compared with those in ovariectomized and ovary-intact FF and ++ ewes. During the 6 weeks before GnRH treatment (Exp. 1), the plasma concentrations of FSH (approximately 1 ng/ml) and LH (less than or equal to 0.8 ng/ml) were not different between the genotypes. After GnRH treatment both the mean FSH and LH concentrations increased significantly (P less than 0.01) above basal values after 2 days with F gene-specific differences being noted for FSH but not LH (FSH; FF greater than ++; P less than 0.05). Thereafter, the mean FSH but not LH concentrations increased at a faster rate in FF than in ++ ewes with the overall mean FSH concentrations between the genotypes being significantly different (P less than 0.05). In Exp. 2 considerable between-animal variation in the pulsatile pattern of FSH but not LH concentrations was seen in ewes of both genotypes during GnRH treatment. The overall mean FSH concentrations were higher in FF than in ++ ewes (P less than 0.05) and the mean FSH response to each GnRH pulse was significantly higher in FF than in ++ ewes (P less than 0.05).(ABSTRACT TRUNCATED AT 400 WORDS)

These studies characterized the secretion of GH-releasing factor (GRF) and somatostatin (SRIF) into the hypophysial portal circulation in ewes after long term restricted feeding. In addition, we examined the temporal relationship between the concentrations of these two hypothalamic peptides in portal blood and the concentration of GH in jugular blood. Six sheep were fed 1000 g hay/day (normal feeding) and 6 sheep were fed 400-600 g hay/day (restricted feeding). This resulted in a wt loss of 35% in restricted animals compared with 6% in control animals after 20 weeks. Fluctuations in portal levels of GRF indicated a pulsatile pattern of secretion with approximately 60% of pulses coincident with, or immediately preceding, a GH pulse. Similarly, 65% of GH pulses were associated with GRF pulses. Restricted feeding increased (P less than 0.01) mean ( +/- SEM) plasma GH levels (9.8 +/- 1.4 vs. 2.9 +/- 0.6 ng/ml) and mean GH pulse amplitude (7.9 +/- 1.8 vs. 2.8 +/- 0.3 ng/ml) but did not affect mean GH pulse frequency (6.0 +/- 1.1 vs. 5.7 +/- 1.1 pulses/8 h). The level of feeding had no effect on mean portal concentration of GRF (restricted: 5.5 +/- 0.8, normal: 6.6 +/- 1.4 pg/ml), GRF pulse amplitude (14.7 +/- 2.3 vs. 13.5 +/- 0.7 pg/ml), or GRF pulse frequency (5.3 +/- 1.1 vs. 6.7 +/- 0.9 pulses/8 h). Portal concentrations of SRIF in sheep on a restricted diet were half (P less than 0.01) those of sheep fed a normal diet (10.2 +/- 2.3 vs. 19.6 +/- 1.6 pg/ml). Pulses of SRIF were not significantly associated with changes in GH or GRF concentrations. These data indicate a functional role for hypothalamic GRF in initiating GH pulses. Furthermore, the increase in GH secretion in underfed sheep was most probably due to a decrease in the release of SRIF into hypophysial portal blood. Restricted feeding had no affect on GRF secretion, but because of the reduced exposure of the pituitary gland to SRIF, it is possible that responsiveness to GRF is enhanced.

To determine whether opiates directly modulate pituitary LH secretion in vivo, morphine was administered to hypothalamo-pituitary-disconnected (HPD) ewes which were receiving exogenous pulses of GnRH. To define the steroidal background which is permissive to a morphine-induced decrease in LH secretion, ovariectomized (OVX) ewes were treated as follows in groups of four: group 1, no implant; group 2, small 17 beta-estradiol (E2) (1 cm long x 0.33 diameter) and progesterone (P) implants; group 3, medium E2 (1 cm long x 0.46 diameter) and P implants, and group 4, medium E2 implants. Jugular blood samples were taken at 10-min intervals for 9 h, during which there was a 3-hour pretreatment period, a 3-hour treatment period when the sheep were given six intravenous injections of 10 mg morphine every 30 min, and a 3-hour run-off period. Morphine inhibited the mean plasma concentrations of LH and LH pulse frequency in group 3 only, and in 2/4 ewes in this group LH secretion was abolished and did not return to a pulsatile mode during the 3-hour run-off sampling period. In a second experiment designed to test the pituitary action of morphine, OVX-HPD ewes were primed with medium E2 and P implants and were given hourly pulses of 250 ng GnRH intravenously. Jugular blood samples were taken around each GnRH pulse over an 8-hour period. The first three pulses served as a control sampling period, after which the sheep were treated with morphine (six intravenous injections of 10 mg morphine every 30 min).(ABSTRACT TRUNCATED AT 250 WORDS)

Long-term (i.e. approximately 70 days) hypophysectomy led to a significant (P less than 0.05) reduction in ovarian weight but no reduction in the total number of antral follicles (greater than 0.1 mm in diameter). In hypophysectomized ++ Booroola ewes (N = 8) follicles were always less than or equal to 3 mm and in hypophysectomized FF Booroola ewes (N = 6) follicles were always less than or equal to 2 mm in diameter; in ewes of both genotypes follicles reached diameters which were approximately 40% of their predicted final size at ovulation. Under in-vitro conditions, follicles from the FF and ++ hypophysectomized ewes produced significant increases in cAMP within 1 h of exposure to gonadotrophins (P less than 0.05) although no genotypic differences in cAMP production were noted. We conclude that ovarian follicles in FF and ++ ewes have absolute requirements for pituitary hormone on reaching diameters of 2 mm and 3 mm respectively and that appreciable numbers of antral follicles in ewes of both genotypes remain responsive to pituitary gonadotrophins despite prolonged deprivation of these hormones.

Studies were performed to determine whether the isolated ovine anterior and intermediate pituitary might rhythmically secrete three POMC peptides, ACTH, ir-beta-endorphin (ir-beta-EP), and ir-alpha-melanocyte stimulating hormone (ir-alpha-MSH) in vivo. When blood was taken at 10-min intervals from four ewes with hypothalamo-pituitary-disconnection (HPD), a distinct POMC-peptide and cortisol ultradian rhythm was noted. A comparison of the four HPD ewes with five nonstressed hypothalamopituitary-intact (HPI) ewes revealed that the mean plasma levels of the three POMC-peptides and cortisol were increased, the mean ACTH and ir-alpha-MSH pulse amplitudes were increased, and the mean ir-beta-EP and ir-alpha-MSH interpulse intervals were decreased. When four HPI ewes were subjected to a mild stress, plasma POMC-peptide and cortisol levels increased significantly when compared with the five unstressed HPI animals. In addition, the ACTH and cortisol pulse amplitudes increased and the ir-beta-EP and ir-alpha-MSH interpulse intervals decreased. Although plasma ACTH levels in the stressed HPI and HPD ewes were comparable, mean plasma cortisol levels were 2-fold greater in the stressed HPI animals. To determine whether the ACTH hypersecretion in the HPD ewe might reflect a net reduction in hypothalamic inhibitory influence over ACTH secretion, we examined the effects of dopamine (DA), somatostatin (SS-14), and rat atrial natriuretic peptide [rANF(1-28)] on the secretion of ACTH from cultured ovine anterior pituitary cells. DA and SS-14 did not exert a discernible effect on basal, CRF-, or arginine vasopressin (AVP)-stimulated ACTH secretion. Although basal ACTH secretion was unaffected by rANF(1-28) (10(-12)-10(-8) M), a significant inhibition of CRF- and AVP-stimulated ACTH release was observed.(ABSTRACT TRUNCATED AT 400 WORDS)

The effects of long term restricted feeding on the synthesis, storage, and release of GH, LH, FSH, and PRL were examined in adult ovariectomized ewes. Two groups of six ewes were fed a diet of either 1000 g/day (normal feeding) or 400-600 g/day (restricted feeding) hay for 20 weeks. Restricted feeding increased mean plasma GH concentrations and the amplitude of GH pulses, but did not affect GH pulse frequency. In contrast, mean plasma LH and FSH concentrations and LH pulse frequency were decreased by restricted feeding. Mean plasma PRL concentrations were unaffected by treatment. The levels of mRNA for GH in pituitary cytosol were increased by restricted feeding, but no changes were seen in mRNA levels of alpha-subunit, LH beta, FSH beta, or PRL. The pituitary contents of hormones measured did not change with the level of feeding. In conclusion, these data show that long term restricted feeding affects anterior pituitary function in adult ewes, presumably reflecting alterations in the secretion of hypothalamic releasing and inhibiting factors.

More (P less than 0.05) post-partum acyclic ewes (8/9) showed evidence of pulsatile LH release than did seasonally anoestrous ewes (2/8). Mean plasma prolactin concentrations were higher (P less than 0.05) in the post-partum ewes than in the seasonally anoestrous ewes. Hypothalamic and pituitary cytosol oestrogen receptor number, median eminence GnRH content and pituitary LH, FSH and prolactin contents were similar in the two groups of ewes. The number of nuclear-bound oestrogen receptors was greater (P less than 0.01) in pituitaries from seasonally anoestrous ewes than in pituitaries from post-partum ewes. These data suggest that the basis of acyclicity in seasonally anoestrous ewes and in post-partum ewes is probably due to their inability to generate LH pulse frequencies similar to that in the follicular phase of the oestrous cycle. The higher LH pulse frequency in post-partum ewes, compared to that in seasonally anoestrous ewes, suggests that pregnancy may partly negate the reduction in LH secretion that is associated with photoperiod-induced acyclicity. The lower number of nuclear-bound oestrogen receptors in post-partum ewes suggests that there may be lower plasma oestrogen levels and less ovarian follicular growth than in non-suckling anoestrous ewes.

A surgical procedure is described for isolating the pituitary gland from hypothalamic influences in sheep. The procedure results in total deafferentation of the stalk and median eminence but maintains the blood supply to the pituitary gland. The median eminence, pituitary stalk and anterior face of the pituitary gland were exposed by a transnasal, transphenoidal approach. In early studies section of the pituitary stalk as close as possible to the pituitary gland caused almost total infarction of the gland. Attempts were made to disconnect the pituitary gland from the hypothalamus by section immediately above the lateral inputs of the superior hypophyseal arteries but variable results were obtained, always with infarction of part of the pars distalis. When the pituitary gland was disconnected from the hypothalamus by entering the median eminence above the portal circulation and evacuating the nervous tissue of the tuber cinereum, (hypothalamo-pituitary disconnection; HPD), a small area of infarction was found in the pars distalis of only 1/10 cases. HPD effectively disconnected the pituitary gland from hypothalamic control whilst the pars distalis was not deprived of its blood supply. Complete severance of hypothalamo-pituitary connections also caused atrophy of the pars nervosa and enlargement of the cells of the pars intermedia. Following HPD, plasma LH and FSH concentrations diminished and plasma prolactin concentrations rose. On the day after surgery there were no LH, FSH or prolactin responses to 50 micrograms (i.m.) of oestradiol benzoate indicating the functional isolation of the pituitary gland from the hypothalamus. The isolated pituitaries were capable of responding to gonadotropin releasing hormone by LH release. Disconnection of the pituitary gland from the hypothalamus by subpial deafferentation provides a good in vivo isolated pituitary model in the sheep.