Harold G. Spies

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.

Oestrogen upregulates noradrenaline release in the mediobasal hypothalamus and tyrosine hydroxylase gene expression in the brainstem of ovariectomized rhesus macaques

Noradrenaline plays a key role in the initiation of ovulation in nonprimate species. A similar noradrenaline role in the primate has not been established experimentally. We utilized the ovariectomized‐oestrogen‐supplemented (OVX + E) rhesus macaque to examine the effects of intravenous (i.v.) infusion of oestradiol‐17β (E2) on the activity of the brain noradrenaline system. Experiment 1 established the induction of a preovulatory surge‐like release of luteinizing hormone in OVX + E monkeys by i.v. infusion of E2 (OVX + E + E2). In experiment 2, a marked increase in hypothalamic microdialysate noradrenaline concentrations occurred after identical E2 infusion into the OVX + E monkeys that were used in experiment 1. In experiment 3, tyrosine hydroxylase (TH) mRNA expression in the locus coeruleus of the brainstem increased at various times after E2 infusion as determined by semiquantitative in situ hybridization. The amount of TH mRNA in OVX + E + E2 animals was higher (P < 0.05) than that in either the OVX + E or OVX monkeys; no difference was found in the latter two groups. Moreover, selected locus coeruleus sections from E2‐infused monkeys were examined for the localization of oestrogen receptors (ER) by in situ hybridization. Both ER‐α and ER‐β mRNAs were expressed in the locus coeruleus, although the expression was greater for ER‐α than for ER‐β. We conclude that i.v. infusion of E2, which induces a preovulatory surge‐like release of LH, stimulates brain noradrenaline activity; this enhanced activity likely involves an ER‐mediated process and is reflected by hypothalamic noradrenaline release and locus coeruleus TH mRNA expression. The results support the concept that noradrenaline can influence the E2‐stimulated ovulation in nonhuman primates and that the brainstem is one of the components in this neuroendocrine process.

HYPOTHALAMIC SITE-DEPENDENT EFFECTS OF NEUROPEPTIDE Y ON GONADOTROPIN-RELEASING HORMONE SECRETION IN RHESUS MACAQUES

In rodents and rabbits, neuropeptide Y (NPY) has a bimodal effect on gonadotropin‐releasing hormone (GnRH) secretion. lntracerebroventricular (icv) administration or direct infusion of NPY into the median eminence (ime) suppresses GnRH release in ovariectomized (OVX) animals, but stimulates GnRH release in intact or OVX animals treated with ovarian steroids. Specific ovarian steroiddependent NPY effects are, however, not obvious in non‐human primates. In OVX rhesus monkeys, icv administration of NPY has been shown to suppress luteinizing hormone (LH) secretion whereas ime infusion of NPY stimulates GnRH pulses. In such animals, estrogen replacement does not reverse the inhibitory NPY effect on LH release, although estrogen enhances the stimulatory NPY effect on GnRH secretion. These observations led us to speculate that the bimodal NPY effects in non‐human primates may depend on either the site of NPY action or the nature of the steroid milieu. This study utilized the push‐pull perfusion (PPP) technique to examine the effects of either ime or icv infusion of NPY on GnRH release in OVX monkeys treated with or without both ovarian steroids. Without exception, irne infusion of NPY increased GnRH concentrations in push‐pull perfusates regardless of the steroid status of the animals. In contrast, GnRH levels were reduced during icv infusion of NPY in both untreated and estrogen/progesterone‐treated, OVX monkeys. These results indicate that, unlike other mammalian species, in the rhesus monkey the stimulatory and inhibitory effects of NPY on GnRH release depend on the site of NPY infusion within the brain rather than the ovarian steroidal environment.

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.

Luteinizing hormone release and ovulation induced by the intraventricular infusion of prostaglandin E1 into pentobarbital-blocked rats

Abstract Five to 20 μg of prostaglandin (PG) E1 infused into the third ventricle of 38 rats on the afternoon of vaginal proestrus reversed the pentobarbital (Pb) blockade of ovulation in 25. Plasma levels of luteinizing hormone (LH) were higher in the PGE1-infused females than in the Pb-blocked controls, but the peak concentrations were only half as high and lasted only half as long as comparable values in normal proestrous females. PGE1 was a more potent stimulator of ovulation in Pb-blocked females than either PGE2 or PGF2α. If infused into the pituitary or injected subcutaneously, however, PGE1 (10 μg) failed to induce ovulation in Pb-blocked animals. The data indicate that PGE1 stimulates LH release and ovulation in the Pb-treated rat by activating a neurally-controlled gonadotropin-releasing mechanism.

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)

Inhibin: Differences in Bioactivity within Human Follicular Fluid in the Follicular and Luteal Stages of the Menstrual Cycle

Summary The presence of a nonsteroidal substance (inhibin) within human follicular fluid (HFF) with the ability to suppress pituitary follicle-stimulating hormone (FSH) but not luteinizing hormone (LH) secretion was determined by two methods. The first method consisted of infusion of HFF directly into the anterior pituitary gland of ovariectomized rhesus monkeys. The second employed a pituitary cell culture system. Release of LH and FSH was stimulated in HFF-treated cells by the addition of luteinizing hormone-releasing hormone (LHRH) to the tissue culture medium. In both methods, to determine the presence of inhibin, we used the criterion of a decreased release of FSH into the medium or serum that was not accompanied by an concomitant decrement in LH concentrations. Both methods detected the presence of inhibin within HFF from women during the follicular but not the luteal phase of the menstrual cycle. This finding suggests that this substance may play a role in the regulation of FSH secretion at a specific stage of the reproductive cycle in women.

Effects of Centrally Acting Drugs on Serum Prolactin Levels in Rhesus Monkeys

Prolactin (Prl) responses to anesthetics, stress of immobilization, and agents that block adrenergic receptors were determined in 13 female rhesus monkeys. The local anesthetic (lidocaine HCl) had no effect, but the general anesthetics (ketamine HCl and sodium pentobarbital) significantly increased serum Prl levels in intact animals. There was no indication of a pentobarbital-induced inhibition of Prl secretion as reported for other species. A combination of halothane anesthesia and immobilization for 30 or 60 min produced significantly greater increases in Prl levels than immobilization alone for similar periods of time. Prl responses to blockers of adrenergic receptors varied in chair-adapted ovariectomized monkeys. The increase in serum Prl concentrations produced by the beta-receptor blocker propranolol was less marked than that induced by the alpha-receptor blockers phentolamine and phenoxybenzamine. Prl elevations of the highest magnitude and longest duration were produced by haloperidol, which blocks both adrenergic and dopaminergic receptors. Pretreatment of these animals with estradiol-17beta had no effect on Prl responses to phentolamine and haloperidol. These results indicate involvement of an adrenergic neurotransmitter system in the control of Prl secretion in primates.