K.-Y. Francis Pau

The Stimulatory Effect of Leptin on the Neuroendocrine Reproductive Axis of the Monkey

Leptin acts as a metabolic activator of the neuroendocrine reproductive axis in several rodent species, but whether leptin plays a similar role in primates is unknown. To explore this question, we examined the effects of leptin on gonadotropin and testosterone secretion in male rhesus monkeys that were fasted for 2 days. Mean plasma levels of LH and FSH, LH pulse frequency, and LH pulse amplitude were significantly higher in leptin-treated animals compared with saline-treated controls during the second day of the fast. To identify targets for leptin’s action, we used in situ hybridization and computerized imaging to map leptin receptor (Ob-R) messenger RNA (mRNA) distribution. Ob-R mRNA was observed in the anterior pituitary and several areas of the brain, including the arcuate and ventromedial nuclei of the hypothalamus. Ob-R mRNA was coexpressed in both POMC and neuropeptide Y neurons in the arcuate nucleus, whereas little or no coexpression of Ob-R mRNA was evident in GnRH neurons. These results sugges...

Putative estrogen receptor β and α mRNA expression in male and female rhesus macaques

Abstract The profound effects of estrogen on different tissues may involve at least two estrogen receptor (ER) subtypes, ERα and the recently discovered ERβ. Where and how the two ER subtypes differentially or cooperatively mediate estrogen actions, however, are still unknown. In this study, we report the cloning of a specific ERβ cDNA fragment and the expression of ERα and ERβ mRNAs in various endocrine and non-endocrine tissues of male and female rhesus macaques. Total RNA from monkey tissues was isolated and subjected to reverse transcription-polymerase chain reaction (RT-PCR) using human-specific ERβ primers. A 126 bp RT-PCR product was identified in ovarian tissue and subsequently transfected into pGEM-T vectors for DNA sequencing. The cloned rhesus monkey ERβ fragment contained a sequence nearly identical to the corresponding sequence in the human (four mismatched nucleotides out of 126). Because complete monkey ERβ and ERα DNA sequences have not been established, the expression of the ERβ and ERα fragments in monkey tissues by RT-PCR reflects ‘putative’ ERβ and ERα mRNA expression, respectively. Both ERβ and ERα mRNAs were present in male and female reproductive organs, in several endocrine and non-endocrine tissues, and in various regions of the brain, whereas several tissues, including liver, frontal cortex, caudate nucleus, locus coeruleus and cerebellum, expressed only ERα message. In some brain regions, i.e. the putamen, internal capsule, hippocampus and paraventricular hypothalamus, the ERβ fragment was expressed in the female but not in the male. These data suggest that ERα mRNA is widely distributed in both female and male tissues, while ERβ mRNA is more widely distributed in female than in male brain.

Release of hypothalamic neuropeptide Y and effects of exogenous NPY on the release of hypothalamic GnRH and pituitary gonadotropins in intact and ovariectomized does in vitro

The effect of NPY on the hypothalamic release of GnRH and pituitary release of gonadotropins was examined in intact and ovariectomized (OVEX) rabbits in a superfusion system. Exposure of mediobasal hypothalami (MBH) from intact rabbits to NPY (8 X 10(-8) M) resulted in a sustained stimulation of GnRH secretion into the medium. The same dose of NPY had no effect on MBH-GnRH release from OVEX rabbits. NPY also produced a sustained stimulation of LH and FSH release by pituitary fragments from intact rabbits, but NPY caused only a transient release of these hormones by pituitaries from OVEX does. Media samples from MBH superfusions were also measured for NPY concentrations. NPY was released episodically into the medium. The amplitude and frequency of NPY pulses in intact and OVEX rabbits did not differ; nor were mean levels of NPY significantly affected by castration. These results suggest that NPY has direct effects on both the hypothalamus and pituitary to modulate the the activities of GnRH neurons and gonadotropes. The pattern of GnRH and gonadotropin response to NPY exposure is determined by ovarian factors.

Characterization of central actions of neuropeptide Y on food and water intake in rabbits

The effects of a 36-amino acid peptide, neuropeptide Y (NPY), on feeding and drinking behaviors were studied in young and adult rabbits. Intraventricular injection of NPY to adult rabbits induced feeding and drinking in a dose-related manner. While the lowest doses tested (0.2 micrograms) was without effect, other doses (0.5 and 1 microgram) elicited feeding and drinking almost instantaneously. When 1, 5 and 10 microgram doses were injected into young rabbits, immediate increases in feeding and drinking were evident, but differences in the magnitude of responses among these dosages were significant only in water consumption. Unlike studies in rats, in these rabbits NPY elicited a more pronounced response in drinking than in feeding. The drinking response after NPY administration was not a consequence of food intake because it occurred in the absence of food. With ad lib feeding, the majority of enhanced food consumption was confined to the first 30-min after NPY injection; however, an increased motivation to eat was retained for at least 2 hr after NPY when food was withheld and then returned. These observations are consistent with specific stimulatory effects of NPY on food and water intake.

Estrogen-dependent effects of norepinephrine on hypothalamic gonadotropin-releasing hormone release in the rabbit

In the rabbit, either coitus or intraventricular administration of norepinephrine (NE) induces gonadotropin release and ovulation. It is hypothesized that ovulation induced with these manipulations involves activation of neuronal pathways that include catecholaminergic and peptidergic neurons. The aim of this study was to examine if perfusion of NE directly through the mediobasal hypothalamus (MBH) stimulates gonadotropin-releasing hormone (GnRH) release from the MBH in ovariectomized (OVEX) and estradiol-treated OVEX does (OVEX/E2). All does were fitted with push-pull (PP) cannulae directed to the MBH and subsequently subjected to PP perfusion at a flow rate of 20 microliters/min for 6 h to measure hypothalamic GnRH release. Five OVEX/E2 and 7 OVEX does received NE that was added to the PP system (intrahypothalamic NE perfusion) at the rate of 2.5 micrograms/min for 2 h during 6 h of PP perfusion. In addition, 6 OVEX/E2 does were given intrahypothalamic perfusion of homovanillic acid (HVA), a metabolite of the adrenergic system, to serve as controls. All PP samples were collected on ice at 10-min intervals, and jugular vein blood samples were obtained at 20-min intervals. The GnRH, luteinizing hormone (LH), and follicle-stimulating hormone (FSH) were measured by specific radioimmunoassays. In OVEX/E2 does, intrahypothalamic perfusion of NE, but not HVA, stimulated a 10-fold increase in peak values of hypothalamic GnRH within 30 min, and a 3-fold increase in peak values of plasma LH within 40 min. Thereafter, both GnRH and LH levels returned to basal values by the end of the NE perfusion.(ABSTRACT TRUNCATED AT 250 WORDS)

Galanin’s Functional Significance in the Regulation of the Neuroendocrine Reproductive Axis of the Monkey

Galanin stimulates the neuroendocrine reproductive axis in the rat, but whether galanin acts similarly in primate species is unknown. To test the hypothesis that galanin acts within the hypothalamo-hypophyseal axis to stimulate luteinizing hormone (LH) and gonadotropin-releasing hormone (GnRH) secretion in the primate, galanin was administered either systemically or directly into the arcuate nucleus-median eminence of ovariectomized macaques (pigtailed or rhesus, respectively) that were maintained on estradiol. The mean plasma levels of LH were significantly elevated in pigtailed macaques after peripheral injection of galanin (2 mg) as compared with vehicle treatment. In rhesus monkeys, galanin (80 µM) administered by push-pull perfusion into the arcuate nucleus-median eminence did not significantly alter either GnRH or LH release. To determine whether in the monkey, as in the rat, subpopulations of medial forebrain GnRH neurons coexpress galanin mRNA, we used single- and double-label in situ hybridization and computerized imaging techniques. GnRH mRNA-containing cells were identified in both the medial and lateral forebrain of the female pigtailed macaque. No galanin mRNA expression was detectable in GnRH neurons located in either the medial preoptic area or mediobasal hypothalamus; however, within the substantia innominata a subset of GnRH mRNA-expressing neurons did coexpress galanin mRNA. Taken together, these results suggest that galanin induces LH release in primates, but galanin may not act directly on hypothalamic GnRH neurons. Presently, we have confirmed in another primate species the existence of GnRH gene expression in the lateral forebrain and discovered that a small subset of these neurons coexpress galanin. These particular cells may have a unique and as of yet undefined physiological function that is distinct from those GnRH neurons serving a hypophysiotropic function.

Effects of Neuropeptide Y on the in vitro Release of Gonadotropin-Releasing Hormone, Luteinizing Hormone, and Beta-Endorphin and Pituitary Responsiveness to Gonadotropin-Releasing Hormone in Female Macaques

The objectives of these studies were to examine the release of gonadotropin-releasing hormone (GnRH) and beta-endorphin-like activity (beta-EP) from macaque hypothalami, and the release of luteinizing hormone (LH) and GnRH-induced LH from macaque anterior pituitaries in response to neuropeptide Y (NPY) treatment. Anterior hypothalamic (AH) and mediobasal hypothalamic (MBH) blocks of tissues and the adenohypophysis were bisected along the midline into two equal-sized fragments. Fragments were superfused with medium for 3 h, followed by 3 h of either NPY (80 nM) or medium alone. In a separate experiment, adenohypophyseal (AP) fragments were superfused in accordance with the same protocol (3 h medium - 3 h NPY or medium) except that exogenous GnRH (352 nM) was added for 30 min at the beginning of hour 3 and again at the beginning of hour 6. Immunoactive GnRH, beta-EP, and LH levels were measured in superfusate samples (400 microliters) collected at 10-min intervals. GnRH levels rose within 20-30 min of initiation of NPY treatment, and elevated GnRH release was sustained for the duration of NPY exposure of both AH and MBH fragments from ovarian intact (INT) rhesus (Macaca mulatta: n = 8; p less than 0.05) or Japanese (Macaca fascicularis; n = 4; p less than 0.01) macaques. NPY treatment had no effect on either AH or MBH fragments isolated from ovariectomized (OVX) rhesus macaques (n = 4 for AH, and n = 5 for MBH). In AP fragments isolated from INT rhesus macaques (n = 8), NPY stimulated LH release within 1 h of treatment (p less than 0.05), whereas NPY had no effect on pituitaries from OVX animals (n = 4).(ABSTRACT TRUNCATED AT 250 WORDS)

Acute administration of estrogen suppresses LH secretion without altering GnRH release in ovariectomized rhesus macaques

The pattern of hypothalamic gonadotropin-releasing hormone (GnRH) release was examined during estrogen (E)-induced suppression of plasma luteinizing hormone (LH) in ovariectomized (OVX) rhesus macaques. In Expt. 1, 4 OVX macaques were fitted with a jugular catheter and a push-pull cannula (PPC) directed into the median eminence (ME). Push-pull perfusion (PPP) was initiated 10 h before and continued for 10 h after subcutaneous estradiol benzoate (EB) injection (42 micrograms/kg b.wt.). In Expt. 2, 4 additional monkeys were subjected to local intrahypothalamic perfusion with estradiol-17 beta (E2, 3 microM) for the last 10 h of a 20-h PPP. In Expt. 2, OVX animals were challenged with 5 micrograms exogenous GnRH 3 h before and 8 h after EB injection to test for changes in altered LH release. Integrated 10-min ME perfusate and intermittent 10- or 60-min peripheral plasma samples were assayed for GnRH and LH by radioimmunoassay and bioassay, respectively. In addition, 2 other OVX macaques that received similar ME-PPC placement were sacrificed 2 days after the completion of a PPP for immunocytochemical labeling of GnRH neurons at the perfusion site. The results show that after EB, hypothalamic GnRH (MBH-GnRH) release remained unaltered while LH levels declined rapidly (Expt. 1). Similarly, intrahypothalamic perfusion of E2 failed to change the pattern of MBH-GnRH release in any of 4 monkeys (Expt. 2). Conversely, plasma LH release in response to exogenous GnRH was greatly reduced after EB (Expt. 3).(ABSTRACT TRUNCATED AT 250 WORDS)

GONADECTOMY ALTERS TYROSINE HYDROXYLASE AND NOREPINEPHRINE TRANSPORTER MRNA LEVELS IN THE LOCUS COERULEUS IN RABBITS

The central noradrenergic system has a major regulatory role on gonadotropin‐releasing hormone/luteinizing hormone (GnRH/LH) secretion in rabbits. Exogenous administration of norepinephrine (NE) alters GnRH/LH release in a sex steroid‐dependent manner, i.e. NE stimulates GnRH/LH release in oestrogen‐primed ovariectomized (OVX) animals but not in non‐primed individuals. To investigate how gonadal steroids influence noradrenergic neuronal activities in the locus coeruleus (LC), mRNA levels of tyrosine hydroxylase (TH) and NE transporter (NET), two key factors regulating NE synthesis and uptake, were compared 3 weeks after gonadectomy (GDX). Intact male (n=5) and female (n=6) New Zealand White rabbits were sacrificed along with castrated males (n=4) and OVX females (n=5). The brainstem from each individual was sectioned and the LC was punched for detection of TH and NET mRNA levels using the ribonuclease protection assay (RPA). Trunk blood was collected to determine immunoactive serum LH values. Levels of LH were elevated in both males and females after GDX. Luteinizing hormone concentrations averaged 0.10±0.05 ng/ml in intact males vs 1.64±0.31 ng/ml in castrated males (P<0.01) and 0.30±0.08 ng/ml in intact vs 9.80±3.50 ng/ml in OVX females (P<0.05), respectively. Removal of the gonads also increased TH mRNA levels in the LC in both males and females. In intact males, TH mRNA levels were 0.796±0.181 pg/&mgr;g total RNA, whereas in castrates mRNA levels averaged 1.667±0.345 pg/&mgr;g total RNA (P<0.05). In intact females, TH mRNA levels were 0.617±0.054 pg/&mgr;g total RNA while the OVX group averaged 1.084±0.202 pg/&mgr;g total RNA (P<0.05). Similar increases in NET mRNA were noted after GDX in both sexes. In males, NET mRNA levels were 1.461±0.401 pg/&mgr;g total RNA in intacts vs 3.666±0.649 pg/&mgr;g total RNA in castrates (P<0.05). In females, NET mRNA levels averaged 1.336±0.212 pg/&mgr;g total RNA and 3.297±0.835 pg/&mgr;g total RNA in the intact and OVX groups, respectively (P<0.05). The data indicate that GDX enhances gene expression of both TH and NET. The results support the hypothesis that the feedback regulation of sex steroids on LH secretion in rabbits of both sexes involves transcriptional/translational processes of at least TH and NET in brainstem NE cells.

Molecular activation of noradrenergic neurons in the rabbit brainstem after coitus

Our previous studies indicate that coitus in female rabbits induces a gonadotropin-releasing hormone (GnRH) surge that is preceded by an increase in hypothalamic norepinephrine (NE) release. The additional findings of an enhanced tyrosine hydroxylase (TH) mRNA expression in the female brainstem after coitus, in addition to the appropriate topographic distribution of TH and dopamine-beta-hydroxylase (DBH), lead us to hypothesize that coital signals are relayed to hypothalamic GnRH-secreting neurons via brainstem NE-containing perikarya. Here we analyzed coitally activated areas in the brainstem by in situ hybridization of the oncogene c-fos, as well as the expression of TH mRNA at 0, 30 and 60 min postcoitus using specific 35S-labeled probes for c-fos and TH. To establish the identity of activated brainstem neurons, we immunocytochemically double-labeled cells with specific antibodies against Fos protein and DBH at 90 min postcoitus. Both c-fos and TH mRNAs were present at 0 min (control) in the A1, A2 and A6 brainstem-noradrenergic areas. At 30 min after coitus the expression of both genes significantly increased (P<0.01) in the A1 and A2 areas. By 60 min postcoitus the expression of c-fos mRNA decreased to control levels, while that of TH mRNA remained stimulated. Double-labeling of Fos and DBH indicated that the number of dual-labeled neurons increased (P<0.05) over control levels only in the A1 and A2 areas (not in A6) at 90 min postcoitus. These findings support the hypothesis that coitus activates transcriptional/translational events within brainstem NE neurons that culminate in the release of hypothalamic NE and hence a GnRH surge.