Stalk-median Eminence Research Articles

Marked changes of arginine vasopressin, oxytocin, and corticotropin-releasing hormone in hypophysial portal plasma after pituitary stalk damage in the rat.

Mechanical compression of the pituitary stalk with the help of a blunt stereotaxic knife results in posterior pituitary denervation (PPD) and sprouting proximal to the injury, leading to formation of an ectopic neurohypophysis in the stalk. This provides an experimental model for those cases in which traumatic damage severs the nerve fibers to the neural lobe but does not obliterate the hypophysial-portal circulation. The effect of PPD on the hypophysial-portal concentration profile of putative ACTH secretagogues as well as basal and stimulated ACTH secretion in vitro were investigated at varying times after PPD. The contents of arginine vasopressin (AVP) and oxytocin (OT) in extracts of the stalk median eminence 1 week after PPD were markedly elevated, whereas corticotropin-releasing hormone (CRH) content was unaffected. Levels of these three neuropeptides in hypophysial-portal blood collected under anesthesia from the proximal stump of the transected stalk (or the ectopic neural lobe) were measured at weekly intervals in groups of rats after sham or PPD surgery. Hypophysial-portal AVP levels showed a monotonic increase with time after PPD from a 1.8-fold elevation at 1 week post-PPD to a maximum concentration 6-fold greater than that in sham groups at 4 weeks post-PPD. Portal plasma OT levels also exhibited extreme elevation. In contrast, portal plasma CRH levels showed an initial 72% decline 1 week post-PPD. We suggest that mechanical damage to the pituitary stalk and the subsequent sprouting redirected secretion of AVP and OT from the neural lobe to the pituitary stalk. This caused sustained elevations of portal plasma concentrations of AVP and OT. The resulting tonic exposure to AVP and/or OT may down-regulate anterior pituitary receptors to these neurohypophyseal peptides and indirectly decrease CRH release into the portal circulation.

Control of luteinizing hormone-releasing hormone pulse generation in nonhuman primates.

1. The pulsatile release of luteinizing hormone-releasing hormone (LHRH) is critical for reproductive function. However, the exact mechanism of LHRH pulse generation is unclear. The purpose of this article is to review the current knowledge on LHRH pulse generation and to discuss a series of studies in our laboratory. 2. Using push-pull perfusion in the stalk-median eminence of the rhesus monkey several important facts have been revealed. There is evidence indicating that LHRH neurons themselves have endogenous pulse-generating mechanisms but that the pulsatility of LHRH release is also modulated by input from neuropeptide Y (NPY) and norepinephrine (NE) neurons. The release of NPY and NE is pulsatile, with their pulses preceding or occurring simultaneously with LHRH pulses, and the neuroligands NPY and NE and their agonists stimulate LHRH pulses, while the antagonists of the ligands suppress LHRH pulses. 3. The pulsatile release of LHRH increases during the estrogen-induced LH surge as well as the progesterone-induced LH surge. These increases are partly due to the stimulatory effects of estrogen and progesterone on NPY neurons. 4. An increase in pulsatile LHRH release occurs at the onset of puberty. This pubertal increase in LHRH release appears to be due to the removal of tonic inhibition from gamma aminobutyric acid (GABA) neurons and a subsequent increase in the inputs of NPY and NE neurons to LHRH neurons. 5. There are indications that additional neuromodulators are involved in the control of the LHRH pulse generation and that glia may play a role in coordinating pulses of the release of LHRH and neuromodulators. 6. It is concluded that the mechanism generating LHRH pulses appears to comprise highly complex cellular elements in the hypothalamus. The study of neuronal and nonneuronal elements of LHRH pulse generation may serve as a model to study the oscillatory behavior of neurosecretion.

Steroid Modulation of Pulsatile LHRH Release in the Rhesus Monkey

Studies using push-pull perfusion of the stalk-median eminence (S-ME) in ovariectomized adult monkeys indicate that input of neuropeptide Y (NPY) neurons is an important modulator for tile pulsatility of LHRH release: (1) NPY release in the S-ME was pulsatile; (2) NPY pulses occurred coupled to LHRH pulses, with NPY pulses preceding LHRH pulses; and (3) infusion of NPY into the S-ME stimulated LHRH release in a dose-responsive manner; whereas (4) infusion of a specific antiserum to NPY into the S-ME suppressed LHRH pulses. Further studies suggest that NPY neurons may mediate the action of steroid hormones. While treatment with a small dose of estrogen did not affect the coupling of NPY and LHRH pulses, it enhanced the sensitivity of LHRH release in response to NPY by 10,000-fold. Progesterone treatment 24 hr after estrogen induced an increase in LHRH release followed by an LH surge: Progesterone increased tile pulse frequencies of berth NPY release and LHRH release, maintaining the tight coupling of the pulses, while increasing LHRH pulse amplitude, but not NPY pulse amplitude. A parallel role of norepinephrine (NE) neurons in the modulation of pulsatile LHRH release was also observed indicating that more than one neuronal system can be simultaneously involved in the regulation of LHRH release and the action of ovarian steroids. Moreover, in the modulation of LHRH pulses, NPY and NE neurons were independent of each other. Further studies indicate that the LHRH neurosecretory system in the sexually immature monkey was insensitive to estrogen. This insensitivity appeared to be due to the tonic inhibition of pulsatile LHRH release by γ-aminobutyric acid (GABA) neurons through GABAA receptors. These results suggest that in vivo LHRH release is modulated by NPY, NE, and GABA neuronal inputs which mediate the action of steroid hormones.

Horse plasma corticotrophin-releasing hormone (CRH): characterisation and lack of a late gestational rise or a plasma CRH-binding protein.

Immunoreactive corticotrophin-releasing hormone (irCRH) was present in methanolic extracts of equine peripheral blood and showed no elevation in maternal peripheral serum in late gestation (0.54 +/- 0.25 pmol/l; mean +/- S.D.) compared with control horses (0.41 +/- 0.15 pmol/l). The irCRH of methanolic extracts of pituitary venous plasma had a similar elution position following reverse-phase HPLC to synthetic human CRH(1-41) and to irCRH released from horse stalk-median eminence tissue incubated in vitro. Gel chromatographic studies showed no evidence for a plasma CRH-binding protein (CRHBP) analogous to that found in human plasma in either peripheral blood from normal or pregnant horses or in pituitary venous plasma sampled from a cannulated horse. CRH-binding activity was detectable in peripheral plasma from one horse, however the molecular size of this was indicative of a gamma-globulin rather than the 37 kDa CRHBP. These studies suggest that, unlike in the human, CRH does not rise to high values in late gestation nor circulate in a bound form in equine plasma.

Posttranslational processing of progonadotropin-releasing hormone (PROGnRH) in ewes

Gonadotropin-releasing hormone (GnRH) is released from hypothalamic neurons into the hypophyseal-portal blood system following enzymatic cleavage of the decapeptide from a large precursor (proGnRH) molecule. The purpose of this study was to determine whether the ability of GnRH-producing neurons to synthesize and/or process proGnRH differed during physiological states associated with a suppressed and enhanced release of GnRH in ewes. Tissues were collected from ovariectomized ewes (OVX, n=4), OVX-estradiol treated ewes (OVX-E, n=5), and ewes (n=7) slaughtered 5 d after parturition (PP). Following euthanasia and exsanguination, stalk-median eminence (SME), medial-basal hypothalamus (MBH) and preoptic areas (POA) were collected. Concentrations of GnRH and proGnRH were determined by radioimmunoassay using specific antisera. Concentrations of GnRH in the SME did not differ (P>.05) between OVX-E and OVX ewes, but both groups contained less (P<.05) GnRH than the SME from PP ewes (4.4 ± 0.7, 12.1 ± 3.8 vs 24.3 ± 5.1 fmol/mg tissue, respectively). Concentrations of proGnRH in SME mimicked those of GnRH and were less (P<.05) in OVX-E ewes than PP ewes, but were not different (P>.05) from those in OVX ewes (.34 ± .34 vs 3.76 ± 1.53 and 1.7 ± .78 fmol/mg, respectively). In the MBH, OVX-E ewes had greater (P<.05) concentrations of GnRH than PP ewes (0.76 ± 0.29 vs 0.24 ± 0.04 fmol/mg) and OVX ewes were intermediate (0.41 ± 0.13 fmol/mg). No differences (P>.05) in concentrations of GnRH in the POA were detected among groups. Concentrations of proGnRH in MBH and POA were not different (P>.05) among groups. In summary, proGnRH is present in the SME which contains nerve terminals of GnRH-producing cells. Although concentrations of proGnRH and GnRH in the SME and MBH were affected by physiological state, ratio's of the prohormone:mature decapeptide remained constant. Therefore, alterations in posttranslational processing of the prohormone leading to formation of the mature GnRH-decapeptide were not demonstrated.

Changes in pulsatile release of neuropeptide-Y and luteinizing hormone (LH)-releasing hormone during the progesterone-induced LH surge in rhesus monkeys.

In previous studies we have shown that the pulsatility of LH-releasing hormone (LHRH) release in gonadectomized monkeys is modulated by input from neuropeptide-Y (NPY) neurons: 1) the endogenous release of NPY in the stalk-median eminence (S-ME) was pulsatile; 2) NPY pulses were temporally correlated with LHRH pulses, with NPY pulses preceding LHRH pulses by approximately 5 min; and 3) infusion of NPY into the S-ME stimulated LHRH release, whereas 4) infusion of antiserum to NPY suppressed endogenous LHRH pulses. It is not known, however, whether ovarian steroid hormones alter the pulsatility of NPY and LHRH release or whether the temporal correlation of NPY and LHRH pulses is maintained during the LH surge. In the present study we examined the changes in pulsatile release of NPY and LHRH in ovariectomized monkeys treated with estradiol benzoate (EB) followed by progesterone or oil. Using push-pull perfusion, perfusate samples from S-ME were collected at 10-min intervals for 15 h. NPY and LHRH concentrations in the perfusates were measured by RIA. Circulating LH levels were also monitored by periodic blood sampling and RIA. Injection of progesterone (sc) after EB induced an LH surge with a peak latency of 7.3 +/- 1.3 h (mean +/- SE) in seven of seven monkeys, whereas oil injection after EB elicited an LH surge in none of seven monkeys. The progesterone-induced LH surge was associated with an increase in LHRH release; the mean, pulse amplitude, and pulse frequency increased significantly (for all, P < 0.05) 4-8 h after progesterone. NPY pulse frequency also increased significantly (P < 0.05) 4-8 h after progesterone treatment, whereas mean release and pulse amplitude did not change in response to progesterone. Oil treatment after EB administration did not alter any parameter of LHRH and NPY pulses. Interestingly, the NPY and LHRH pulses were highly correlated (P < 0.001) in monkeys treated with either EB-progesterone or EB-oil, and NPY pulses preceded LHRH pulses by 4.8 +/- 0.7 and 5.1 +/- 0.6 min, respectively. In summary, 1) an episode of increased LHRH release occurs before and during the progesterone-induced LH surge; 2) acceleration of LHRH pulse frequency and the increase in LHRH pulse amplitude after progesterone are accompanied by acceleration of NPY pulse frequency; and 3) ovarian steroids do not affect the temporal correlation between NPY and LHRH pulses.(ABSTRACT TRUNCATED AT 400 WORDS)

Pituitary adenylate cyclase-activating polypeptide (PACAP) increases prolactin release and tuberoinfundibular dopaminergic neuronal activity

Pituitary adenylate cyclase activating polypeptide (PACAP) is a hypothalamic peptide that affects anterior pituitary cell function. This study examined the effects of PACAP on prolactin (PRL) release in vivo and on tyrosine hydroxylase (TH) activity in tuberoinfundibular dopaminergic neurons in vivo and in vitro. In ovariectomized rats, intravenous injection of PACAP increased circulating PRL levels 3-fold and TH activity in the stalk-median eminence (SME) by 30%. Incubation of the SME with 1 μM PACAP in vitro increased TH activity 2-fold. Intravenous infusion of ovine PRL (oPRL) by an osmotic mini-pump for 2 days in ovariectomized rats increased TH activity in the SME 1.7-fold and reduced circulating concentrations of endogenous rat PRL to 20% of control levels. PACAP induced a 4-fold rise in endogenous rat PRL levels in oPRL-treated rats and a 30% increase in TH activity that was additive to the elevation caused by hyperprolactinemia. In suckled lactating rats, PACAP did not alter circulating PRL levels or TH activity in the SME. When pups were removed from the dams for 4–5 h, systemic injection of PACAP stimulated PRL release without altering TH activity. However, PACAP, when administered in vitro, stimulated TH activity in the SME of lactating rats separated from their pups. These data indicate that PACAP may play a role in augmenting PRL release in female rats. The PACAP-induced rise in PRL release is modest and not due to a decrease in tuberoinfundibular dopaminergic neuronal activity. PACAP increases TH activity in the tuberoinfundibular dopaminergic neurons, possibly by a direct action on nerve terminals within the median eminence.

Progesterone suppresses tyrosine hydroxylase messenger ribonucleic acid levels in the arcuate nucleus on proestrus.

This study examined the intracellular mechanisms for the regulation of tyrosine hydroxylase in the tuberoinfundibular dopaminergic neurons of cycling female rats. It also evaluated the hormonal influences that contribute to the control of this enzyme on proestrus. Tyrosine hydroxylase messenger RNA (mRNA) levels in the arcuate nucleus of the hypothalamus were assessed by in situ hybridization. Tyrosine hydroxylase activity in the stalk-median eminence was determined from the in vitro or in vivo rate of 3,4-dihydroxyphenylalanine (DOPA) accumulation after inhibiting DOPA decarboxylase with brocresine or m-hydroxybenzylhydrazine, respectively. Tyrosine hydroxylase mRNA levels and in vitro DOPA accumulation were similar on diestrous day 2 and proestrous mornings, but were reduced by 50% on estrus. Although circulating PRL concentrations were similar on the morning of each day of the estrous cycle, a broad preovulatory PRL surge was observed on the afternoon of proestrus. In vitro DOPA accumulation was similar at 1000 h before the PRL surge and at 1330 h during the peak phase of the PRL surge, but was reduced during the plateau phase of the PRL surge (1700 and 2200 h) coincident with the preovulatory progesterone rise and remained low on estrus. However, in vivo DOPA accumulation was transiently decreased only at 1700 h on proestrus. Tyrosine hydroxylase mRNA levels were similar at 1000, 1330, and 1700 h on proestrus, were reduced by 50% at 2200 h on proestrus subsequent to the decrease in enzyme activity, and remained low on the morning of estrus. Okadaic acid, a protein phosphatase-1 and -2A inhibitor, induced a similar increase in tyrosine hydroxylase activity in vitro at 1330 and 2200 h on proestrus and at 1100 h on estrus, indicating that tyrosine hydroxylase was capable of being activated in spite of decreased mRNA levels. Ovariectomy between 1100-1200 h on proestrus prevented the decrease in tyrosine hydroxylase mRNA levels and in vitro DOPA accumulation at 2200 h. The effects of ovariectomy were completely reversed by progesterone, whereas estradiol had no effect. Circulating PRL levels at 2200 h were suppressed to basal levels after ovariectomy, but were increased by progesterone treatment at 1530 h to levels similar to those in the plateau phase of the PRL surge in control rats. Administration of the progesterone antagonist RU486 at 1200 h on proestrus did not alter tyrosine hydroxylase activity, tyrosine hydroxylase mRNA levels, or circulating PRL concentrations at 2200 h.(ABSTRACT TRUNCATED AT 400 WORDS)

Secretion of gonadotropins and estimated releasable pools of gonadotropin-releasing hormone and gonadotropins during establishment of suckling-induced inhibition of gonadotropin secretion in the sow.

Two experiments were conducted to evaluate relationships between changes in hypothalamic GnRH, pituitary and serum gonadotropins, and ovarian function during the first week postpartum in lactating multiparous sows. In experiment 1, our hypothesis was that sows would have active LH secretion immediately after parturition and that suckling would inhibit LH concentrations within the first 3 days postpartum. Sows were killed at Day 1 (n = 5) or Day 7 (n = 5) of lactation, and blood samples were taken every 6 h from 48 h before parturition until 24 h (Day 1 sows) or 156 h (Day 7 sows) after parturition. Preoptic area (POA), medial basal hypothalamus (MBH), stalk median eminence (SME), anterior pituitary (AP), and ovaries were collected at slaughter. On Day 1, sows had fewer (p < 0.05) small follicles (< 4 mm diameter), but more (p < 0.05) medium (4-6 mm) and large (> 6 mm) follicles, than sows on Day 7 (20 +/- 2, 6.5 +/- 1.1, 2.5 +/- 0.7 vs. 26.3 +/- 3, 0, and 0, respectively). Serum LH and FSH decreased (p < 0.04) during the period from 24 to 48 h postpartum and remained low through Day 7. GnRH concentrations in POA, MBH, and SME and the proportion of residual GnRH released in vitro in response to K+ did not differ between Day 1 and 7. Weights of AP were not different, but pituitary concentrations (microgram/mg) of LH and FSH increased (p < 0.05) from Day 1 to 7 (LH: 0.27 +/- 0.03 vs. 0.44 +/- 0.01; FSH: 2.6 +/- 0.07 vs. 7.8 +/- 0.8, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)

Gamma-Aminobutyric acid is an inhibitory neurotransmitter restricting the release of luteinizing hormone-releasing hormone before the onset of puberty.

To test the hypothesis that the pubertal increase in luteinizing hormone-releasing hormone (LHRH) release is withheld by a dominant inhibitory neuronal system, the role of gamma-aminobutyric acid (GABA), a known inhibitory neurotransmitter, in the control of LHRH release was examined in conscious female monkeys at the prepubertal and pubertal stages using a push-pull perfusion method. GABA, bicuculline (a GABAA receptor blocker), and 2-hydroxysaclofen (a GABAB receptor blocker) were directly infused into the stalk-median eminence while perfusates were collected for LHRH determination. Bicuculline, but not saclofen, induced a large and prompt increase in LHRH release in prepubertal monkeys, whereas it stimulated LHRH release slightly in pubertal monkeys. In contrast, GABA suppressed LHRH release in pubertal, but not prepubertal, monkeys. These differential effects of GABA and the GABA antagonist on LHRH release in the two developmental stages were due to an age factor rather than to the steroid hormonal background. Moreover, GABA release in the stalk-median eminence of prepubertal monkeys was much higher than that in pubertal monkeys. Thus, the results suggest that in the prepubertal period there is a powerful GABA inhibition of the LHRH neurosecretory system: infusions of GABAA, but not GABAB, antagonists stimulate LHRH release by removal of the endogenous GABA inhibition, whereas exogenous GABA is ineffective because of high endogenous GABA levels. The decrease of this tonic inhibition may be a key factor for the onset of puberty in non-human primates.

Effects of progesterone and cortisol on the release of gonadotropin-releasing hormone from the perifused pituitary stalk-median eminence and on luteinizing hormone release from the pituitary of cows

The direct effects of progesterone and cortisol on the release of gonadotropin-releasing hormone (GnRH) and luteinizing hormone (LH) were examined by perifusing tissues from the bovine pituitary stalk-median eminence and the anterior pituitary. These tissues were isolated and perifused for 160 min and 8 h, respectively. To assess the effect of progesterone and cortisol, from 0 to 10 μM of progesterone or cortisol was added separately to the tissues and the amounts of GnRH and LH assayed in the effluent medium. The release of GnRH from the hypothalamic fragments was significantly reduced within 20 min after the addition of 0.1 and 10 μM of progesterone when compared with the values for the control group ( P<0.05), but GnRH release was not markedly affected by the addition of up to 10 μM cortisol. Spontaneous release of LH from the anterior pituitary was not markedly affected by the addition of 0.1 or 10 μM progesterone, although the GnRH-stimulated release of LH was significantly higher than that of the control group ( P<0.05). GnRH-stimulated release of LH was not markedly affected by addition of cortisol. These results suggest that high concentrations of progesterone act directly on the bovine pituitary stalk-median eminence to inhibit the release of GnRH, but they enhanced the release of LH from the anterior pituitary in response to the same GnRH stimulus.

Hypothalamic and Hypophyseal Receptors for Estradiol in High and Low Sexually Performing Rams

Rams, characterized over a 3-year period as being high ( n = 8) or low ( n = 10) sexual performers, were euthanized to determine if differences in sexual behavior were associated with differences in hypophyseal and hypothalamic receptors for estradiol. Rams were exposed to estrous ewes and courtship behavior was recorded for 5 hr prior to tissue collection. Blood samples for determination of serum concentrations of luteinizing hormone (LH) were collected every 15 min during the behavior testing period. Hourly blood samples were analyzed for serum concentrations of estradiol (E) and testosterone (T). Rams characterized as high (HP) or low (LP) sexual performers had similar LH profiles; there were no differences noted in basal concentration, number of pulses, or pulse amplitude of LH. There were no differences in serum concentrations of T or E during the behavior testing period between HP and LP rams. Unoccupied receptors for E (moles E bound x 10 -15) in the stalk-median eminence (SME) ( P < 0.05) in HP rams (15.1 ± 3.1) were greater than those in LP rams (4.6 ± 1.9). Occupied receptors for E (moles E bound x 10 -15) in the preoptic area (POA) ( P < 0.1) in HP rams (6.0 ± 0.8) tended to be greater than those in the LP rams (3.8 ± 0.8). The proportion of total E receptors (occupied + unoccupied) in the POA that were occupied was greater ( P < 0.05; 0.41 ± 0.03 vs 0.23 ± 0.04) for HP than for LP rams. In contrast, HP rams had less ( P < 0.05) occupied E receptors (moles E bound x 10 -15) in the anterior pituitary gland (AP) than LP rams (152.0 ± 38.5 vs 337.3 ± 51.7). The proportion (%) of E receptors that were occupied in the AP in HP rams also tended to be less ( P < 0.06) than that in LP rams (8 ± 2 vs 14 ± 1). No differences were detected in occupied or unoccupied E receptors in the medial basal hypothalamus, medial amygdala, or stalk-median eminence. In conclusion, differences in occupied receptors for E in the POA and the AP, and unoccupied receptors for E in the SME, were associated with differences in ram sexual behavior.

Relationship of secretion of GnRH in vitro to changes in pituitary concentrations of LH and FSH and serum concentrations of LH during lactation in sows.

The objectives of this study were (1) to determine whether release of GnRH in vitro was related to concentrations of LH and FSH in serum and pituitaries and to oestradiol in serum of sows at mid- or late lactation, and (2) to determine whether weaning at mid-lactation changes concentrations of these hormones from values expected at mid-lactation to values expected at late lactation. Multiparous crossbred sows were killed on day 14 (n = 5) or day 28 (n = 5) of lactation or on day 14 post partum after litters had been weaned on day 10 (n = 5). Blood samples were taken every 6 h for 4 days before sows were killed, and the preoptic suprachiasmatic area, medial basal hypothalamus, stalk median eminence, anterior pituitary and ovaries were collected at slaughter. Sows killed on day 14 after having their litters weaned on day 10 had more (P < 0.01) preovulatory follicles (> 6 mm in diameter) than lactating sows killed on day 14 or 28 (7.0 +/- 1.2 versus 0.2 +/- 0.1 and 1.5 +/- 0.8, respectively). Concentrations of LH, FSH and oestradiol in serum during 90 h before slaughter were greater (P < 0.05) in weaned sows and lactating sows killed on day 28 than in lactating sows killed on day 14 (LH: 0.72 +/- 0.3 and 0.68 +/- 0.3 versus 0.45 +/- 0.2 ng ml-1; FSH: 39.3 +/- 2.7 and 57.3 +/- 4.0 versus 28.8 +/- 1.6 ng ml-1; oestradiol: 10.9 +/- 1.6 and 5.6 +/- 0.7 versus 2.7 +/- 0.2 pg ml-1, respectively).(ABSTRACT TRUNCATED AT 250 WORDS)

Ovariectomy increases in vivo luteinizing hormone-releasing hormone release in pubertal, but not prepubertal, female rhesus monkeys.

In pubertal, but not prepubertal, monkeys ovariectomy (OVX) results in an elevation of circulating luteinizing hormone (LH) levels. To determine if the castration-induced LH increase in pubertal monkeys is due to an increase in pulsatile LH-releasing hormone (LHRH) release, effects of OVX on in vivo LHRH release in the stalk-median eminence were examined in fully conscious monkeys using a push-pull perfusion method. The average ages (+/- SEM) of female rhesus monkeys in each group at OVX were 14.5 +/- 0.6 months (n = 6; prepubertal), 25.0 +/- 1.3 months (n = 5; early pubertal) and 37.8 +/- 2.1 months (n = 6; midpubertal). Perfusate samples from the stalk-median eminence were obtained in 10-min fractions for 6 h in the morning (0600 to 1200 h) and 6 h in the evening (1800 to 2400 h), from the same subjects before OVX, and at 29 days and approximately 100 days after OVX. LHRH levels in perfusates were measured by radioimmunoassay. LH levels throughout the experiment were monitored by periodic blood sampling. OVX resulted in a significant LH increase in early and midpubertal monkeys (P < 0.001 for both), but not in prepubertal monkeys. Similarly, OVX in early and midpubertal monkeys increased mean LHRH release when examined 29 days after surgery (P < 0.05 and P < 0.01, respectively). The OVX-induced LHRH increases in early and midpubertal monkeys remained elevated at approximately 100 days postcastration. Furthermore, it was found that effects of OVX on the increased LHRH release were primarily due to the elevation of basal release and pulse amplitude, but not pulse frequency. In contrast, OVX did not cause any significant effects on pulsatile LHRH release in prepubertal monkeys. The results indicate that an increase in LHRH release and a concomitant increase in circulating LH occurs after OVX in pubertal monkeys, but not in prepubertal monkeys. These data are consistent with the hypothesis that the low level of LH in circulation before the onset of puberty is due to a low amount of LHRH release which is independent of ovarian steroid feedback and that the maturity of the neuronal control system for the pulsatile LHRH release is responsible for the onset of puberty. After the onset of puberty, the negative feedback of ovarian steroid hormones becomes important to the regulation of gonadotropin release.

Negative feedback effects of estrogen on luteinizing hormone-releasing hormone release occur in pubertal, but not prepubertal, ovariectomized female rhesus monkeys.

In a previous study we found that ovariectomy resulted in an increase in both LHRH release and LH release in pubertal monkeys but not in prepubertal monkeys. To determine whether this castration-induced LHRH increase is due to the removal of estrogen, in the present study, the effects of estradiol benzoate (EB, 30 micrograms/kg body wt) on in vivo LHRH release were examined using a push-pull perfusion method in prepubertal (age 15-19 months, n = 5), early pubertal (24-29 months, n = 5), and midpubertal (36-48 months, n = 5) female rhesus monkeys that were ovariectomized 3 to 5 months earlier. LHRH in 10-min perfusate fractions from the stalk-median eminence was measured from -6 to +24 h after EB injection. Circulating LH levels were also monitored over the same period at various intervals. EB decreased LH levels in early pubertal and midpubertal monkeys, whereas it did not cause any significant effects on LH release in the prepubertal monkey. EB also resulted in suppression of LHRH release in both early and midpubertal monkeys; mean LHRH release before EB in the early and midpubertal groups was 6.6 +/- 0.6 and 7.0 +/- 0.6 pg/ml.10 min, respectively. EB decreased mean LHRH release beginning 3 h after EB with the nadir occurring at 18-21 h after EB (1.0 +/- 0.2 pg/ml.10 min) in early pubertal monkeys and 21-24 h after EB (1.2 +/- 0.1 pg/ml.10 min) in midpubertal monkeys. Decrease of mean LHRH release was due to a decrease in LHRH pulse amplitude and basal release but not pulse frequency. Oil injection alone (control) failed to suppress LHRH and LH release. In contrast to the results in pubertal monkeys, mean LHRH release in prepubertal monkeys was not altered by EB (before EB, 1.1 +/- 0.2 pg/ml.10 min; 18-21 h after EB, 1.5 +/- 0.3 pg/ml.10 min). These results suggest that the LHRH neurosecretory system in pubertal monkeys is responsive to the negative feedback effects of estrogen. However, the fact that estradiol failed to suppress LHRH release in prepubertal monkeys indicates that the LHRH neurosecretory system and/or its regulatory systems are not sensitive to estradiol before the onset of puberty. These findings are consistent with the hypothesis that the increase in pulsatile LHRH release at the onset of puberty is not dependent on changes in ovarian steroid feedback mechanisms.

Negative feedback effects of estrogen on luteinizing hormone-releasing hormone release occur in pubertal, but not prepubertal, ovariectomized female rhesus monkeys

In a previous study we found that ovariectomy resulted in an increase in both LHRH release and LH release in pubertal monkeys but not in prepubertal monkeys. To determine whether this castration-induced LHRH increase is due to the removal of estrogen, in the present study, the effects of estradiol benzoate (EB, 30 micrograms/kg body wt) on in vivo LHRH release were examined using a push-pull perfusion method in prepubertal (age 15-19 months, n = 5), early pubertal (24-29 months, n = 5), and midpubertal (36-48 months, n = 5) female rhesus monkeys that were ovariectomized 3 to 5 months earlier. LHRH in 10-min perfusate fractions from the stalk-median eminence was measured from -6 to +24 h after EB injection. Circulating LH levels were also monitored over the same period at various intervals. EB decreased LH levels in early pubertal and midpubertal monkeys, whereas it did not cause any significant effects on LH release in the prepubertal monkey. EB also resulted in suppression of LHRH release in both early and midpubertal monkeys; mean LHRH release before EB in the early and midpubertal groups was 6.6 +/- 0.6 and 7.0 +/- 0.6 pg/ml.10 min, respectively. EB decreased mean LHRH release beginning 3 h after EB with the nadir occurring at 18-21 h after EB (1.0 +/- 0.2 pg/ml.10 min) in early pubertal monkeys and 21-24 h after EB (1.2 +/- 0.1 pg/ml.10 min) in midpubertal monkeys. Decrease of mean LHRH release was due to a decrease in LHRH pulse amplitude and basal release but not pulse frequency. Oil injection alone (control) failed to suppress LHRH and LH release. In contrast to the results in pubertal monkeys, mean LHRH release in prepubertal monkeys was not altered by EB (before EB, 1.1 +/- 0.2 pg/ml.10 min; 18-21 h after EB, 1.5 +/- 0.3 pg/ml.10 min). These results suggest that the LHRH neurosecretory system in pubertal monkeys is responsive to the negative feedback effects of estrogen. However, the fact that estradiol failed to suppress LHRH release in prepubertal monkeys indicates that the LHRH neurosecretory system and/or its regulatory systems are not sensitive to estradiol before the onset of puberty. These findings are consistent with the hypothesis that the increase in pulsatile LHRH release at the onset of puberty is not dependent on changes in ovarian steroid feedback mechanisms.

A possible role of neuropeptide Y in the control of the onset of puberty in female rhesus monkeys.

The onset of puberty is heralded by an increase in pulsatile LHRH release. Since neuropeptide Y (NPY) has been implicated as a major regulator in the control of pulsatile LHRH release in mature monkeys, we have hypothesized that maturational changes in the NPY neuronal system play an important role in puberty. To test this hypothesis, three experiments were conducted in female rhesus monkeys using a push-pull perfusion method. In the first experiment, changes in NPY release in the stalk-median eminence (S-ME) during puberty were determined in 9 prepubertal, 7 early pubertal and 8 midpubertal monkeys. NPY and LHRH levels were measured in aliquots of the same perfusate samples obtained from the S-ME. NPY release was pulsatile in all three groups. Mean NPY release and pulse frequency increased significantly from the prepubertal through the midpubertal stage. These developmental changes in NPY release were parallel to those observed for LHRH release in the same monkeys. In order to examine whether NPY infusion into the S-ME influences LHRH release during puberty, in the second experiment, NPY (10(-6) or 10(-8) M) or vehicle was infused into the S-ME for 10 min at 90-min intervals in 5 prepubertal and 9 midpubertal monkeys. In the midpubertal stage, infusion of NPY at doses of 10(-8) and 10(-6) M resulted in significant (p < 0.01) increases in LHRH release, while vehicle administration had no effect. In contrast, in prepubertal monkeys, neither NPY nor vehicle infusion altered LHRH release. In order to test whether endogenous NPY plays a role in the maintenance of pulsatile LHRH release, in the third experiment, a specific antiserum to NPY (aNPY) was infused into the S-ME of 6 prepubertal and 8 midpubertal monkeys. Infusion of aNPY (1:100, 1:1,000 dilution) significantly suppressed LHRH release in midpubertal but not prepubertal monkeys. The results are summarized as follows. (1) In prepubertal monkeys, NPY release is low, and the presence of NPY in the S-ME does not influence LHRH release. (2) At the onset of puberty, NPY release begins to increase, and NPY probably starts to stimulate LHRH release. (3) In the midpubertal period, NPY release increases further, and NPY in the S-ME is highly stimulatory to LHRH release.(ABSTRACT TRUNCATED AT 400 WORDS)

Progesterone reverses the estradiol-induced decrease in tyrosine hydroxylase mRNA levels in the arcuate nucleus.

Estradiol (E2) and progesterone (P4) interact to influence tuberoinfundibular dopaminergic neuronal activity and contribute to the control of prolactin (PRL) release. This study examined tyrosine hydroxylase mRNA signal levels in the arcuate nucleus of the hypothalamus and tyrosine hydroxylase activity in the stalk-median eminence after 1 week of steroid treatment and related these to circulating PRL levels. Ovariectomized rats were untreated (control) or were implanted with E2, P4 or both E2 and P4 pellets and were sacrificed after 7 days at either 10:00 or 18:00 h. Some E2 + P4-treated rats were injected with either RU 486 or its vehicle at 12-hour intervals for the last 3 of the 7 days of steroid treatment. Tyrosine hydroxylase mRNA signal levels in the arcuate nucleus were decreased by 70% at both 10:00 and 18:00 h in the E2-treated rats compared to control rats. P4 alone had no effect on tyrosine hydroxylase mRNA levels, but reversed the E2-induced decrease so that mRNA levels in the E2 + P4-treated rats were similar to control levels. The progesterone antagonist RU 486 blocked this effect of P4, supporting the observation of decreased mRNA levels in E2-treated rats. Steroid treatment had no effect on tyrosine hydroxylase mRNA levels in the medial zona incerta. Tyrosine hydroxylase activity in the stalk-median eminence was similar at 10:00 and 18:00 h in control rats, and was decreased by 25 and 36% at 10:00 and 18:00 h, respectively, in E2-treated rats. P4 alone had no effect on tyrosine hydroxylase activity, but reversed the E2-induced decrease in enzyme activity to control levels at both 10:00 and 18:00 h. In contrast to the effect of RU 486 on tyrosine hydroxylase mRNA, tyrosine hydroxylase activity in E2 + P4-treated rats was not significantly altered by RU 486 at either 10:00 or 18:00 h. Circulating PRL levels were elevated in the E2-treated and E2 + P4-treated rats. A diurnal PRL rise was evident at 18:00 h in E2-treated rats, but was abolished by concomitant treatment with P4. The diurnal PRL surge was re-established in E2 + P4-treated rats after administration of RU 486, whereas basal circulating PRL levels were not altered by RU 486. These data indicate that P4 antagonizes the profound inhibitory effect or E2 on tyrosine hydroxylase mRNA content in the tuberoinfundibular dopaminergic neurons.(ABSTRACT TRUNCATED AT 400 WORDS)

Effects of season and estradiol on concentrations of histamine within discrete brain regions of ovariectomized ewes

Histamine has been implicated as a neuromodulator of secretion of gonadotropins in several species. Concentrations of histamine were analyzed within discrete brain regions and endocrine tissues to help determine whether this amine has the potential to exert a similar function in ewes expected to have dramatically different serum concentrations of LH. Following collection of blood samples at 12-min intervals for 4 hr, ovariectomized (OVX) and OVX-estradiol treated (OVX-E) ewes were slaughtered during the breeding and anestrous seasons (five animals/group). Concentrations of LH were depressed by treatment with estradiol (E; P<.01), but to a greater extent (P<.05) during the anestrous season compared to the breeding season. Concentrations of histamine in tissues (ng/mg) differed (P<.01) between the breeding and anestrous seasons, in the medial thalamus (39.2 ± 14.1 vs 109.9 ± 13.0), posterior pituitary gland (247.6 ± 50.7 vs 23.0 ± 9.1) and midbrain tegmentum (10.4 ± 5.6 vs 50.7 ± 3.9). Estradiol containing implants decreased (P<.05) concentrations of histamine in the midbrain tegmentum (20.3 ± 7.1 vs 37.7 ± 7.8) and posterior pituitary gland (87.3 ± 24.0 vs 258.2 ± 67.5) compared to non-estradiol treated controls. Histamine concentrations in the pineal and anterior pituitary glands and brain regions; stalk-median eminence, medial basal hypothalamus, preoptic area, cerebellum, parietal neocortex, were not (P>.05) affected solely by either season or E. An interaction between effects of season and estradiol on concentrations of histamine occurred (P<.05) in the posterior pituitary gland and the preoptic area. Mean concentrations of histamine (ng/mg) in the posterior pituitary gland of OVX ewes during the anestrous and breeding seasons were 23.3 ± 10.3 and 376.1 ± 77.7, respectively, and were 23.7 ± 15 and 119.1 ± 30.7 in OVX-E ewes during the same periods. Mean concentrations of histamine in the preoptic area of OVX ewes were 63.3 ± 10.5 and 12.5 ± 7.3 during the anestrous season and breeding seasons and 29.8 ± 8.5 and 42.9 ± 12.8, respectively, in OVX-E ewes. In a second experiment, OVX (n=5) and OVX-E (n=5) ewes were slaughtered during the breeding season and their brains dissected into micro-punches to determine effects of E on concentration of histamine in more discrete loci. Differences (P<.05) in concentrations of histamine were noted only in the posterior thalamus (144.1 ± 7.8 vs 79.0 ± 10.0) in OVX and OVX-E ewes, respectively. In conclusion histamine may function as a intermediary neuromodulator in the neural circuitry which regulates secretion of gonadotropins in ewes. Neither season or estradiol alone, however, significantly influenced concentrations of histamine in brain regions which closely regulate release of GnRH.

Neuropeptide Y System of the Female Monkey Hypothalamus: Retrograde Tracing and Immunostaining

Neuropeptide Y (NPY) stimulates the release of hypothalamic gonadotropin-releasing hormone (GnRH) as well as pituitary gonadotropins in the presence of ovarian steroids, but inhibits release in their absence. In primates, however, the effects of NPY depend largely upon the site and method of administration. In ovariectomized monkeys, NPY infusion into the stalk-median eminence reportedly causes a dose-response increase in GnRH secretion in the absence of gonadal steroids. To help elucidate these findings, we investigated the NPY system and its neuroendocrine (NEU) component in the primate brain by retrograde tracing and immunostaining. One adult female and 1 juvenile female cynomolgus monkey were given microinjections of retrograde tracer into the median eminence (ME). Two weeks later, they were perfused with fixative, and series of 40-microns frontal vibratome sections were collected at 500-microns intervals through 4 mm of the forebrain. Injection sites were not visible in the juvenile female monkey ME, so this animal served as a neurosurgical and injection control. Sections were immunostained using a polyclonal NPY antiserum and the peroxidase antiperoxidase (PAP) technique. NPY immunostaining in another adult female cynomolgus monkey and in a late fetal female and a neonatally castrated adult male rhesus monkey gave essentially similar results. NPY-immunoreactive (NPY-IR) neurons were widely distributed throughout the caudate nucleus, but appeared concentrated within specific hypothalamic areas. Their number, as well as the number of NEU neurons, was nearly equal in bilaterally paired areas and on both sides of the hypothalamus overall. Ratios of retrogradely labeled NPY-IR neurons to the number of NPY-IR somata were expressed as percentages of NEU NPY-IR neurons for each side and in each area. These averaged 65% in the supraoptic nucleus (SON), 41% in the paraventricular nucleus (PVN), 32% in the medial preoptic area (MPOA), which has only one quarter of their number of NPY-IR cells, and 11% in the medial basal hypothalamus (MBH). NPY-IR fiber densities were highest in the area olfactoria, medial septal and ventromedial nuclei. They were high in the tuberculum olfactorium, lateral septum, nucleus accumbens, MPOA, PVN, dorsomedial nucleus and regions of the MBH including the arcuate nucleus, tuber cinereum and ventral hypothalamic tract (VHT). NPY fiber densities were moderate in the vertical portion of the diagonal band of Broca, the ventral part of the caudate nucleus, the anterior commissural nucleus and the lateral preoptic area, as well as the anterior and lateral hypothalamic areas, the anterior ventral periventricular area, the suprachiasmatic nucleus and the dorsolateral SON.(ABSTRACT TRUNCATED AT 400 WORDS)