Lamprey gonadotropin hormone-releasing hormone-III has no selective follicle-stimulating hormone-releasing effect in rats.

Lamprey gonadotropin releasing-hormone (LGnRH)-III, a hypothalamic neurohormone recently isolated from sea lamprey, was reported to have a selective stimulatory effect on follicle-stimulating hormone (FSH) release in rats and suggested to be the mammalian FSH-releasing factor. In this study, we determined the relative luteinizing hormone (LH)- and FSH-releasing potency of LGnRH-III compared to mammalian gonadotropin-releasing hormone (LHRH) in normal female rats, ovariectomized (OVX) and oestrogen/progesterone substituted rats and the superfused rat-pituitary cell system. The specificity of LGnRH-III for the mammalian LHRH receptor was investigated by blocking the receptor with an LHRH antagonist, MI-1544. In vitro, LGnRH-III dose-dependently stimulated both LH and FSH secretion from rat pituitary cells at 10(-7) to 10(-5) M concentrations, while LHRH stimulated gonadotropin secretion at a 1000-fold lower doses (10(-10) to 10(-8) M). The difference between its LH- and FSH-releasing potency was similar to that of LHRH. LGnRH-III bound to high affinity binding sites on rat pituitary cells with a Kd of 6.7 nM, B(max)=113 +/- 27 fmol/mg protein. In vivo, LGnRH-III also stimulated both LH and FSH secretion in a dose-dependent manner and, similar to LHRH, induced a greater rise in the serum LH than the FSH level. In normal cycling rats, it showed 180-650-fold weaker potency than LHRH in stimulating LH secretion and 70-80-fold weaker effect in stimulating FSH secretion. In OVX rats, LGnRH-III demonstrated a similarly weak effect on both gonadotropins. It was found to be 40-210-fold less potent than LHRH regarding LH release and 50-160-fold weaker regarding FSH release. LHRH-receptor antagonist MI-1544 prevented both the LH- and the FSH-releasing effect of LGnRH-III both in vitro and in vivo. These results do not support the hypothesis that LGnRH-III might be the mammalian FSH-releasing factor but demonstrate that it is a weak agonist for the pituitary LHRH receptor and stimulates both gonadotropins in a dose-dependent fashion.

Increase in mRNA concentrations of pituitary receptors for growth hormone-releasing hormone and growth hormone secretagogues after neonatal monosodium glutamate treatment.

Previous studies have demonstrated that neonatal monosodium glutamate (MSG) treatment destroys growth hormone releasing-hormone (GHRH) neurones within the hypothalamic arcuate nucleus, decreases serum GH and insulin-like growth factor (IGF-I) concentrations, and retards linear growth. In the present study we investigated whether expression of pituitary GH, GHRH receptors (GHRH-R), growth hormone secretagogue receptors (GHS-R) and liver IGF-I is altered in this model of GHRH deficiency. In addition, we investigated if treatment of MSG-lesioned rats with the GHRH agonist, JI-38, would 'normalise' the GH-axis. Serum GH and IGF-I concentrations were determined by RIA, GH mRNA levels were evaluated by Northern blotting, and GHRH-R, GHS-R and IGF-I mRNA levels were measured by semiquantitative RT-PCR. In accord with previous reports, neonatal MSG treatment caused 50% and 76% decreases in serum GH and IGF-I concentrations, respectively, at 8 weeks of age. The decline in circulating GH was accompanied by a 56% reduction in total pituitary GH content, which was a reflection of the decrease in total pituitary protein. However, GH concentration (per mg protein) was unaltered. Despite the maintenance of a normal GH concentration, GH mRNA concentration (per microg total RNA) was suppressed by 42%, compared to saline-treated controls (P<0.05). These data indicate that a post-transcriptional mechanism, such as a reduction in the GH secretory rate, acts to maintain intracellular GH concentrations. The fall in circulating concentrations of GH leads to a 42% decrease in liver IGF-IB mRNA levels, while liver IGF-IA transcripts showed only a 27% suppression. In contrast, pituitary GHRH-R and GHS-R mRNA levels (per microg total RNA) were increased in MSG-lesioned rats by 96% and 180% of normal values (P<0.01), respectively. Twice daily treatment of MSG-lesioned rats (for 2 weeks) with the GHRH agonist, JI-38, increased serum GH and IGF-I levels, as measured 20 h after the last agonist injection. However, GH, IGF-I, GHRH-R and GHS-R mRNA levels were not altered at this time. These results demonstrate that intermittent GHRH agonist treatment stimulates pituitary GH secretion and GH in turn stimulates hepatic IGF-I but that effects on gene expression are not sustained. Collectively, our observations demonstrate a complex interplay between transcriptional, translational and post-translational mechanisms within each level of the GH-axis following destruction of GHRH neurones by neonatal MSG treatment.

Electrochemical stimulation of the median eminence evokes FSH but not LH release after LHRH antagonist treatment in vivo and in vitro.

Experimental data suggest that a follicle stimulating hormone-releasing factor (FSH-RF) distinct from luteinizing hormone-releasing hormone (LHRH) exists. In the present study, we investigated, in short-term ovariectomized (OVX) rats, whether FSH-RF(s) can be released from nerve terminals by electrochemical stimulation (ECS) of the median eminence. To prevent the effect of LHRH liberated by ECS, 100 microg of a potent LHRH antagonist (MI-1544) was administered to one group of OVX rats 60 min before ECS. Two groups of OVX rats were used as controls. One group was treated with the solvent of the LHRH antagonist 60 min before the ECS; the other group received sham-ECS only. In-vitro experiments using a hypothalamus-pituitary coperifusion system were also performed to investigate the direct effect of ECS of the median eminence on LH and FSH release from pituitary cells. ECS in vivo induced 4.6-fold (P<0.01) and 10.2-fold (P<0.01) elevation of serum LH concentration, measured by RIA at 10 min and 60 min after ECS, respectively. Serum FSH concentrations increased 1.35-fold at 10 min (P<0.01) and 1.50-fold at 60 min (P<0.01) after ECS, compared with sham-stimulated controls. Administration of LHRH antagonist attenuated the ECS-induced release of LH by 44% at 10 min and prevented it entirely at 60 min after ECS. However, the ECS-induced release of FSH was not modified by the antagonist at 10 min and was diminished by only 17% at 60 min after ECS, compared with solvent-treated and stimulated controls. Immunohistological examination of the hypothalami showed that LHRH-immunoreactivity was depleted in the region of ECS. In the study in vitro, substances released from the fragments of mediobasal hypothalami bearing ECS in the median eminence induced significant release of both LH and FSH, and the induced release of LH, but not FSH, was prevented by the LHRH antagonist. The present study suggests that FSH-releasing factor(s) different from LHRH can be released from the median eminence and that a significant portion of FSH secretion is independent of the control of LHRH.

Gonadectomy modifies the gender specific pattern of desensitization of pituitary cells by gonadotropin-releasing hormone in the superfusion system.

The effect of a 6-h infusion of 1 nM gonadotropin-releasing hormone (GnRH) on LH release from dispersed anterior pituitary cells from rats in metoestrus ("metoestrus cells"), intact male rats ("male cells"), ovariectomized rats ("ovx cells"), and from orchidectomized rats ("orchidex cells") was investigated. Using metoestrus cells the initial amplitude of the response was high but desensitization was strong: the amount of LH secreted in the last 90-min period of the 6-h incubation was less than 20% of the initial value. In male cells, the immediate response was weaker; however, in the second 90-min of the 6-h incubation a 20% to 30% increase was observed and even in the fourth 90-min period the amount of LH secreted was more than 70% of the initial levels. Gonadectomy diminished the gender specific differences. Desensitization became less pronounced when ovx cells were used and its level was found to increase in orchidex cells, as compared to metoestrus cells and male cells, respectively. The responses to 3-min pulses of GnRH and KCl given after the 6-h incubation were strongly reduced in metoestrus cells but increased in male cells, compared to the initial peaks. Gonadectomy nullified these differences, too. Our data show that gonadectomy causes alterations in the intracellular pools of LH available for immediate and prolonged release in male and female rats. Decrease of estrogen in the sex steroid balance of female rats reduces the amount of LH available for immediate release and relatively increases the rate of replenishment into the immediate release pool. This allows a more stable secretion of LH during the 6-h stimulation. Decrease in androgens causes opposite changes. These phenomena could be observed in vitro, after keeping the pituitaries in a hormone free milieu for 16-28 h.

Effects of continuous and repetitive administration of a potent analog of GH-RH(1-30)-NH2 on the GH release in rats treated with monosodium glutamate.

To assess the efficacy of a potent GH-releasing hormone (GH-RH) analog (D-Ala2,Nle27,Gaba30-GH-RH-(1-30)-amide) in the treatment of GH deficiency, we investigated the effects of chronic administration of this analog (A-495) on growth responses in monosodium glutamate (MSG)-lesioned rats. Basal serum GH concentrations, GH responses to bolus injections of GH-RH, as well as acceleration of body gain and linear growth were compared after long-term continuous and repetitive administration of A-495. The effects of continuous and repetitive administration of the analog on GH responses in vitro were also compared using the superfused pituitary cell system method. Treatment with MSG reduced the body weight and linear growth of the animals (-22% and -11%, respectively), the basal serum GH concentration (-66%), and the GH-RH-induced absolute GH responses (-61%) but did not alter the relative GH responses (to basal GH concentrations). Repetitive administration of 10 micrograms daily doses of A-495 at 24 h intervals for 2 weeks highly increased the GH responsiveness to GH-RH and induced catch-up growth, by which MSG-treated animals achieved the growth rate of normal controls. However, basal serum GH concentrations were only modestly enhanced. Continuous infusion of A-495 at the same daily dose resulted in slight increases in the GH-RH-induced GH rises, moderate acceleration of body gain, and no change in linear growth. Basal serum GH concentrations were not significantly influenced by this treatment. These results demonstrate that exogenous GH-RH pulses administered at lower frequency than the frequency of the physiological GH secretion are able to fully restore the normal growth rate of the GH deficient rats. The effectivity of the treatment is rather dependent on the magnitude of GH rises than the basal GH level. Although continuous administrations of the GH-RH is also have some effect on the body gain, repetitive administration is more effective at the same daily dose. Our results from in vitro experiments show that, in addition to the low magnitude of the GH-RH-stimulated GH rises, desensitization of the GH secretory response might also be accounted for the low effectivity of the continuously administered GH-RH. Present results demonstrate the therapeutic usefulness of our new GH-RH analog and are the first to evidence that GH-RH need not be administered as frequently as the appearance of the endogenous GH pulses to restore the normal growth of the GH deficient rats.

Effects of continuous and repetitive administration of a potent analog of GH-RH(1-30)NH2 on the GH release in rats.

We examined the desensitization and/or sensitization phenomenon in the pituitary GH responsiveness induced by continuous infusion and multiple pulses at different frequencies of a potent GH-RH analog [D-Ala2, Leu15, Nle27, GABA30-GH-RH(1-30)amide]. Further, we investigated the correlation between doses and GH responses, as well as between pulse frequency and GH responses in male rats in vivo and in vitro. Long-term, continuous administration was attained by osmotic minipumps releasing low and high doses of the analog for 14 days. The effects of repetitive administration of the GH-RH analog on the pituitary GH release was investigated by injecting 4-6 pulses of the analog at different doses and pulse frequencies. The in vitro experiments were performed in the superfused rat anterior pituitary cell system. Pituitary cells were challenged with continuous, repetitive and simultaneous continuous and repetitive perfusion of the analog. Continuous infusion with low doses of the GH-RH analog in vivo induced sensitization of the pituitary GH-secretory responsiveness and resulted in moderately increased GH releases (129% of the control) to additional bolus injections of the same analog, whereas continuous stimulation of the pituitary with high doses of the GH-RH analog evoked desensitization and resulted in blunted GH responses (29% of the control). Despite the desensitization of the pituitary GH-secretory responsiveness, high doses of the analog elevated the serum GH concentration to 310% and induced acceleration of body weight gain (160% of the control). Repetitive pulsatile administration of the GH-RH analog evoked both sensitization and desensitization of the pituitary GH-secretory responsiveness, depending on the dose and pulse frequency administered.(ABSTRACT TRUNCATED AT 250 WORDS)

Antiovulatory doses of antagonists of LH-RH inhibit LH and progesterone but not FSH and estradiol release.

The differential regulation of immunoactive FSH and LH secretion by endogenous LH-RH was studied using LH-RH antagonists (Ac-D-Trp1,2, D-Cpa2, D-Lys6, D-Ala10LH-RH (MI-1544) and (Ac-D-Nal1, D-Phe(pCl2), D- Trp3, D-Cit6, D-Ala10LH-RH (SB-030) in ovariectomized (OVX) and regularly cycling rats. Single injections of 10 micrograms and 100 micrograms doses and long-term treatment with 10 micrograms doses of MI-1544 were used in OVX animals. Serum and pituitary LH and FSH, as well as serum estradiol and progesterone was determined by RIA during and/or after the treatment. Single injections of MI-1544 in OVX animals caused prompt (in 2 h) and long-lasting (for more than 24 h) suppression of the serum LH, while no or late decrease (after more than 6 h) of the serum FSH. Long-term treatment with the same analog decreased the serum LH (by 50%) and moderately increased the pituitary LH (by 21%) but did not change the serum and the pituitary FSH concentrations. In normal rats, long-term treatment with both of our analogs also resulted in divergent alterations in the LH and FSH concentrations. Serum LH dropped to undetectable levels,while serum FSH did not change significantly. Pituitary LH increased (by 31 to 41%), while FSH decreased (by 27 to 38%). Marked depression was found in the serum progesterone (by 64%) but no significant change in the serum estradiol levels, after the long-term treatment for 21 days. The ovarian cycles were interrupted, and no ovulation appeared during the treatment. Significant decrease was detectable in the weight of the ovaries (by 46%), whereas the weight of the uteri did not change or slightly elevated (by 22%), after the treatment with SB-030 or MI-1544, respectively.

New Gaba-containing analogues of human growth hormone releasing hormone (1-30)-amide: II. Detailed in vivo biological examinations.

Analogues of human growth hormone-releasing hormone-(1-30)-amide [GH-RH(1-30)-amide] were tested for their ability to stimulate GH release in vivo by injecting the peptides intravenously (iv), subcutaneously (sc), and intramuscularly (im). The analogues involved derivatization with Nle27 and Gaba substituents at the C-terminus with or without D-amino acid(s) in the peptide chain. The potency of the analogues was compared to that of GH-RH(1-29)-amid testing their ability to release GH at 5, 15 and 30 min after the administration. In iv test the potency of the analogues was 1.2-2 times higher than that of the GH-RH(1-29)-amide, and no significant differences were detected between the potencies of the analogues with or without D-amino acid. In the sc test the analogue with D-Ala2, Nle27, and Gaba30 substitutions expressed 8.0-51.7 times higher potency than the GH-RH(1-29)-amide, however, the analogue with similar modifications but with L-Ala2 showed the same low potency (1.2-2.1) as in the iv test. Results from the im experiments were similar to those of SC test. The most potent analogues were those which had D-Ala2, Nle27, and Gaba30 substitutions with Gly15 or Leu15. Circular dichroism (CD) spectra of the analogues showed that Leu in position 15 increased the stability of the predominant alpha-helix conformation, which improved the absorption of the molecule.(ABSTRACT TRUNCATED AT 250 WORDS)

Immunocytochemical characterization of afferents to estrogen receptor-containing neurons in the medial preoptic area of the rat.

Double-label immunocytochemistry has been employed to elucidate the chemical nature of the afferent neuronal projections to the estrogen receptor-containing neurons located in the medial preoptic area of the rat brain. To ensure a clear separation of the immunolabelled afferent profiles from the estrogen receptors, the former were visualized first and the diaminobenzidine reaction product was silver-gold intensified. Using a monoclonal antibody raised against purified human estrogen receptors, we observed an intense nuclear immunoreactivity in Vibratome, semithin and ultrathin sections. Neuropeptide-Y, serotonin-, phenylethanolamine N-methyltransferase- and adrenocorticotrophin-immunoreactive axons and varicosities were observed in close apposition to the estrogen receptor-positive cells. At the ultrastructural level, neuropeptide-Y-immunoreactive boutons were seen in synaptic contact with cells showing estrogen receptor immunoreactivity in their nucleus. These results indicate that neurons located in the medial preoptic area, one of the principal sites for the control of female reproductive function, may be influenced by both estrogen and neurotransmitters/neuropeptides via, respectively, nuclear receptors and synaptic contacts.

Desensitization of pituitary cells by gonadotrophin-releasing hormone or its analogues in the superfusion system: different pattern for males and females.

The effect of a 6-h infusion of gonadotrophin-releasing hormone (GnRH) or its analogues on dispersed anterior pituitary cells from male or female rats was investigated. The cells were stimulated with 3-min pulses of K(+) and GnRH. Thereafter GnRH (1 nM) or GnRH analogues ([D-Trp(6) ]GnRH-ethylamide ([D-Trp(6) ]GnRH, 50 pM), [D-Phe(6) , Gln(8) ]GnRH-ethylamide (Folligen, 100 pM) and [Asu(6) ]GnRH-ethylamide ([Asu(6) ]GnRH, 33 pM)) were applied for 6 h. In cells from female rats this treatment resulted in a 20-fold increase in luteinizing hormone (LH) secretion during the first 90-min period of the 6-h incubation. Following this a gradual decrease in LH release occurred, and during the fourth 90-min period the amount of LH secreted was only one-third or less of the initial value. The pituitary cells of male rats responded to the same treatment with only a 7-fold rise of LH secretion during the first period. In the second 90-min of the 6-h incubation a 20% to 30% increase was observed. Even in the fourth 90-min period the amount of LH secreted was two-thirds or more greater than that of the first 90-min period. When using 10-fold greater concentrations of the same peptides in males, the increase in hormone secretion in the second 90-min was not seen and the hormone release decreased to around 50%. We found definite differences in the responses of male and female rat pituitary cells to the 6-h infusion of GnRH or its analogues: the initial amplitude of the response in females was higher but desensitization was stronger. In males, the initial response was weaker; however, even using doses one magnitude greater, the level of desensitization did not reach the values obtained in females. The results were similar both with GnRH and the analogues. The responses to 3-min K(+) and GnRH stimuli given after the 6-h incubation were strongly reduced in cells from female rats compared to the initial responses; however, in cells from male rats the reaction was higher or unchanged. The ratio of LH released by the final K(+) stimulus relative to the actual LH content of the cells decreased in females but increased in males. Our data show that the differences between the pattern of desensitization in cells from male and female rats may be caused by the differences in the amount and ratio of immediately releasable hormone and the hormone replenishment into these pools.

Ultrastructural analysis of estrogen receptor immunoreactive neurons in the medial preoptic area of the female rat brain.

Neurons of the medial preoptic area were studied in the brain of the female rat by means of ultrastructural immunocytochemistry using a monoclonal antibody generated against purified estrogen receptor (ER), in order to delineate the morphological correlates of estrogen feedback mechanisms. In addition to the preoptic area, the bed nucleus of the stria terminalis, the arcuate and ventromedial nuclei of the hypothalamus exhibited an intense labelling for estrogen receptor. At the light microscopic level, the cell nuclei were immunoreactive. No major alterations were detected in the ER expression of medial preoptic neurons sampled during the estrous cycle, but proestrous rats did exhibit a slightly increased intensity of staining. At the ultrastructural level, the ER immunoreactivity was primarily confined to the nuclei and associated with the chromatin. Long term steroid deprivation elicited by either ovariectomy or ovariectomy plus adrenalectomy resulted in a marked intensity of nuclear labelling. This pattern was not influenced by acute estradiol replacement. These morphological data indicate that neurons of the medial preoptic area have the capacity to detect estrogens via receptor mechanisms and that changes in the level of the circulating ligand are manifested in an alteration in the staining for the estrogen receptor. The study also supports the revised concept of estrogen receptor action by demonstrating the presence of receptors in the nuclei of the cells, whether or not they are occupied by their ligand.

Combined retrograde tracing and immunocytochemical identification of luteinizing hormone-releasing hormone- and somatostatin-containing neurons projecting to the median eminence of the rat.

LHRH and somatostatin or somatotropin-release inhibiting factor (SRIF) are produced by neurons whose cell bodies are located in telencephalic and diencephalic regions in the rat. Many, but not all, of these neurons project to the external zone of the median eminence (ME), where the peptides are released from the nerve terminals into hypophysial portal vessels. In the present study, we identified these neurons by in vivo injection of a retrograde tracer, the lectin wheat germ agglutinin (WGA), into the external zone of the ME. Subsequently, colchicine was given into the lateral ventricle 10-24 h after the WGA injection. The animals were killed 24-48 h after the WGA injection. Vibratome sections of the brains were stained for both WGA and LHRH or SRIF with a dual immunocytochemical technique. Approximately 70% of the LHRH neurons in the septum and the anterior hypothalamus and about 70% of the SRIF neurons in the medial preoptic area, the anterior periventricular area, and the paraventricular nucleus were double labeled, indicating that they projected to the ME. None of the SRIF neurons in the ventromedial and arcuate nuclei were labeled with WGA. Double labeled LHRH cells were either smooth and fusiform or spiny. WGA-accumulating LHRH or SRIF perikarya were intermixed with single labeled LHRH or SRIF cells, which apparently did not project to the ME. The results indicate that there are at least two populations of LHRH neurons in the preoptic-septal region and two populations of SRIF neurons in the medial preoptic and anterior periventricular areas and the paraventricular nucleus of the rat brain: one with access to the portal capillaries of the ME and, therefore, functionally related to the regulation of the pituitary, and another without access to portal capillaries, perhaps functionally related to intracerebral neurotransmission or modulation. Moreover, some hypophysiotropic LHRH and SRIF neurons may have axon collaterals reaching multiple targets within the central nervous system.

Identification of hypophysiotropic luteinizing hormone-releasing hormone (LHRH) neurons by combined retrograde labeling and immunocytochemistry.

The decapeptide luteinizing hormone-releasing hormone (LHRH) is produced by telencephalic and diencephalic neurons and transported to the median eminence (ME). After having been released from nerve terminals, it is carried by the hypophysial portal vessels to the anterior pituitary, where it stimulates the production and release of luteinizing hormone (LH) and follicle stimulating hormone (FSH). Those LHRH neurons which project to the ME represent the final common pathway for the regulation of the pituitary/gonadal axis. We identified these neurons by injecting a retrograde tracer, the lectin wheat germ agglutinin (WGA), into the external zone of the ME. Eight to 24 hours later colchicine was given into the lateral ventricle and 24-48 hours after the WGA injection the animals were sacrificed. Vibratome sections of the brains were stained simultaneously for WGA and LHRH with a dual immunocytochemical technique. Approximately 70% of the LHRH neurons in the septum and the anterior hypothalamus were double-labeled, indicating that they projected to the ME. Double labeled LHRH cells were either smooth, fusiform or "spiny". WGA-accumulating LHRH perikarya were intermixed with single-labeled LHRH cells. The remaining 30% of the LHRH neurons which were not labeled with WGA appeared to project to different hypothalamic and extrahypothalamic areas of the brain. Our results suggest that there are at least two populations of LHRH neurons, one with access to the portal capillaries of the ME and functionally related to the regulation of the pituitary, and one without access to capillaries of the ME, functionally probably related to intracerebral neurotransmission or modulation.(ABSTRACT TRUNCATED AT 250 WORDS)

Effects of long-term administration of a superactive agonistic and an antagonistic GnRH analog on the pituitary-gonad system.

A powerful GnRH antagonist: [Ac-D-Trp1,3,D-Cpa2,D-Lys6,D-Ala10]-GnRH (MI-1544) and a superactive GnRH agonist: [D-Phe6,desGly10]-GnRH(1-9)EA (OVURELIN) were used in long-term administration to compare their effects on the inhibition of ovulation, LH and progesterone (P) release, LH content of pituitaries as well as on the recovery period. Both analogs showed 100% inhibitory effects on ovulation in very low doses during the daily treatment for 21 days. The antagonist prevented LH release already after the first injection, decreased the serum P level to 40%, and increased the LH content of the pituitary up to 180%, inhibiting only the release but not the synthesis of LH. The agonist showed marked LH-releasing effects on the first day of the treatment, which were reduced to 12% on the 7th day. Serum P concentration was dropped to 68% by the end of the treatment. No change was found in the LH content of pituitaries in the group treated with the agonist. Ovaries showed polifollicular pictures in the antagonist-treated group, and persistent corpora lutea were seen in the ovaries from the agonist-treated group. Regular estrous cycles returned 13-15 days after ceasing the treatment with the antagonist and 3-5 days after ceasing the treatment with the agonist. No edema-inducing effect was observed after the injections of the antagonist in doses of 100 times higher than the single antiovulatory dose.

Immunocytochemical localization of the gonadotropin-releasing hormone-associated peptide portion of the LHRH precursor in the hypothalamus and extrahypothalamic regions of the rat central nervous system.

The gonadotropin-releasing hormone-associated peptide (GAP) of the LHRH precursor and the decapeptide LHRH were localized in the rat brain by immunocytochemistry in 12- to 18-day-old animals, by use of thick Vibratome sections and nickel intensification of the diaminobenzidine-reaction product. Our results indicate that the GAP portion of the LHRH precursor is present in the same population of neurons that contain LHRH in the rat brain. An important difference observed was that the GAP antiserum, in contrast to LHRH antisera, stained several perikarya in the medial basal hypothalamus. GAP-immunoreactive perikarya were observed in the following regions: the olfactory bulb and tubercle, diagonal band of Broca, medial septum, medial preoptic and suprachiasmatic areas, anterior and lateral hypothalamus, and several regions of the hippocampus. In addition to the preoptico-terminal and the septo-preoptico-infundibular pathways, we also observed GAP-immunopositive processes in several major tracts and areas of the brain, including the amygdala, stria terminals, stria medullaris thalami, fasciculus retroflexus, stria longitudinalis medialis, periventricular plexus, periaqueductal gray of the mesencephalon and extra-cerebral regions, such as the nervus terminalis and its associated ganglion. These results confirm the specificity of previous immunocytochemical results obtained with antisera to LHRH. The presence of GAP immunoreactivity in nerve terminals of the rat brain indicates that GAP or a GAP-like peptide is located in the proper site to serve as a hypophysiotropic substance and/or as a neurotransmitter or neuromodulator.

The Pro-LHRH system of the rat brain. Effects of changes in the endocrine background.

The gonadotropin-releasing hormone-associated peptide (GAP) and luteinizing hormone-releasing hormone (LHRH) portions of the LHRH precursor were localized by immunocytochemistry in prepubertal female rats, in adult female rats at different stages of the estrous cycle, and in ovariectomized rats. Our results indicate that GAP is present in the same population of neurons as LHRH in the rat brain. These results confirm the specificity of previous immunocytochemical studies which used antisera to LHRH alone. The endocrine status of the animal was demonstrated to affect the immunocytochemical appearance of the GAP system. The number of GAP immunopositive cells and terminals is highest during diestrus II and lowest on the day of estrus, suggesting either a role in and/or a dependence upon the endocrine changes associated with the estrous cycle. Ovariectomy results in a gradual decrease in GAP immunoreactivity in the median eminence. This observation, in concert with other recent studies, suggests that ovarian factors may be acting to maintain the LHRH system and that ovariectomy may result in decreased synthesis and/or processing of the LHRH system and that ovariectomy may result in decreased synthesis and/or processing of the LHRH precursor.