GnRH Peptide Research Articles

THE SELECTIVE-INHIBITION OF VASCULAR-PERMEABILITY FACTOR (VPF) EXPRESSION IN OVARIAN-CARCINOMA CELL-LINES BY GONADOTROPIN-RELEASING-HORMONE (GNRH) AGONIST

GnRH agonists have been found to be clinically useful in several hormone-sensitive conditions, including cancer. However, there is controversy regarding a direct action of these agents on the pathologic tissue of a given disease process, in particular, tumors. In this study, we examined the effects of D-Trp(6) -LHRH, a potent GnRH agonist, on the expression of an increasingly important angiogenesis factor, VPF (vascular permeability factor)/VEGF (vascular endothelial growth factor). Ovarian carcinoma cell lines exposed to D-Trp(6) -LHRH in culture demonstrated a reversible inhibition of VPF mRNA expression and a parallel decrease in the endothelium-specific mitogenic activity of the conditioned media from these treated cultures. This study reports a novel activity of a GnRH agonist and provides a starting point to investigate the in vivo anti-angiogenic properties of GnRH peptide analogs.

Mating alters gonadotropin-releasing hormone cell number and content.

Female musk shrews (Suncus murinus) are induced ovulators, which lack a behavioral and ovarian estrous cycle. Females mate the first time they are introduced to a male, but a second or third mating, at least 24 h later, is usually required to induce ovulation. Because GnRH-immunoreactive (GnRH-ir) cell numbers increase during and after exposure to a male, we hypothesized that mating promotes synthesis of this important peptide. To test this hypothesis, we examined changes in GnRH-ir cell number and GnRH-ir content at select time points after mating and ovulation. One hour after mating, GnRH-ir cell numbers in olfactory-related regions of the forebrain were increased. By 15 h after mating, just before ovulation, GnRH-ir cell number and content were increased. Twenty-four hours after mating, GnRH-ir cell numbers in the tenia tecta and medial septum/diagonal band were lower in females that ovulated compared with females that did not ovulate. By 40 h postmating, females that ovulated had fewer GnRH-ir neurons and lower GnRH content in the entire brain than females that did not ovulate. In addition, we found significant negative correlations between plasma estradiol concentrations and both GnRH-ir cell numbers and content in the preoptic area of animals killed around the time of ovulation. Interestingly, significantly more GnRH-ir neurons and a greater content of GnRH peptide were observed in several forebrain nuclei of females that did not ovulate 40, compared to 24, h after mating. In contrast, numbers of GnRH-ir neurons in the midbrain declined 40 h postmating in ovulated females. These results suggest that mating stimulates activity in GnRH-ir neurons, and that ovulation is correlated with a decline in GnRH-ir cell number and content. In this species, mating can be used as an external trigger to activate GnRH neurons and examine the regulation and production of GnRH in heterogenic neuronal populations.

Inhibition of human hepatocarcinoma cell proliferation by mammalian and fish gonadotropin-releasing hormones.

This study provides the first demonstration that human hepatocarcinoma-derived cell line HepG2 contains GnRH receptors and responds to various molecular forms of GnRH in terms of inhibition of proliferation in a time- and dose-related manner. In addition, GnRH peptides also significantly inhibited the uptake of [3H]thymidine by these cells. The effect of GnRH was specific because GnRH antagonists reversed the GnRH-mediated inhibition, and non-GnRH peptides had no effect on HepG2 cell proliferation. An important finding is that certain fish GnRH molecules such as lamprey GnRH, which has little gonadotropin (LH) release activity in mammals, suppress hepatocarcinoma cell proliferation with similar potency to a superactive mammalian GnRH analog, [D-Lys6]GnRH. These findings may have profound implications in the development of an effective chemotherapy for treatment of human liver cancer. An added advantage is that fish GnRH forms will likely exert little side effect in terms of human pituitary gonadotropin release, gonadal steroidogenesis, and reproductive functions in general.

Inhibition of human hepatocarcinoma cell proliferation by mammalian and fish gonadotropin-releasing hormones

This study provides the first demonstration that human hepatocarcinoma-derived cell line HepG2 contains GnRH receptors and responds to various molecular forms of GnRH in terms of inhibition of proliferation in a time- and dose-related manner. In addition, GnRH peptides also significantly inhibited the uptake of [3H]thymidine by these cells. The effect of GnRH was specific because GnRH antagonists reversed the GnRH-mediated inhibition, and non-GnRH peptides had no effect on HepG2 cell proliferation. An important finding is that certain fish GnRH molecules such as lamprey GnRH, which has little gonadotropin (LH) release activity in mammals, suppress hepatocarcinoma cell proliferation with similar potency to a superactive mammalian GnRH analog, [D-Lys6]GnRH. These findings may have profound implications in the development of an effective chemotherapy for treatment of human liver cancer. An added advantage is that fish GnRH forms will likely exert little side effect in terms of human pituitary gonadotropin release, gonadal steroidogenesis, and reproductive functions in general.

In Vivo Actions of a Gonadotropin-Releasing Hormone (GnRH) Antagonist on Gonadotropin-II and Growth Hormone Secretion in Goldfish, Carassius auratus

In our previous in vitro studies, [Ac-Δ 3-Pro 1, 4FD-Phe 2, D-Trp 3,6]-mGnRH (analog E) suppressed both gonadotropin-II (GTH-II) and growth hormone (GH) release stimulated by sGnRH and cGnRH-II. In the present study analog E significantly inhibited the increases in plasma GTH-II levels stimulated by sGnRH in sexually mature female and sexually recrudescent goldfish. Treatment of goldfish with α-methyl- p-tyrosine methyl ester (α-MPT) inhibits dopamine synthesis and abolishes the inhibitory actions of dopamine on GTH-II release, resulting in a potentiation of the GTH-II response to sGnRH. Following α-MPT pretreatment, analog E significantly reduced basal plasma GTH-II levels, and suppressed both sGnRH and cGnRH-II actions on GTH-II release. Analog E also inhibited the increase in plasma GTH-II levels in sexually mature male goldfish exposed to the female sexual pheromone, 17α,20β-dihydroxy-4-pregnen-3-one (17α20β-P), demonstrating that the increase in plasma GTH-II levels is due to release of endogenous GnRH. Analog E significantly inhibited the increases in plasma GH levels stimulated by treatment with sGnRH. Implantation of estradiol pellets increases basal plasma GH levels and increases the GH responsiveness to sGnRH in sexually recrudescent goldfish; analog E also suppressed the increase in plasma GH levels stimulated by injection of sGnRH in estradiol-treated fish. Analog E suppressed basal GTH-II and GH levels in fish that were unhandled prior to injection; however, analog E was not effective in reducing basal plasma GTH-II or GH levels in experiments in which the fish were blood sampled or subjected to some experimental manipulation prior to injection of analog E. Analog E also suppressed basal levels of GTH-II in α-MPT-treated fish, suggesting that stress inhibition of GTH-II release may be mediated by the dopaminergic system. In summary, the results demonstrate that (i) analog E can suppress the actions of exogenous sGnRH and cGnRH-II on GTH-II and GH release in vivo , (ii) the GnRH system mediates, at least in part, the plasma GTH-II response in sexually mature male goldfish following exposure to the female sexual pheromone 17α20βP, and (iii) endogenous GnRH peptides are important in the regulation of basal plasma levels of GTH-II as well as GH, particularly in low stress conditions.

Molecular Cloning of a cDNA Encoding the Prepro-Salmon Gonadotropin-Releasing Hormone of the Red Seabream

Complementary DNA fragments encoding the prepro-salmon gonadotropin-releasing hormone ([Trp7, Leu8]GnR, sGnRH) of the red seabream Pagrus major were amplified from mRNA of the olfactory bulbs using a reverse transcriptase-polymerase chain reaction (RT-PCR), and the full-length cDNA was cloned from a cDNA library using the PCR-amplified cDNA as a probe. The cDNA consisted of 442 bp, including an open reading frame of 270 bp which encoded the prepro-sGnRH (90 amino acid residues). The prepro-sGnRH had the same architecture as that reported in other species. It was composed of a signal peptide, sGnRH and a GnRH-associated peptide (GAP), which was connected to sGnRH by a Gly-Lys-Arg sequence. The prepro-sGnRH of the red seabream had 90% amino acid identity to the prepro-sGnHR from an African cichlid Haplochromis burtoni which belongs to the same suborder as the red seabream; however, identity was lower to the prepro-sGnRH from Atlantic salmon Salmo salar (74%), and masu salmon Oncorhynchus mason (70%). The GnRH peptide itself and the Gly-Lys-Arg sequence in the prepro-GnRH are highly conserved among vertebrates. The red seabream GAP also shows significant amino acid identity to the GAPs of the African cichlid (89%), Atlantic salmon (74%), and masu salmon (67%), but exhibits no significant identity to chicken or mammalian GAP.

Seasonal Variations in Gonadotropin Responsiveness, Self-Priming, and Desensitization to GnRH Peptides in the Common Carp Pituitary in Vitro

Seasonal variations of the GtH release response to salmon GnRH (sGnRH) and [d-Arg6, Pro9NEt]-sGnRH (sGnRH-A) were investigated in female common carp at different stages of the reproductive cycle using perifused pituitary fragments. The responsiveness to sGnRH and sGnRH-A varied seasonally in common carp pituitaries in vitro, with the greatest GtH release response in pituitaries from sexually mature (preovulatory) fish compared to pituitaries from sexually regressed fish. The magnitude of this seasonal change in the GtH release response was greater for sGnRH-A than for sGnRH, and sGnRH-A has a higher potency than sGnRH, particularly in pituitaries from sexually mature fish. Desensitization of perifused pituitary fragments to sGnRH and sGnRH-A, and a self-priming effect of sGnRH-A on the GtH release response, caused by repeated pulse administrations of the GnRH peptides, varied with the stage of reproductive cycle of the common carp. Using pituitaries from sexually regressed female common carp, desensitization occurred only when a high dose of sGnRH or sGnRH-A was given as repeated pulses at short time intervals, and no self-priming was observed by repeated administrations of sGnRH and sGnRH-A. Using pituitaries from sexually mature female common carp, desensitization occurred when a high dose of sGnRH and both high and low dosages of sGnRH-A were given as repeated pulses at short time intervals. Self-priming, largely due to the increase in basal GtH levels, occurred in response to repeated pulses of low dosages of sGnRH-A given at long intervals.

Efficient immunocastration of male piglets by immunoneutralization of GnRH using a new GnRH-like peptide

Active immunization to immunomodulate regulatory processes suffers from the disadvantage that the antigen is usually ‘self’ and therefore poorly immunogenic. This has been well illustrated by the long-standing experience with immunocastration vaccines targeting GnRH, a ten amino acid peptide. Not all animals vaccinated with these vaccines are equally affected, even after multiple vaccinations. This is a severe handicap when immunocastration vaccines are applied to male piglets to circumvent surgical castration. Surgical castration is universally practised to prevent boar taint, produced in the testicles of mature boars. Alternative immunocastration is only acceptable if all animals are equally affected using a minimum of vaccinations. Vaccines based on the GnRH peptide itself cannot meet these goals. We showed that using a GnRH-like peptide, a 20 amino acid tandem repeat of the amino acid sequence of the GnRH peptide, these goals can be attained. Using the tandem GnRH peptide to vaccinate male piglets completely abolished the development and endocrinological functioning of the testicles, in contrast to monomer GnRH. These results show that superior antigens can be made for effective immunomodulation by appropriate alteration of the antigen.

Two gonadotropin-releasing hormones in the African catfish, Clarias gariepinus: localization, pituitary receptor binding, and gonadotropin release activity.

Two GnRH peptides have recently been identified in brain extracts of the African catfish, chicken-II GnRH ([His5,Trp7,Tyr8]GnRH, cGnRH-II) and catfish GnRH ([His5,Asn8]GnRH, cfGnRH). Using three experimental approaches, we investigated whether both peptides are involved in the regulation of pituitary gonadotropin secretion. First, the presence of cfGnRH and cGnRH-II in the pituitary was studied by biochemical and immunocytochemical techniques, as GnRH reaches the pituitary via axonal transport in teleost fish. Pituitary extracts contained cfGnRH- and cGnRH-II-immunoreactive material, showing the same HPLC retention times as the respective synthetic GnRH peptides; cfGnRH was present in 37-fold higher amounts than cGnRH-II. Using single and double labeling immunocytochemical techniques, both peptides were localized in the same peptidergic nerve fibers and often within the same secretory granules in the vicinity of the gonadotropes. Second, the two peptides were tested for their capacity to induce an increased secretion of the LH-like gonadotropin-II (GTH-II). In vivo studies showed that both GnRHs released GTH-II, but 100-fold higher cfGnRH than cGnRH-II doses were necessary to induce similar increases in circulating GTH-II levels. In vitro experiments using pituitary tissue fragments in a perifusion system also revealed a clearly higher GTH-II-releasing capacity of cGnRH-II compared to that of cfGnRH. Third, the peptides were tested for their ability to displace [125I]salmon GnRH analog ([D-Arg6,Trp7,Leu8,Pro9-NEt] GnRH, sGnRHa), a high affinity GnRH receptor ligand, from catfish pituitary membrane preparations. Chicken GnRH-II competed with [125I]sGnRHa for pituitary GnRH-binding sites, whereas cfGnRH did so only slightly. The present data show that cGnRH-II is the more potent GTH-II secretagogue, although a role for cfGnRH in the regulation of GTH-II secretion cannot be excluded. The high biological activity of cGnRH-II may be related to the regulation of GTH-II secretion surges, such as those associated with spawning, whereas cfGnRH may be involved in regulating moderate changes in GTH-II plasma levels. The peptides' potency differences appear to be related to their different binding affinities for the pituitary GnRH receptor.

In vitro characterization of gonadotropin-releasing hormone antagonists in goldfish, Carassius auratus.

The two native forms of GnRH, salmon GnRH and chicken GnRH-II, in the brain and pituitary of goldfish are both active in stimulating gonadotropin-II (GTH-II) and GH release. The objective of the present study was to characterize GnRH antagonists for their ability to inhibit sGnRH- and cGnRH-II-induced GTH-II and GH release in goldfish using a pituitary fragments perifusion system. Contrary to expectations, putative GnRH antagonists with D-Arg6 stimulated GTH-II and GH release in nearly all cases. [Ac-delta 3-Pro1,4FD-Phe2,D-Trp3,6]mammalian (m) GnRH inhibited sGnRH- and cGnRH-II-stimulated GTH-II release in a dose-dependent manner, with ED50 values of 242 +/- 48 and 169 +/- 17 nM, respectively. [Ac-delta 3-Pro1,4FD-Phe2,D-Trp3,6]mGnRH also inhibited GH release stimulated by sGnRH (ED50, 128 +/- 74 nM) and cGnRH-II (ED50, 157 +/- 67 nM). The degree of inhibition was higher in sexually regressed fish compared to postspawning fish. [D-p-Glu1,D-Phe2,D-Trp3,6]mGnRH suppressed both sGnRH- and cGnRH-II-induced GTH-II release with ED50 values of 326 +/- 96 and 249 +/- 74 nM, respectively. [Ac-delta 3-Pro1,4FD-Phe2,D-Trp3,6]sGnRH inhibited sGnRH and cGnRH-II stimulated GTH-II release, but stimulated GH release. On the other hand, [Ac-D(2)-Nal1,4Cl-D-Phe2,D-(3)Pal3,6,Arg5,D-A la10]mGnRH weakly stimulated GTH-II release, but strongly inhibited basal GH release. These results indicate that [Ac-delta 3-Pro1,4FD-Phe2,D-Trp3,6]mGnRH has clear antagonistic activity on sGnRH and cGnRH-II stimulation of GTH-II and GH release in vitro. The differential actions of a few GnRH analogs on GTH-II and GH release indicate that the properties of the GnRH receptors on GTH and GH cells may be different. The amino acid in position 6 plays an important role in determining the nature of intrinsic activity of GnRH peptides, and substitution of D-Arg6 normally produces agonistic analogs.

In vitro characterization of gonadotropin-releasing hormone antagonists in goldfish, Carassius auratus

The two native forms of GnRH, salmon GnRH and chicken GnRH-II, in the brain and pituitary of goldfish are both active in stimulating gonadotropin-II (GTH-II) and GH release. The objective of the present study was to characterize GnRH antagonists for their ability to inhibit sGnRH- and cGnRH-II-induced GTH-II and GH release in goldfish using a pituitary fragments perifusion system. Contrary to expectations, putative GnRH antagonists with D-Arg6 stimulated GTH-II and GH release in nearly all cases. [Ac-delta 3-Pro1,4FD-Phe2,D-Trp3,6]mammalian (m) GnRH inhibited sGnRH- and cGnRH-II-stimulated GTH-II release in a dose-dependent manner, with ED50 values of 242 +/- 48 and 169 +/- 17 nM, respectively. [Ac-delta 3-Pro1,4FD-Phe2,D-Trp3,6]mGnRH also inhibited GH release stimulated by sGnRH (ED50, 128 +/- 74 nM) and cGnRH-II (ED50, 157 +/- 67 nM). The degree of inhibition was higher in sexually regressed fish compared to postspawning fish. [D-p-Glu1,D-Phe2,D-Trp3,6]mGnRH suppressed both sGnRH- and cGnRH-II-induced GTH-II release with ED50 values of 326 +/- 96 and 249 +/- 74 nM, respectively. [Ac-delta 3-Pro1,4FD-Phe2,D-Trp3,6]sGnRH inhibited sGnRH and cGnRH-II stimulated GTH-II release, but stimulated GH release. On the other hand, [Ac-D(2)-Nal1,4Cl-D-Phe2,D-(3)Pal3,6,Arg5,D-A la10]mGnRH weakly stimulated GTH-II release, but strongly inhibited basal GH release. These results indicate that [Ac-delta 3-Pro1,4FD-Phe2,D-Trp3,6]mGnRH has clear antagonistic activity on sGnRH and cGnRH-II stimulation of GTH-II and GH release in vitro. The differential actions of a few GnRH analogs on GTH-II and GH release indicate that the properties of the GnRH receptors on GTH and GH cells may be different. The amino acid in position 6 plays an important role in determining the nature of intrinsic activity of GnRH peptides, and substitution of D-Arg6 normally produces agonistic analogs.

Hypothalamo-anterior pituitary gonadotropin-releasing hormone and substance-P systems during the 17 beta-estradiol-induced plasma luteinizing hormone surge in the ovariectomized female monkey.

The present study examined the effects of 17 beta-estradiol benzoate (E2B) on the hypothalamic (HT) and anterior pituitary (AP) content of GnRH precursor (pro-GnRH-GAP), GnRH, GnRH-associated peptide (GAP), and substance-P (SP) during the various phases of the E2B-induced LH surge in cynomolgus monkeys. Changes in GnRH and GnRH-associated peptide (GAP) at both hypothalamic and AP levels were closely related at all times after E2B treatment. However, the pattern of change in the AP was very different from that in the HT. In the HT, pro-GnRH-GAP levels did not change significantly throughout the experimental period. In the AP, the pro-GnRH-GAP increased 48 h post-E2B treatment, the time of initiation of the LH surge. An 8-fold increase in AP GnRH occurred 30 h post-E2B treatment. There were no significant changes in the HT content of SP at any time after E2B treatment. However, there was a depletion of AP content by 48 h post-E2B, the time of the LH surge. These results demonstrate that E2 activates and deactivates in a coordinated manner the GnRH and SP systems of the HT-AP complex during initiation of the E2-induced LH surge. The observation that more significant changes occur in the AP than in the HT suggests that an important component of the E2 effect in inducing the LH surge may be directly at the AP level. This action involves changes in the contents of GnRH and SP in the AP.

Effects of sex steroid treatments on gonadotropin-releasing hormone-stimulated gonadotropin secretion from the goldfish pituitary.

The effects of gonadal steroids on the gonadotropin (GTH) release response to salmon gonadotropin-releasing hormone (sGnRH), chicken gonadotropin-releasing hormone-II (cGnRH-II), and the sGnRH analogue, [D-Arg6, Trp7, Leu epsilon, Pro9]-N-ethylamide-GnRH (sGnRH-A), were investigated using an in vitro perifusion system for goldfish pituitary fragments. Gonad-intact male and female goldfish were implanted intraperitoneally (i.p.) with silastic pellets containing no steroid (blank), testosterone (T; 100 micrograms/g), or estradiol (E2; 100 micrograms/g); pituitaries were removed 5 days later for perifusion experiments. In vivo treatment with T or E2 potentiates sGnRH-, cGnRH-II-, and sGnRH-A-induced GTH secretion from pituitary fragments of sexually regressed and sexually recrudescent goldfish in vitro. Testosterone (100 nM; 24 h) treatment in vitro has a direct effect on the pituitary to increase sGnRH responsiveness, and this potentiating effect of T was blocked by the protein synthesis inhibitor cycloheximide (25 microM). In sexually regressed goldfish, in vivo T implantation enhanced the serum GTH response to sGnRH-A (0.01 microgram/g; 6 h) 7-fold. ED50 estimates for in vitro pituitary GTH responsiveness to sGnRH-A were 1.0 +/- 0.1 nM and 0.1 +/- 0.1 nM (p < 0.05) for blank and T-implanted groups, respectively. Radioligand (125I-sGnRH-A) binding studies demonstrated that enhanced pituitary responsiveness was independent of changes in pituitary GnRH receptor affinity or number. These results demonstrate that sex steroids increase pituitary sensitivity to GnRH peptides in the goldfish.

Evolution of Gonadotropin-Releasing Hormone (GnRH) Neuronal Systems (Part 2 of 2)

The neuropeptide gonadotropin-releasing hormone (GnRH, LHRH) serves as both a hormone and a neurotransmitter, and it has multiple actions on reproductive physiology and behavior. At least seven different molecular forms of GnRH have evolved, and nearly all vertebrates studied express at least two different forms of GnRH: chicken GnRH II, and a second form that varies across classes. The GnRH cell bodies span a broad region of the forebrain and midbrain, and processes project to virtually every region of the CNS, to the vasculature, and to the cerebrospinal fluid. Comparative evidence supports the model that gnathostomic vertebrates possess two principle GnRH systems, with different embryonic and, probably, evolutionary origins, expressing different molecular forms of GnRH and projecting to different targets. The terminal nerve-septo-preoptic system serves as the principle regulator of gonadotropin release in most vertebrates. Neurons originate in the embryonic olfactory placode and migrate centrally during development, and it is proposed that ontogeny of the TN-septo-preoptic system reflects its evolutionary origins as a peripheral endocrine organ associated with the olfactory system. The second GnRH system, which arises from non-placodal precursors, comprises cell bodies in periventricular regions of the posterior diencephalon and/or midbrain. Although much less is known about the posterior GnRH system, evidence suggests that these cells served as the ancestral brain GnRH system and are the cellular locus of the chicken GnRH peptide. The GnRH system of lampreys does not appear to be homologous to either the TN-septo-preoptic or posterior GnRH system, and it is suggested that GnRH in agnathans represents a third evolutionary event.

Evolution of Gonadotropin-Releasing Hormone (GnRH) Neuronal Systems

The neuropeptide gonadotropin-releasing hormone (GnRH, LHRH) serves as both a hormone and a neurotransmitter, and it has multiple actions on reproductive physiology and behavior. At least seven different molecular forms of GnRH have evolved, and nearly all vertebrates studied express at least two different forms of GnRH: chicken GnRH II, and a second form that varies across classes. The GnRH cell bodies span a broad region of the forebrain and midbrain, and processes project to virtually every region of the CNS, to the vasculature, and to the cerebrospinal fluid. Comparative evidence supports the model that gnathostomic vertebrates possess two principle GnRH systems, with different embryonic and, probably, evolutionary origins, expressing different molecular forms of GnRH and projecting to different targets. The terminal nerve-septo-preoptic system serves as the principle regulator of gonadotropin release in most vertebrates. Neurons originate in the embryonic olfactory placode and migrate centrally during development, and it is proposed that ontogeny of the TN-septo-preoptic system reflects its evolutionary origins as a peripheral endocrine organ associated with the olfactory system. The second GnRH system, which arises from non-placodal precursors, comprises cell bodies in periventricular regions of the posterior diencephalon and/or midbrain. Although much less is known about the posterior GnRH system, evidence suggests that these cells served as the ancestral brain GnRH system and are the cellular locus of the chicken GnRH peptide. The GnRH system of lampreys does not appear to be homologous to either the TN-septo-preoptic or posterior GnRH system, and it is suggested that GnRH in agnathans represents a third evolutionary event.

In vitro/in vivo correlations in the kinetics of poly (lactide-co-glycolide) (PLG) delivery systems of GnRH peptides

In vitro/in vivo correlations in the kinetics of poly (lactide-co-glycolide) (PLG) delivery systems of GnRH peptides

Immunocytochemical study of GnRH and GnRH-associated peptide in male Syrian hamsters as a function of photoperiod and gonadal alterations.

Hypothalamic luteinizing-hormone-releasing hormone (GnRH) and gonadotropin-releasing-hormone-associated peptide (GAP) biosynthesis and storage were estimated by immunocytochemistry in male golden hamsters maintained in different photoperiods. Intact or castrated male hamsters with subcutaneously inserted testosterone implants were exposed to long-day (14:10) or short-day photoperiods (10:14) for 4-8 weeks. Exposure to short photoperiod for 4 weeks, an interval characterized by a suppression of gonadotropin secretion but not gonadal regression, was associated with an increase in the number of GnRH- and GAP-immunoreactive cells in the diagonal band of Broca/medial septum. Furthermore, morphometric analysis revealed that these animals displayed significantly more GnRH but not GAP immunoreactivity in the median eminence as opposed to hamsters exposed to long-day photoperiods. In additional studies, gonadally regressed hamsters exposed to short day lengths for 8 weeks had equal numbers of GnRH cells as did the long-day controls. These patterns suggest that reproductive quiescence in golden hamsters is not the result of depletions of neuronal GnRH stores available for secretion.

Age-Related Decreases in Serum Gonadotropin Levels and Gonadotropin-Releasing Hormone Gene Expression in the Medial Preoptic Area of the Male Rat Are Dependent upon Testicular Feedback*

In the male rat, age-associated reproductive decline is thought to be due in part to diminished GnRH secretion. We tested the hypothesis that the age-related decrease in GnRH secretion is due to decreased GnRH gene expression by comparing GnRH mRNA and peptide content in the anterior forebrain of intact young and old male rats. Since sex steroids modulate GnRH secretion, we also determined hypothalamic-pituitary responsiveness to removal of testicular feedback by comparing GnRH mRNA and gonadotropin levels in intact and orchidectomized young and old rats. In an initial study, 10 20-micron coronal sections from the medial preoptic area (MPOA) were anatomically matched and compared in intact young (3-month-old) and old (24-month-old) male F344 rats (n = 5/group). In another group of young and old male rats (n = 8-12/group), animals were randomly assigned to be either orchidectomized or sham operated. Rats were killed 21 days after surgery, and comparisons were made in 12 anatomically matched sections of MPOA and 4 matched sections of diagonal band of Broca. In both studies, GnRH mRNA was quantitated by in situ hybridization, using a 35S-labeled oligodeoxynucleotide probe complementary to rat prepro-GnRH mRNA and a computerized image analysis system. In a third study, GnRH content was measured by RIA in microdissected regions of the arcuate nucleus and median eminence in intact 3- and 24-month-old male rats (n = 10 and 8, respectively). Serum LH, FSH, and testosterone (T) levels were measured by RIA in trunk blood of all animals. The number of neurons expressing the GnRH gene in the MPOA was significantly lower in sham-operated old rats (mean +/- SEM, 10.5 +/- 0.5 cells/section) than in young rats (13.7 +/- 0.7 cells/section; P less than 0.01), while cellular GnRH mRNA content was unchanged with age (103 +/- 1 vs. 103 +/- 2 grains/cell). Similar results were obtained in intact old and young rats. GnRH peptide content was significantly decreased in the arcuate nucleus of intact old (0.5 +/- 0.08 ng/mg protein) compared to young animals (2.3 +/- 0.7 ng/mg protein; P less than 0.05), with a trend toward a decrease in the median eminence of old (53 +/- 2 ng/mg protein) vs. young rats (69 +/- 7 ng/mg protein; P = 0.06).(ABSTRACT TRUNCATED AT 400 WORDS)

Lack of involvement of arachidonic acid metabolism in chicken gonadotropin-releasing hormone II (cGnRH-II) stimulation of gonadotropin secretion in dispersed pituitary cells of goldfish, Carassius auratus. Identification of a major difference in salmon GnRH and chicken GnRH-II mechanisms of action

Gonadotropin (GTH) release in static incubations of dispersed goldfish pituitary cells was stimulated by chicken GTH-releasing hormone II (cGnRH-II), salmon (s)GnRH, phospholipase A 2, phospholipase C, phospholipase D, and arachidonic acid (AA). Coincubations with nordihydroguaiaretic acid (NDGA), 5,8,11,14-eicosatetraenoic acid, and indomethacin did not alter the GTH responses to cGnRH-II. In contrast, NDGA reduced sGnRH-stimulated GTH release. Incubation with Ca 2+-deficient medium abolished the GTH responses to cGnRH-II, reduced sGnRH-stimulated GTH release, but did not alter AA actions on GTH secretion. Apomorphine, a dopamine agonists that had been shown to partially inhibit the GTH responses to sGnRH and to abolish those induced by cGnRH-II, did not affect the hormone response to AA. However, the partial inhibitory actions of NDGA and apomorphine on sGnRH-induced GTH release were additive. These findings suggest the existence of a major difference in cGnRH-II and sGnRH stimulation of GTH release — AA metabolism is not involved in cGnRH-II, as opposed to sGnRH actions. This difference in second messenger activation may also explain the differential sensitivity of the two GnRH peptides to dopamine inhibition and manipulations of extracellular Ca 2+ availability. The results further suggest that dopamine and AA affect GTH release via non-overlapping signal transduction pathways.

Postnatal Development of Gonadotropin-Releasing Hormone and Cyclophilin Gene Expression in the Female and Male Rat Brain*

The rat preoptic area-anterior hypothalamic continuum (POA-AH) contains about 400-800 neurons that express the decapeptide GnRH and the 56-amino-acid GnRH-associated peptide. Originating from the olfactory placode, these neurons migrate and establish their final distribution and connections in the POA-AH several days before birth. The aim of the present study was to examine whether the biosynthesis of the mRNA encoding the precursor (proGnRH) common to GnRH and GnRH-associated peptide undergoes postnatal changes corresponding to the development of sexual maturation. The POA-AH content of proGnRH messenger RNA (mRNA) was followed from postnatal day 1 to day 90 in female and male Sprague-Dawley rats killed by decapitation between 1000-1200 h. Cytoplasmic RNA fractionated from individual POA-AH homogenates was purified using proteinase K digestion. Cytoplasmic proGnRH mRNA was quantitated simultaneously with cyclophilin mRNA (an internal standard control) using solution hybridization-RNase protection assay, with the protected fragments separated through polyacrylamide gel electrophoresis. In the POA-AH, the concentrations of proGnRH mRNA (femtograms mRNA per microgram total RNA) increased significantly with age in both sexes (P less than 0.001). In males, proGnRH mRNA levels increased by day 30 some 2-fold over the values of days 5 and 10, and the levels established on day 30 were maintained through adulthood. In females, the first rise in proGnRH mRNA levels occurred on day 30, followed by an additional increase on day 45 to levels seen in adulthood. Levels of proGnRH mRNA established in adulthood were significantly higher in females than in males (P less than 0.03). The concentrations of cyclophilin mRNA (picograms mRNA per microgram total RNA) remained essentially unchanged in both sexes during the same period of time when proGnRH mRNA levels were increasing. These results provide evidence for postnatal sex-related increases in the levels of proGnRH mRNA in the rat POA-AH, which are likely to reflect differential regulation by gonadal steroids.