Mammalian Luteinizing Hormone-releasing Hormone Research Articles

Gonadotropin-releasing hormone neurons in the preoptic-hypothalamic region of the rat contain lamprey gonadotropin-releasing hormone III, mammalian luteinizing hormone-releasing hormone, or both peptides.

This study utilized a newly developed antiserum, specific for lamprey gonadotropin-releasing hormone III (l-GnRH-III), to determine the following: in which regions of the rat hypothalamus the neuronal perikarya producing l-GnRH-III are localized; and whether this peptide, known to selectively induce follicle-stimulating hormone release, is coexpressed in neurons containing mammalian luteinizing hormone-releasing hormone (m-LHRH). Double-label immunocytochemistry was performed by using an l-GnRH-III polyclonal antiserum and an LHRH monoclonal antiserum. Immunopositive neurons for l-GnRH-III, m-LHRH, or neurons coexpressing both peptides were detected within the organum vasculosum lamina terminalis (OVLT) region of the preoptic area (POA). Caudal to the OVLT, l-GnRH-III-positive neurons were also observed dorso-medially, above the third ventricle in the medial POA. The m-LHRH neurons were not observed in this area. The lateral POA region contained neurons positive for both peptides along with single-labeled neurons for each peptide. Importantly, neurons that expressed l-GnRH-III, m-LHRH, or both peptides were also detected in the ventral regions of the rostral hypothalamus, dorsolateral to the borders of the supraoptic nuclei. In both of these latter areas, neurons containing l-GnRH-III were slightly dorsal to neurons containing only m-LHRH. The l-GnRH-III perikarya and fibers were eliminated by absorption of the primary antiserum with l-GnRH-III, but not by l-GnRH-I, chicken-GnRH-II, or m-LHRH. These results indicate that, unlike other isoforms of GnRH found in the mammalian brain, l-GnRH-III neurons not only are observed in regions that control follicle-stimulating hormone release but also are colocalized with m-LHRH neurons in areas primarily controlling LH release. These findings suggest an interrelationship between these two peptides in the control of gonadotropin secretion.

Presence of luteinizing hormone-releasing hormone fragments in the rhesus monkey forebrain.

Previously, we have shown that two types of luteinizing hormone-releasing hormone (LHRH) -like neurons, "early" and "late" cells, were discernible in the forebrain of rhesus monkey fetuses by using antiserum GF-6, which cross-reacts with several forms of LHRH. The "late" cells that arose from the olfactory placode of monkey fetuses at embryonic days (E) 32-E36, are bona fide LHRH neurons. The "early" cells were found in the forebrain at E32-E34 and settled in the extrahypothalamic area. The molecular form of LHRH in "early" cells differs from "late" cells, because "early" cells were not immunopositive with any specific antisera against known forms of LHRH. In this study, we investigated the molecular form of LHRH in the "early" cells in the nasal regions and brains of 13 monkey fetuses at E35 to E78. In situ hybridization studies suggested that both "early" and "late" LHRH cells expressed mammalian LHRH mRNA. Furthermore, "early" cells predominantly contain LHRH1-5-like peptide and its cleavage enzyme, metalloendopeptidase E.C.3.4.24.15 (EP24.15), which cleaves LHRH at the Tyr5-Gly6 position. This conclusion was based on immunocytochemical labeling with various antisera, including those against LHRH1-5, LHRH4-10, or EP24.15, and on preabsorption tests. Therefore, in primates, a group of neurons containing mammalian LHRH mRNA arises at an early embryonic stage before the migration of bona fide LHRH neurons, and is ultimately distributed in the extrahypothalamic region. These extrahypothalamic neurons contain LHRH fragments, rather than fully mature mammalian LHRH. The origin and function of these neurons remain to be determined.

Ultrastructural changes in granulosa cells and plasma steroid levels after administration of luteinizing hormone-releasing hormone in the Western painted turtle, Chrysemys picta

In this study we investigated the effects of treatment by luteinizing hormone-releasing hormone (LHRH) on the morphology and steroid release of ovarian tissues in the Western painted turtle, (Chrysemys picta). In Experiment I, four adult female turtles were injected with synthetic mammalian LHRH (i.p., 500pg/g bodyweight) and four with saline 2–3 weeks prior to ovulation. Granulosa cells from LHRH-treated turtles vs controls contained both preovulatory follicles (16–20mm in diameter) and small follicles (0.5–1.00mm in diameter) with increased RER, free ribosomes and mitochondria with swollen cristae. An increase in the amount of cytoskeletal material (microfilaments) was observed in granulosa cells of the experimental turtles compared to the controls. Cytoplasmic extensions of the oocyte and granulosa cells were longer in the small follicles of treated animals, accounting for the observed increase in the thickness of the zona pellucida (ZP) over the controls. In Experiment II, administration of LHRH (i.p.) to 10 turtles during the same period triggered a substantial increase in plasma progesterone and estradiol-17β levels over the 10 saline-injected controls. This supports the idea that in this species, as in mammals, steroidogenic activity in the ovarian follicles are under the control of the hypothalamic–pituitary axis. The ultrastructure and hormonal levels of the experimental animals were typical of untreated turtles just prior to ovulation. In this species the development of follicles and steroidogenesis can be stimulated prematurely by a releasing hormone from a nonreptilian origin.

Localization of immunoreactive lamprey gonadotropin-releasing hormone in the rat brain☆

A highly specific antiserum against lamprey gonadotropin-releasing hormone (GnRH) was used to localize l-GnRH in areas of the rat brain associated with reproductive function. Immunoreactive l-GnRH-like neurons were observed in the ventromedial preoptic area (POA), the region of the diagonal band of Broca and the organum vasculosum lamina terminalis, with fiber projections to the rostral wall of the third ventricle and the organum vasculosum lamina terminalis. Another population of l-GnRH-like neurons was localized in the dorsomedial and lateral POA, with nerve fibers projecting caudally and ventrally to terminate in the external layer of the median eminence. Other fibers apparently projected caudally and circumventrically to terminate around the cerebral aqueduct in the mid-brain central gray. By using a highly specific antiserum directed against mammalian luteinizing hormone-releasing hormone (m-LHRH), the localization of the LHRH neuronal system was compared to that of the l-GnRH system. There were no LHRH neurons in the dorsomedial or the lateral region of the POA that contained the l-GnRH neurons. As expected, there was a large population of LHRH neurons in the ventromedial POA associated with the diagonal band of Broca and organum vasculosum lamina terminalis. In both of these regions, there were many more LHRH neurons than l-GnRH neurons and the LHRH neurons extended more dorsally and laterally than the l-GnRH neurons. The LHRH neurons seemed to project to the median eminence in the same areas as those that were innervated by the l-GnRH neurons. Absorption studies indicated that l-GnRH cell bodies were eliminated by adding 1 μg of either l-GnRH-I or 1-GnRH-III, but not m-LHRH to the antiserum before use. Fibers were largely eliminated by the addition of 1 μg l-GnRH-III to the antiserum. No chicken GnRH-II neurons or nerve fibers could be visualized by immunostaining. Because the antiserum recognized GnRH-I and GnRH-III equally, we have visualized an l-GnRH system in rat brain. The results are consistent with the presence of either one or both of these peptides within the rat hypothalamus. Because l-GnRH-I has only weak nonselective gonadotropin-releasing activity, whereas l-GnRH-III is a highly selective releaser of follicle-stimulating hormone, and because l-GnRH neurons are located in areas known to control follicle-stimulating hormone release selectively, our results support the hypothesis that l-GnRH-III, or a closely related peptide, may be mammalian follicle-stimulating hormone-releasing factor.

Hypothalamic control of gonadotropin secretion by LHRH, FSHRF, NO, cytokines, and leptin

Gonadotropin secretion by the pituitary gland is under the control of luteinizing hormone-releasing hormone (LHRH) and the putative follicle stimulating hormone-releasing factor (FSHRF). Lamprey III LHRH is a potent FSHRF in the rat and seems to be resident in the FSH controlling area of the rat hypothalamus. It is an analog of mammalian LHRH and may be the long sought FSHRF. Gonadal steroids feedback at hypothalamic and pituitary levels to either inhibit or stimulate the release of LH and FSH, which is also affected by inhibin and activin secreted by the gonads. Important control is exercised by acetylcholine, norepinephrine (NE), dopamine, serotonin, melatonin, and glutamic acid (GA). Furthermore, LH and FSH also act at the hypothalamic level to alter secretion of gonadotropins. More recently, growth factors have been shown to have an important role. Many peptides act to inhibit or increase release of LH and the sign of their action is often reversed by estrogen. A number of cytokines act at the hypothalamic level to suppress acutely the release of LH but not FSH. NE, GA, and oxytocin stimulate LHRH release by activation of neural nitric oxide synthase (nNOS). The pathway is as follows: oxytocin and/ or GA activate NE neurons in the medial basal hypothalamus (MBH) that activate NOergic neurons by alpha 1 (α 1) receptors. The NO released diffuses into LHRH terminals and induces LHRH release by activation of guanylate cyclase (GC) and cyclooxygenase. NO not only controls release of LHRH bound for the pituitary, but also that which induces mating by actions in the brain stem. An exciting recent development has been the discovery of the adipocyte hormone, leptin, a cytokine related to tumor necrosis factor (TNF) α. In the male rat, leptin exhibits a high potency to stimulate FSH and LH release from hemipituitaries incubated in vitro, and increases the release of LHRH from MBH explants. LHRH and leptin release LH by activation of NOS in the gonadotropes. The NO released activates GC that releases cyclic GMP, which induces LH release. Leptin induces LH release in conscious, ovariectomized estrogen-primed female rats, presumably by stimulating LHRH release. At the effective dose of estrogen to activate LH release, FSH release is inhibited. Leptin may play an important role in induction of puberty and control of LHRH release in the adult as well.

Luteinizing Hormone-Releasing Hormone (LHRH) Neurons Maintained in Hypothalamic Slice Explant Cultures Exhibit a Rapid LHRH mRNA Turnover Rate

Evidence indicates that neuropeptide gene expression is tightly coupled to biosynthesis and secretion. Moreover, rhythmic gene expression often accompanies rhythmic secretion. Luteinizing hormone-releasing hormone (LHRH) neurosecretion, which regulates gonadal function, is pulsatile, with interpulse intervals of approximately 1 hr and pulse decays of <30 min in rats. As a basis for a rapid fall in peptide secretion, we hypothesize that LHRH mRNA levels rapidly decay. To address this hypothesis, we examined LHRH mRNA turnover in primary postnatal LHRH neurons maintained in long-term hypothalamic/preoptic area slice explant cultures, using in situ hybridization histochemistry (ISHH). Relative LHRH mRNA content per cell was quantitated by single-cell analysis after transcription inhibition with 5, 6-dichloro-1-D-ribofuranosyl-benzimidazole (DRB) or actinomycin D. Cultures were maintained in serum-free medium with tetrodotoxin to suppress spontaneous electrical activity and hence assess only intrinsic cellular activity. A plot of LHRH mRNA level per cell versus DRB treatment time showed a rapid initial decay of LHRH mRNA (t1/2, 5-13 min), followed by a slower decay rate (t1/2, 329-344 hr). LHRH cell number after drug treatment as determined by immunocytochemistry did not change. Comparison of mammalian LHRH mRNA 3'-untranslated regions showed two conserved regions. These data indicate that, in primary LHRH neurons, LHRH mRNA has an intrinsically high rate of turnover and a mRNA stabilization component. Foremost, decay of LHRH mRNA, the fastest reported for a neuropeptide to date, corresponds to the decay of LHRH peptide pulses.

A hypothalamic follicle-stimulating hormone-releasing decapeptide in the rat.

Previous studies indicated that there is a separate hypothalamic control of follicle-stimulating hormone (FSH) release distinct from that of luteinizing hormone (LH). An FSH-releasing factor (FSHRF) was purified from rat and sheep hypothalami, but has not been isolated. We hypothesized that FSHRF might be an analogue of mammalian luteinizing hormone-releasing hormone (m-LHRH) and evaluated the activity of many analogues of m-LHRH and of the known LHRHs found in lower forms. Here we demonstrate that lamprey (l) LHRH-III has a potent, dose-related FSH- but not LH-releasing action on incubated hemipituitaries of male rats. l-LHRH-I on the other hand, had little activity to release either FSH or LH. m-LHRH was equipotent to l-LHRH-III to release FSH, but also had a high potency to release LH in contrast to l-LHRH-III that selectively released FSH. Chicken LHRH-II had considerable potency to release both LH and FSH, but no selectivity in its action. Salmon LHRH had much less potency than the others tested, except for l-LHRH-I, and no selectivity in its action. Because ovariectomized, estrogen, progesterone-treated rats are a sensitive in vivo assay for FSH- and LH-releasing activity, we evaluated l-LHRH-III in this assay and found that it had a completely selective stimulatory effect on FSH release at the two doses tested (10 and 100 pmols). Therefore, l-LHRH-III is a highly potent and specific FSH-releasing peptide that may enhance fertility in animals and humans. It may be the long sought after m-FSHRF.

LHRH-I Release from Quail Hypothalamic Slices Measured by Specific EIA

A competitive enzyme immunoassay (EIA) was developed for luteinizing hormone-releasing hormone (LHRH), which measures either mammalian luteinizing hormone-releasing hormone or chicken LHRH-I (cLHRH-I). There is negligible cross-reactivity with chicken LHRH-II. Assay sensitivity is 1 pg/ml and the intra- and interassay variation are 3.4 and 10.0%, respectively. The assay was validated for measuring cLHRH-I by parallelism and quantitative recovery. Using this E1A, cLHRH-I content was measured in microdissected samples of median eminence from mature quail and chickens. Mean cLHRH-I concentrations were 1.25 ± 0.35 and 2.10 ± 0.25 ng/mg protein in quail and chickens, respectively. In vitro release of cLHRH-I was studied by perifusion of quail medial basal hypothalamus-preoptic area (MBH-POA) slices. Challenge with increasing concentrations of K + resulted in significant ( P < 0.05) release of cLHRH-I. The release of cLHRH-I from MBH-POA slices was also measured in response to norepinephrine (NE), epinephrine (E), and isoproterenol (ISO). Chicken LHRH-I was released in a dose-dependent manner; maximal release occurred with 10 -7 M NE, 10 -8 M E, and 10 -7 M ISO. Tissue response was optimal for experimental manipulation 6-25 hr postcollection; thereafter, the response deteriorated until 60 hr postcollection. These data extend previous studies in birds by detailed characterization of responses to neurochemical challenge and description of optimal parameters for tissue response during perifusion.

Ovarian involvement in the suppression of luteinizing hormone in the incubating turkey (Meleagris gallopavo).

A series of experiments were conducted to elucidate the role of the ovary in incubation behavior-associated luteinizing hormone (LH) suppression. Ovariectomy (Ovx) increased (p < 0.05) serum LH levels in nonphotostimulated, laying and photorefractory turkeys but not in incubating birds (p > 0.05). Ovx had no effect on intramuscularly injected mammalian luteinizing hormone-releasing hormone (4 micrograms/kg i.m.)-induced LH in incubating hens, but enhanced the LH response in laying hens. Serum LH and prolactin (Prl) were unaffected by Ovx, and nest deprivation (ND) decreased Prl levels (p < 0.05) with no effect on serum LH of incubating turkeys. However, serum LH increased (p < 0.05) and Prl decreased in Ovx-ND birds. Prl mRNA abundance (11.9 +/- 1.2 ng/microgram total RNA) decreased following Ovx (3.4 +/- 0.4 ng/microgram total RNA) or ND (3.6 +/- 0.5 ng/microgram total RNA). Nest deprivation increased LH beta mRNA (2.5-fold) which was further increased (4.8-fold) by Ovx. Hypothalamic GnRH-I and GnRH-II contents increased (p < 0.05) in Ovx-ND turkeys. We conclude that serum LH suppression during incubation behavior requires ovarian participation acting synergistically with elevated Prl and/or nesting stimulus on hypothalamic GnRH, and that the concentration fo LH beta mRNA may be a limiting factor in LH secretion.

Comparison of in vivo biological activities of luteinizing hormone releasing hormone (LHRH) analogues in 60-day-old cockerels

The in vivo biological activities of various luteinizing hormone releasing hormone (LHRH) analogues in immature 60-day-old cockerels were compared, based on their capacity to increase circulating luteinizing hormone (LH) concentrations. Intravenous injections of 50, 100, or 500 ng of [Gln 8]LHRH (cGnRH-I) and [His 5,Trp 7,Tyr 8]LHRH(cGnRH-II) induced significant increases in the plasma LH level. The maximum response was always observed 2 min after the injection. Mammalian LHRH(mGnRH) significantly increased plasma LH, but only at the 500 ng dosage level. [Ile 8]LHRH, [D-Ala 6]LHRH, [Ala 4]LHRH, [Leu 8]LHRH, and [Ile 5]LHRH increased plasma LH at 50–500 ng. [Ile 7]LHRH, [Phe 3]LHRH, and [Phe 2]LHRH did not significantly increase plasma LH. Relative in vivo biological activities, calculated from dose (30–100 ng)-response curves of mGnRH-I, cGnRH-I, cGnRH-II, [Ile 8]LHRH, [D-Ala 6]LHRH, [Ala 4]LHRH, [Leu 8]LHRH, [Ile 5]LHRH, [Ile 7]LHRH, [Phe 3]LHRH, and [Phe 2]LHRH were 1.00, 1.40, 1.81, 1.50, 4.59, 1.90, 1.28, 1.56, 0.57, 0.67, and 0.57, respectively. These results demonstrate that avian LHRHs have higher biological activities than mammalian LHRH in the chicken. Further-more, cGnRH-II is more potent than cGnRH-I, and [D-Ala 6]LHRH has a very high activity while [Ile 7]LHRH and [Phe 2]LHRH have very low activities.

(Hydroxyproline9) luteinizing hormone-releasing hormone: A novel peptide in mammalian and frog hypothalamus

Luteinizing hormone-releasing hormone (LHRH)-like immunoreactive material was detected in mammalian and frog hypothalamic extracts after high performance liquid chromatography. Radioimmunological detection was performed with three different antibodies directed respectively against the N-(N-antibody) or the C-terminus (C-antibody) as well as the conformational structure (W-antibody) of synthetic mammalian LHRH (mLHRH). Under the chromatographic conditions used, two immunoreactive peptides could be separated. One of those was detected in a homothetic manner by the three antibodies. It coeluted with synthetic mLHRH, had the same amino acid composition as mLHRH, and was shown to correspond to authentic endogenous LHRH. The other peptide was more efficiently detected by the N- than by the two other antibodies. It was eluted prior to mLHRH on reverse phase chromatography but did not correspond to LHRH fragments or to any other known LHRH sequences. Its molecular weight was 1198 Da. Amino acid analysis and enzymatic digestion indicated a very close LHRH sequence homology, with a substitution of proline (Pro) by a hydroxyproline (Hyp) residue in position 9. The new peptide was found in extracts of human, rodent, ovine and amphibian hypothalamus, with a markedly higher concentration in fetal than in adult rats. Various extraction and chromatographic control procedures led to the conclusion that the new peptide was genuinely endogenous and did not result from artifactual modification(s) of LHRH during the purification process. It is concluded that, in the mammalian as in the frog hypothalamus, LHRH-like material corresponds to at least two native molecular forms: (Pro(9)) and (Hyp(9))LHRH.

Degradation of gonadotropin-releasing hormones in the gilthead seabream, Sparus aurata: II. Cleavage of native salmon GnRH, mammalian LHRH, and their analogs in the pituitary, kidney, and liver

The pattern and kinetics of degradation of native salmon gonadotropin-releasing hormone (sGnRH), mammalian luteinizing hormone-releasing hormone (LHRH), and some of their analogs by cytosolic enzymes of pituitary, kidney, and liver were studied in the gilthead seabream, Sparus aurata. The native peptides sGnRH and LHRH are rapidly degraded by all three tissues, LHRH being degraded faster than sGnRH. The kinetics of production of the peptide fragments suggest that initial cleavage of sGnRH and LHRH in the three studied tissues occurs at the 5–6 and 9–10 bonds. This indicates the initial activity of a Tyr 5-Gly 6 endopeptidase and a Pro 9-Gly 10NH 2 peptidase or postproline cleaving enzyme. Secondary degradation of the main initial fragments (1–5, 6–10, and 1–9) is more intensive in the kidney than in the pituitary or liver. Substitution of the position 6 amino acid glycine by a dextrorotatory ( d) amino acid such as in the d-Trp 6-LHRH renders the 5–6 bond resistant to cleavage. However, whereas [ d-Trp 6]-LHRH is intensively cleaved at the Pro 9-Gly 10NH 2 bond by the pituitary, its cleavage at this site by the kidney and liver is slow. This suggests a low activity of the Pro 9-Gly 10NH 2 peptidase in the kidney and liver as compared to the pituitary. When, in addition to the position 6 substitution, the carboxy terminus Pro 9-Gly 10NH 2 is modified to Pro 9NET, such as in the [ d-Ala 6-Pro 9NET]-LHRH and the [ d-Arg 6-Pro 9NET]-sGnRH, the 9–10 cleavage site is also blocked, resulting in GnRH analogs highly resistant to degradation. The relationships between susceptibility of the different forms of GnRH to enzymatic degradation by the pituitary, kidney, and liver and their relative biological activities in S. aurata are discussed. We conclude that increased resistance of GnRH analogs to enzymatic degradation contributes to their superactivity.

Degradation of gonadotropin-releasing hormones in the gilthead seabream, Sparus aurata: I. Cleavage of native salmon GnRH and mammalian LHRH in the pituitary

The pattern and kinetics of degradation of native salmon gonadotropin-releasing hormone (sGnRH) and mammalian luteinizing hormone-releasing hormone (LHRH) by pituitary bound enzymes were studied in the gilthead seabream, Sparus aurata. sGnRH and LHRH were incubated for different periods of time with membrane or cytosolic fractions of pituitary homogenates. At the end of the incubation, the degradation mixture was fractionated on reverse-phase high-pressure liquid chromatography. The degradation products were identified by comparing their retention times to those of synthesized GnRH fragments and by analyzing their amino acid composition. The main GnRH degradative activity resides in the cytosolic fraction of the pituitary homogenate. Both sGnRH and LHRH are rapidly degraded by pituitary cytosol, with 78.3 and 87.7% of the peptides, respectively, cleaved after 3 hr of incubation. Maximal degradation of sGnRH occurred at a pH range of 7 to 8. The main initial products of degradation of sGnRH and LHRH are the 1–5, 6–10, and 1–9 fragments. This suggests the involvement of two site-specific peptidases, a Tyr 5-Gly 6 endopeptidase and a Pro 9-Gly 10NH 2 peptidase or postproline cleaving enzyme. While the 1–6 and 1–9 fragments undergo rapid secondary degradation, the 1–5 is relatively stable. Competition experiments suggest that the endopeptidase cleaving the sGnRH at the Tyr 5-Gly 6 bond is not specific to the neuropeptide and is probably a general proteolitic enzyme. However, the cleavage at the 9–10 bond has a high degree of specificity to the Pro 9-Gly 10NH 2 sequence found in sGnRH. The two proposed pituitary peptidases of S. aurata have some characteristics similar to those of rat hypophyseal and hypothalamic GnRH cleaving enzymes. No differences are found in hypophyseal GnRH degradative activity between females with occytes undergoing previtellogenesis or advanced stages of vitellogenesis.

Chicken II luteinizing hormone-releasing hormone inhibits the M-current of bullfrog sympathetic neurons

The M-current of dissociated bullfrog sympathetic neurons, measured by the whole-cell patch clamp technique, is powerfully inhibited by chicken II LH-RH (luteinizing hormone-releasing hormone), with 50% inhibition near 1 nM. Chicken II LH-RH is approximately 100 times more potent than salmon LH-RH, and at least 1000 times more potent than other known naturally occurring LH-RH analogs (chicken I LH-RH, mammalian LH-RH, and lamprey LH-RH). This high potency makes chicken II LH-RH a candidate for the endogenous transmitter mediating the late, slow excitatory postsynaptic potential (EPSP) in bullfrog sympathetic ganglia.

Immunocytochemical evidence for peptidergic (GnRH) and dopaminergic innervation of the gonadotropic cells in the pituitary of the African catfish, Clarias gariepinus

The proximal pars distalis (PPD) of the pituitary of the African catfish, Clarias gariepinus, was studied with immunocytochemical methods at the ultrastructural level. Antiserum raised against synthetic mammalian luteinizing hormone-releasing hormone (LHRH) was applied on Lowicryl-embedded pituitaries and the antigenic sites were visualized with protein A-gold. In nerve fibers contacting the gonadotropic cells, granulated vesicles with a diameter of 90–120 nm were labeled after this procedure, whereas the glandular cells were not labeled. For the immunocytochemical demonstration of dopaminergic fibers, the preembedding method was performed on Vibratome sections, using highly specific antibodies against dopamine. Immunoreactivity was restricted to fibers containing granulated vesicles with a diameter of approximately 80 nm and terminating on gonadotropic cells. The present data support the results of earlier in vivo and in vitro studies on the catfish pituitary, indicating a dual neuroendocrine regulation of the gonadotropic cells.

A muscarine-resistant M-current in C cells of bullfrog sympathetic ganglia

C cells of bullfrog sympathetic ganglia have an M-current similar to that of the larger B cells. However, the M-current of C cells is much less sensitive to muscarinic agonists. The M-current of C cells is blocked by barium and by LHRH (mammalian luteinizing hormone-releasing hormone). LHRH sometimes also decreases a different conductance, with an extrapolated reversal potential near 0 mV. These results demonstrate that the coupling of receptors to ion channels can vary between cell types, even within a single ganglion.

Effects of two kinds of chicken luteinizing hormone-releasing hormone (LH-RH), mammalian LH-RH and its analogs on the release of LH and FSH in Japanese quail and chicken

A newly isolated and characterized chicken luteinizing hormone-releasing hormone-II (chicken LH-RH-II, Miyamoto et al., 1984) had luteinizing hormone (LH) and follicle-stimulating hormone (FSH) releasing activity in vitro and in vivo in Japanese quail; the activity was almost equal to chicken LH-RH-I and mammalian LH-RH. These three LH-RHs induced the release of LH several times higher than that of FSH in vitro and also in vivo. No significant difference between chicken LH-RH-I and LH-RH-II was observed in LH releasing activity in vitro using chicken pituitary gland in the same incubating condition as in quail. Another experiment indicated that no synergism existed between chicken LH-RH-I and -II and that there was neither LH nor FSH releasing activity in [ d-Phe 2, Pro 3, d-Phe 6]-LH-RH or in mesotocin. However, the same potency as in the chicken LH-RH-II was observed in [ d-Ala 6, des-Gly 10]-LH-RH ethylamide, a superactive analog in mammals. The results indicate that an avian adenohypophysis differs from a mammalian adenohypophysis in its responsiveness to LH-RH suggesting that an avian LH-RH receptor may have a lower specificity in ”recognition” of LH-RH molecules than a mammalian LH-RH receptor has.

Effects of estradiol and mammalian LHRH on the ultrastructure of the pars distalis of the eel.

Male silver eels were injected with estradiol-17 beta (E2) to induce the development of gonadotropic (GTH) cells. They were subsequently injected with luteinizing hormone-releasing hormone (LHRH). Exocytotic figures and the lysis of some large globules and granules were observed. Morphometric studies showed a significant increase in the percentage of vacuoles after 4 and 6 injections of LHRH and a slight but significant decrease of granules. This response did not, however, occur in all GTH cells which never appeared completely degranulated and did not reach a vesicular stage. Hemi-pituitaries of E2-pretreated eels were incubated with or without LHRH (20 min to 2 h). Although typical exocytoses were not detected, an increased number of small granules near the basal lamina and lytic processes (globules with a raspberry-shaped structure, granules with variable electron density) were observed in the LHRH-incubated hemi-pituitaries compared with those kept in a control medium. The structure of GTH cells and their response to LHRH has been studied only under conditions of artificial stimulation, and their functional similarity to GTH cells of spontaneously maturing eels is discussed. Large female eels had unstimulated GTH cells. Growth hormone (STH), thyrotropic (TSH) and prolactin (PRL) cells were stimulated after E2 and LHRH. As with GTH cells, they regressed slowly after treatment was discontinued.

Purification and characterization of luteinizing hormone-releasing hormone from codfish brain

We have purified luteinizing hormone-releasing hormone (LH-RH) from codfish brain and have demonstrated its identity with salmon LH-RH (sLH-RH). An antiserum raised against sLH-RH was used in a specific radioimmunoassay (RIA) to monitor purification and to manufacture an immunoaffinity chromatography column for the initial purification step. The cross-reactivity of the sLH-RH RIA with mammalian LH-RH was 0.1%. Acid extracts of codfish brains were sequentially purified by immunoaffinity chromatography, gel-filtration chromatography, and three steps of reverse-phase HPLC. The purified material and synthetic sLH-RH coeluted on reverse-phase HPLC and exhibited similar biological activity in a dispersed pituitary cell bioassay. Furthermore, the amino acid composition of the purified material was identical to salmon LH-RH. These results suggest that there is structural conservation of LH-RH between these species of teleost fish.

Relationship between brain gonadotrophin-releasing hormone (GnRH) and seasonal reproductive cycle of “caribe colorado,” Pygocentrus notatus

Immunoreactive gonadotrophic hormone-releasing hormone (ir-GnRH) was detected in hypothalamic and telencephalic extracts of the Venezuelan freshwater fish “caribe colorado,” Pygocentrus notatus. Hypothalamic ir-GnRH from female fish demonstrated displacement curves parallel to those of synthetic mammalian luteinizing hormone-releasing hormone (LHRH). The content of hypothalamic and telencephalic ir-GnRH from female fish was more than four-fold greater than that of male animals. Also, fluctuations that depended on the reproductive state and environmental conditions (rainfall) occurred in females but not in males. Thus, ir-GnRH levels were higher in hypothalamic and telencephalic extracts from sexually mature females than in those from fish sampled outside the climatically determined breeding season.