Judith Molnár

The role of PACAP in gonadotropic hormone secretion at hypothalamic and pituitary levels

The presence of pituitary adenylate cyclase-activating polypeptide (PACAP) and its mRNA in the three levels of the hypothalamo-hypophyseal-ovarian axis was previously demonstrated using immunohistochemistry, in situ hybridization, and reverse transcriptase polymerase chain reaction (RT-PCR). In the hypothalamus, PACAP is present in neuroendocrine effector cells and in the median eminence. In the anterior pituitary and ovary, PACAP is transiently present during the proestrous stage of the estrous cycle. In the pituitary, PACAP was observed in gonadotropes. In the ovary, PACAP was demonstrated in the granulosa cells of the preovulatory ovarian follicles. The effect of PACAP on luteinizing hormone (LH) secretion was demonstrated in in vivo and in vitro models. In our work we have studied the role of PACAP in gonadotropic hormone secretion at hypothalamic and pituitary levels. At the hypothalamic level, PACAP, administered intracerebroventricularly to female rats before the critical period of the proestrus stage, can inhibit LH release and ovulation. Its inhibiting effect is mediated through corticotropin-releasing factor (CRF) and endogenous opioids. PACAP administered to neonatal female rats delayed the onset of puberty by influencing the luteinizing hormone-releasing hormone (LHRH) neuronal system. In the pituitary gland, the release of PACAP depended on the stage of the estrous cycle and on the time of day the animals were sacrificed. On the day of proestrus, the number of PACAP-releasing cells showed a diurnal change with two peaks (in the morning and in the evening). The peak was much higher in the evening at the end of the LH surge than in the morning.

(D-Met2, Pro5)-Enkephalinamide-Induced Blockade of Ovulation and its Reversal by Naloxone in the Rat

The effects of (D-Met2,Pro5)-enkephalinamide--a superactive enkephalin analogue--on the preovulatory discharge of gonadotrophic hormones and on ovulation were investigated. Injection of the opioid (16 nmol) into a lateral cerebral ventricle of regularly cycling female rats immediately before the critical period on the day of proestrus resulted in a blockade of ovulation and in a concomitant depression of the preovulatory plasma luteinizing hormone and follicle-stimulating hormone levels. A smaller dose of the opioid (2 nmol) did not inhibit ovulation. The inhibition of the preovulatory gonadotrophic hormone discharge and the blockade of ovulation by the opioid were reversed by naloxone administration. Our data are consistent with the view that the endogenous opioid peptides may be involved in the physiological regulation of the central neural events which lead to ovulation in the rat.

Effect of Various Hypothalamic Deafferentations Injuring Different Parts of the GnRH Pathway on Ovulation, GnRH Content of the Median Eminence, and Plasma LH and FSH Levels

Various supra- and retrochiasmatic cuts injuring different parts of the septo-preoptico-infundibular GnRH pathway were made in adult female rats, and their effects on ovulation, median eminence (ME) GnRH content and on plasma LH and FSH levels were studied. Extended retrochiasmatic frontal cut just behind the optic chiasm, or a frontal cut in front of the suprachiasmatic nucleus presumably interrupting the whole GnRH pathway blocked ovulation, led to persistent estrus with polyfollicular ovaries, and reduced the ME GnRH content to 10 and 32%, respectively, expressed in percentage of unoperated control value. Severance of the GnRH pathway on one side or partial interruption of the pathway on the two sides in the retrochiasmatic area did not interfere with ovulation, and the ME GnRH content was 50% or more of the control value. Disconnection of the GnRH fibers coming from the septum resulted in a more than 30% decrease in the GnRH content of the ME, but did not block ovulation. Two separate symmetrical lateral cuts in the suprachiasmatic area leaving intact the presently known GnRH pathway reduced the ME GnRH content to 40% of the intact value, but did not interfere with ovulation. Plasma LH and FSH levels of the animals with different knife cuts were considerably dissociated and there were no apparent correlations between brain interventions and the concentrations of these two hormones in the blood. Our previous findings together with the present data suggest that: half of the GnRH pathway, medial or lateral bundles of the pathway on the two sides are sufficient for ovulation and cyclic gonadotrophic function; persistent estrus develops if the ME GnRH content is below 40% of the control value, and about 60% of the ME GnRH originates outside the preoptic, supra- and retrochiasmatic region. Half of this 60% may come from the septum and the vertical part of the diagonal band of Broca, the other half from the region in front of the preoptic area. The remaining 40% presumably arises from the preoptic (7-10%), supra- (15-20%) and retrochiasmatic region (8-10%).

Morphine amplifies norepinephrine (NE)-induced LH release but blocks NE-stimulated increases in LHRH mRNA levels: comparison of responses obtained in ovariectomized, estrogen-treated normal and androgen-sterilized rats

In these studies we examined the temporal effects of intracerebroventricular (i.c.v.) infusions of norepinephrine (NE) on plasma LH and on LHRH mRNA levels in the organum vasculosum of the lamina terminalis (OVLT) and in neurons located in the rostral (r), middle (m) and caudal (c) preoptic areas (POA) of ovariectomized, estrogen-treated rats. Thereafter, we compared these responses to those which occur in androgen-sterilized rats (ASR). NE infusions not only increased plasma LH concentrations but within 1 h after NE, LHRH mRNA levels also were increased significantly in the OVLT and rPOA but not in the mPOA or cPOA. By 4 h, these message levels still were elevated in the OVLT and rPOA and they now also were significantly higher than control values in the mPOA and cPOA. While NE also increased LH secretion in ASR, the plasma LH concentrations obtained were markedly blunted compared to control values. Moreover, NE infusions did not alter single cell levels of LHRH mRNA in any region of the rostral hypothalamus. Previously, we have reported that morphine (s.c.) markedly amplifies NE-induced LH release and questioned whether these responses are accompanied by concomitant augmented increases in LHRH mRNA levels. Morphine alone did not affect basal LHRH mRNA or plasma LH levels. However, when rats were pretreated with morphine (-15 min) and NE was infused i.c.v. at 0 time, significant amplification of LH release occurred but, unexpectedly, morphine completely blocked NE-induced increases in LHRH mRNA levels in all of the neurons we examined. Morphine also amplified LH release in ASR but these responses were significantly less than those obtained in control rats.(ABSTRACT TRUNCATED AT 250 WORDS)

Regulation of the gonadotropin-releasing hormone (GnRH) neuronal system: morphological aspects.

This paper gives a brief survey of the structural organization of the GnRH neuronal system and of the chemically identified axons impinging directly on GnRH neurons. There are great species differences in the localization of the GnRH nerve cells. In the human and monkey brain, they are concentrated in the medial basal hypothalamus. GnRH neurons project not only to the median eminence, but terminate also on other nerve cells including GnRH elements. Under physiological circumstances the GnRH neurons are influenced by nervous structures in- and outside the medial basal hypothalamus and by ovarian and other hormones. Recent, primarily immunocytochemical studies indicate that catecholaminergic, serotoninergic, GABA-ergic, opioid peptidergic, immunoreactive substance-P and corticotropin releasing factor-immunoreactive axons synapse on GnRH neurons. These findings provide morphological basis for the view that neurons operating with these compounds can act directly on GnRH release. Direct connections of the GnRH neurons demonstrated so far does not exclude the possibility that these neurotransmitters or neuromodulators influence GnRH release also at other levels of the CNS.

Further studies on circadian hormone rhythms after local pharmacological destruction of the serotoninergic innervation of the rat suprachiasmatic region before the onset of the corticosterone rhythm

In previous studies we observed that local pharmacological destruction of the serotoninergic innervation of the hypothalamic suprachiasmatic nucleus before the onset of the circadian plasma corticosterone rhythm interferes with the appearance of the corticosterone rhythm up to the age of two months. In the present investigations we studied other hormone rhythms in such rats and tested animals for corticosterone rhythm when they were older than two months. We found normal circadian fluctuations in plasma testosterone and prolactin levels, variations in growth hormone concentrations, but no changes in plasma corticosterone levels of 63-day-old rats receiving 5,7-dihydroxytryptamine, a neurotoxin selectively destroying the serotoninergic structures, into the suprachiasmatic nucleus at the age of 16 days. Rats did show circadian variations in plasma corticosterone concentrations when tested 3 months after treatment with the neurotoxin. In these latter animals, a significant amount of serotonin-immunoreactive fibers and terminals were evident in the suprachiasmatic nucleus. Only a very few of such elements were seen in rats with a shorter postoperative survival. Our data support the view that serotoninergic innervation of the suprachiasmatic nucleus is essential for the onset of the circadian fluctuations of plasma corticosterone concentrations.

PACAP38 and PACAP27 administered intracerebroventricularly have an opposite effect on LH secretion.

The effect of PACAP38 on the LH surge and ovulation was compared with that of PACAP27 and VIP in the same model. The peptides were administered intracerebroventricularly before the critical period of the proestrous stage. PACAP38 was able to inhibit ovulation and to prevent the preovulatory LH surge; however, PACAP27 did not inhibit the ovulation and VIP inhibited the ovulation in 2/11 animals. In those animals of the last two groups in which ovulation occurred, the preovulatory LH surge was higher than in control rats. It is speculated that the opposite effect of PACAP38 and PACAP27 on the preovulatory LH surge and ovulation is possibly mediated through different receptors.

New Aspects of the Neuroendocrine Role of PACAPa

The presence of PACAP was revealed in the anterior pituitary with RIA, HPLC, and with the demonstration of its mRNA. The level of PACAP mRNA in the anterior pituitary is the highest during the proestrous LH surge. In our immunohistochemical studies we were able to demonstrate PACAP immunoreactive cells in the anterior pituitary. The shape and the distribution of PACAP immunoreactive cells were very similar to that of the gonadotropes; however, the number of PACAP cells was less than that of LH cells. Additionally, another PACAP-positive cell population with small diameter appeared in the proestrous stage, during pregnancy and lactation. Double labeling revealed that the major part of large PACAP cells exhibited LH immunoreactivity and those with a small diameter contained PRL. It is not clear whether the pituitary- or the hypothalamic-born PACAP, or both, influence pituitary LH and PRL secretion. I.c.v. administration of PACAP just prior to the critical period in the proestrous stage inhibited the expected ovulation and blocked the proestrus LH and PRL surge, although i.v. administration of PACAP had no effect. PACAP antiserum did not interfere with ovulation when i.c.v. or i.v. injection was used. Our results support the view that PACAP has a role in the control of LH and PRL secretion during the estrous cycle, pregnancy, and lactation. The inhibitory effect of PACAP on ovulation is mediated through the hypothalamus.

Study on the hypothalamic factors mediating the inhibitory effect of PACAP38 on ovulation.

1. IntroductionThe pituitary adenylate cyclase activating polypeptide(PACAP) was first isolated from ovine hypothalami in anattempt to discover new hypothalamic factors [25]. It ispresent in two bioactive amidated forms: PACAP38 andPACAP27 with 38 and 27 amino acid residues, respectively[25,26]. In the brain, PACAP38 is the predominant form[4]. Immunohistochemical studies have shown PACAP im-munoreactive cell bodies in the hypothalamus and fibers inthe median eminence and in the posterior pituitary [16,17].Gradually accumulating observations suggest that PACAPis a hypophysiotropic factor:1. PACAP immunoreactive fibers can be found in theexternal zone of the median eminence around theportal capillaries [15,16].2. PACAP is released into the portal blood where itsconcentration is higher than in the general circulation[9].3. PACAP receptors are present in the anterior pituitary[12].4. PACAP influences the anterior pituitary functions (seeRawlings and Hezareh for review [29]).Although a lot of data suggests the role of PACAP as ahypophysiotropic factor, there is also evidence which showsthat PACAP may act as a neurotransmitter or neuromodu-lator, influencing other hypothalamic factors which are in-volved in the control of anterior pituitary functions. Ourprevious experiments have shown that PACAP38 givenintracerebroventricularly (ICV) before the critical period ofthe proestrous afternoon was able to block the ovulation andthe preovulatory LH surge, while the i.v. administration hadno effect [19]. It was concluded that PACAP38 does notexert its inhibitory effect at the pituitary level, rather it ismediated by other hypothalamic factors. This hypothesis issupported by in vitro experiments. In a superfusion systemPACAP38 stimulated rather than inhibited the LH release[25]. With the use of a special cell culture and immunohis-tochemistry (cell immunoblot assay, CIBA) it was alsofound that PACAP38 stimulated the LH release from ante-rior pituitary cells taken from proestrous rats [20]. On thebasis of the above mentioned results, it is obvious that IVadministered PACAP38 has no inhibitory effect on the LHrelease in proestrous female rats. The in vitro results furthersupport the hypothesis that PACAP38 exerts its inhibitoryeffect on the gonadotrop hormone secretion through hypo-thalamic factors. Our candidates were the endogenous opi-oids, melatonin, somatostatin, CRF, and GABA, all of themwith well known inhibitory effect on LH secretion.1.1. Opiates and endogenous opioidsThe inhibitory action of opiates on LH secretion wasshown by Barraclough and Sawyer nearly five decades ago[5]. The major site of action seems to be the hypothalamus,since morphine inhibits the firing frequency of the LHRHneurons [14] and the LHRH release into the hypophysialportal blood [7]. (D-Met

The inhibitory effect of PACAP38 on ovulation is mediated by CRF and endogenous opioids

In our previous experiments 1,2 it was demonstrated that PACAP38 given intracerebroventricularly ( icv ), before the critical period of the proestrous stage, was able to block the expected ovulation and the preovulatory LH surge, whereas intravenous ( i.v. ) administration had no inhibitory effect. It was concluded that the inhibitory effect of PACAP38 on the ovulation might be mediated via the hypothalamus rather than directly through the pituitary gland. In the work reported here our aim was to elucidate these factors.