Identification of diverse molecular forms of GnRH in reptile brain

Gonadotropin-releasing hormone (GnRH) molecular forms in the brains of three reptiles, Alligator mississippiensis (alligator), Calcides ocellatus tiligugu (skink) and Podarcis s. sicula (lizard) were characterized by high performance liquid chromatography (HPLC) and radioimmunoassay with region-specific antisera, and by assessment of luteinizing (LH)-releasing activity in chicken dispersed pituitary cells. In alligator brain two GnRHs had identical properties to the two known forms of chicken hypothalamic GnRH (Gln⁸-GnRH and His⁵,Trp⁷,Tyr⁸-GnRH) in their elution on two reverse phase HPLC systems, cross-reaction with region-specific GnRH antisera, and ability to release LH. In skink brain, one immunoreactive and bioactive GnRH form, which eluted in the same position as His⁵,Trp⁷,Tyr⁸-GnRH on reverse phase HPLC, was identified. Three bioactive and immunoreactive GnRHs were detected in lizard brain. One form had similar properties to salmon brain GnRH (Trp⁷,Leu⁸-GnRH). The other two GnRH-like peptides are novel forms. One of these forms eluted in the same position as Gln⁸-GnRH on HPLC but had different immunological properties, while the third form was a rather hydrophobic species which appeared to be modified in the middle region of the molecule.     

Differential regional distribution of gonadotropin-releasing hormones in amphibian (clawed toad, Xenopus laevis) brain

In most vertebrate species two forms of gonadotropin-releasing hormone (GnRH) are present in the brain, and their differential distribution suggests they have different functional roles. The regional distribution and relative concentrations of GnRH molecular forms in the brain of adult clawed toad (Xenopus laevis) were determined using high performance liquid chromatography and radioimmunoassay with a library of region-specific GnRH antisera. Four immunoreactive forms of GnRH were detected: mammalian, hydroxyproline mammalian, chicken II, and an unidetified form of GnRH. Mammalian GnRH was distributed throughout the brain, and hydroxyproline mammalian was present in the forebrain, midbrain (excluding hypothalamus), and hypothalamus. Chicken GnRH II also occurred throughout the brain, but was present in greater amounts in the hindbrain and midbrain (excluding hypothalamus). An unidentified form of GnRH with properties of salmon GnRH was detected in the forebrain. Considering the relative proportions of mammalian GnRH and chicken GnRH II in the major brain areas, the concentration of mammalian GnRH was high in the forebrain, midbrain (excluding hypothalamus), and in particular in the hypothalamus, and very little chicken GnRH II was present in these areas. In the hindbrain, chicken GnRH II predominated and the concentration of chicken GnRH II was highest in the medulla. These findings suggest: (1) mammalian GnRH is the prime regulator of gonadotropin release from the pituitary, and (2) chicken GnRH II has an extrapituitary role.     

Increased expression of kisspeptin and GnRH forms in the brain of scombroid fish during final ovarian maturation and ovulation

ABSTRACT: BACKGROUND: Kisspeptins (Kiss) are prime players in the control of reproductive function through their regulation of gonadotropin-releasing hormone (GnRH) expression in the brain. The experimental scombroid fish, chub mackerel (Scomber japonicus) expresses two kiss (kiss1 and kiss2) and three gnrh (gnrh1, gnrh2, and gnrh3) forms in the brain. In the present study, we analyzed expression changes of kiss and gnrh mRNAs in the brain and corresponding GnRH peptides in the brain and pituitary during final ovarian maturation (FOM) and ovulation. METHODS: Female fish possessing late vitellogenic oocytes were injected with GnRH analogue to induce FOM and ovulation. Fish were observed for daily spawning activities and sampled one week post-injection at germinal vesicle migration (GVM), oocyte hydration, ovulation, and post-ovulatory time periods. Changes in relative mRNA levels of kiss and gnrh forms in the brain were determined using quantitative real-time PCR. Changes in GnRH peptides in the brain and pituitary were analyzed using time-resolved fluoroimmunoassay. RESULTS: Both kiss1 and kiss2 mRNA levels in the brain were low at late vitellogenic stage and increased significantly during the GVM period. However, kiss1 mRNA levels decreased during oocyte hydration before increasing again at ovulatory and post-ovulatory periods. In contrast, kiss2 mRNA levels decreased at ovulatory and post-ovulatory periods. Levels of gnrh1 mRNA in the brain increased only during post-ovulatory period. However, levels of gnrh2 and gnrh3 mRNAs were elevated during GVM and then, decreased during oocyte hydration before increasing again at ovulatory period. During post-ovulatory period, both gnrh2 and gnrh3 mRNA levels declined. Peptide levels of all three GnRH forms in the brain were elevated during GVM and oocyte hydration; their levels were significantly lower during late vitellogenic, ovulatory, and post-ovulatory periods. In contrast, pituitary GnRH peptide levels did not show any significant fluctuations, with the GnRH1 peptide levels being many-fold higher than the GnRH2 and GnRH3 forms. CONCLUSION: The results indicate increased expression of multiple Kiss and GnRH forms in the brain and suggest their possible involvement in the regulation of FOM and ovulation in captive female chub mackerel.     

Gonadotropin-releasing hormone in ratfish (Hydrolagus colliei): distribution between the sexes and possible relationship with chicken II and salmon II forms

1. Brain extract from the spotted ratfish, Hydrolagus colliei, contains gonadotropin-releasing hormone (GnRH)-like peptides in both sexes. 2. The dominant form occurs with a concentration of 0.5-1.7 ng/g frozen brain tissue in males, and 1.3-2.5 ng/g in females. 3. A similar pattern of GnRH immunoreactivity and chromatographic behaviour are found in both sexes. 4. A semipurified extract of this peptide could not be distinguished chromatographically from either chicken II or salmon II forms of the peptide. 5. The ratfish represents the most primitive organism that contains a form of GnRH that coelutes with chicken II and salmon II GnRH.     

Four functional GnRH receptors in zebrafish: analysis of structure, signaling, synteny and phylogeny

Reproduction in all vertebrates requires the brain hormone gonadotropin-releasing hormone (GnRH) to activate a cascade of events leading to gametogenesis. All vertebrates studied to date have one to three forms of GnRH in specific but different neurons in the brain. In addition, at least one type of GnRH receptor is present in each vertebrate for activation of specific physiological events within a target cell. Humans possess two types of GnRH (GnRH1 and GnRH2) but only one functional GnRH receptor. Zebrafish, Danio rerio, also have two types of GnRH (GnRH2 and GnRH3), although in contrast to humans, zebrafish appear to have four different GnRH receptors in their genome. To characterize the biological significance of multiple GnRH receptors within a single species, we cloned four GnRH receptor cDNAs from zebrafish and compared their structures, expression, and cell physiology. The zebrafish receptors are 7-transmembrane G-protein coupled receptors with amino-acid sequence identities ranging from 45 to 71% among the four receptors. High sequence similarity was observed among the seven helices of zebrafish GnRHRs compared with the human GnRHR, the green monkey type II GnRHR, and the two goldfish GnRHRs. Also, key amino acids for putative ligand binding, disulfide bond formation, N-glycosylation, and G-protein coupling were present in the extracellular and intracellular domains. The four zebrafish receptors were expressed in a variety of tissues including the brain, eye, and gonads. In an inositol phosphate assay, each receptor was functional as shown by its response to physiological doses of native GnRH peptides; two receptors showed selectivity between GnRH2 and GnRH3. Each of the four receptor genes was mapped to distinct chromosomes. Our phylogenetic and syntenic analysis segregated the four zebrafish GnRH receptors into two distinct phylogenetic groups that are separate gene lineages conserved throughout vertebrate evolution. We suggest the maintenance of four functional GnRH receptors in zebrafish compared with only one in humans may depend either on subfunctionalization or neofunctionalization in fish compared with mammalian GnRH receptors. The differences in structure, location, and response to GnRH forms strongly suggests that the four zebrafish GnRH receptors have novel functions in addition to the conventional activation of the pituitary gland in the reproductive axis.     

Two gonadotropin-releasing hormones from African catfish (Clarias gariepinus).

Two forms of gonadotropin-releasing hormone (GnRH) have been purified from brain extracts of the African catfish, Clarias gariepinus, using reverse-phase high performance liquid chromatography (HPLC) and radioimmunoassay (RIA). The amino acid sequences of both forms of African catfish GnRH were determined using Edman degradation after digestion with pyroglutamyl aminopeptidase. In addition, both GnRHs were studied by mass spectrometry. The primary structure of African catfish GnRH I is identical to Thai catfish GnRH I, pGlu-His-Trp-Ser-His-Gly-Leu-Asn-Pro-Gly-NH2, and the primary structure of African catfish GnRH II is identical to the widely distributed and highly conserved chicken GnRH II, pGlu-His-Trp-Ser-His-Gly-Trp-Tyr-Pro-Gly-NH2.     

Identification of Gln8-GnRH and His5,Trp7,Tyr8-GnRH in the hypothalamus and extrahypothalamic brain of the ostrich (Struthio camelus)

Gonadotropin-releasing hormone (GnRH) molecular forms were studied in extracts of ostrich hypothalamus and extrahypothalamic brain using high performance liquid chromatography, radioimmunoassay with region-specific antisera and assessment of luteinizing hormone (LH)-releasing activity using chicken dispersed pituitary cells. Two molecular forms of GnRH with chromatographic, immunological and biological properties identical to those of Gln8-GnRH and His5,Trp7,Tyr8-GnRH were demonstrated in both the hypothalamic and extrahypothalamic brain extracts. A greater proportion of His5,Trp7,Tyr8-GnRH was present in the hypothalamus than in extrahypothalamic brain. It is likely that these two forms of GnRH are present in all bird species, since the chicken and the ostrich have evolved separately.     

Partial characterization of four forms of immunoreactive gonadotropin-releasing hormone in the brain and terminal nerve of the spiny dogfish (Elasmobranchii; Squalus acanthias).

Four forms of immunoreactive GnRH have been detected in tissue extracts of both whole brains and terminal nerves from the spiny dogfish (Squalus acanthias). The GnRH forms were characterized using reverse-phase high pressure liquid chromatography (HPLC) and immunological recognition with four different antisera. Three of these forms possess immunological and chromatographic properties consistent with known forms of GnRH: mammalian GnRH, chicken GnRH-II and salmon GnRH. An additional form, with an HPLC elution position intermediate between chicken GnRH-II and salmon GnRH appears to be a new structure of GnRH. The presence of all four GnRH forms in the terminal nerve suggests a lack of regional specificity of the expressed forms of GnRH in the brain.     

Differential distribution of two molecular forms of gonadotropin-releasing hormone in discrete brain areas of goldfish (Carassius auratus)

Two molecular forms of gonadotropin-releasing hormone (GnRH) were identified in the extracts of various brain areas, spinal cord and pituitary in female and male goldfish and had chromatographic and immunological properties similar to [His5, Trp7, Tyr8]-GnRH (cGnRH-II) and [Trp7,Leu8]-GnRH (sGnRH). Radioimmunoassay using different GnRH antisera after high pressure liquid chromatography did not reveal significant peaks of mammalian GnRH, [Gln8]-GnRH and [Tyr3,Leu5,Glu6,Trp7,Lys8]-GnRH in the brain extracts. The proportion of cGnRH-II-like immunoactivity to sGnRH-like immunoactivity was higher in the caudal brain areas compared to the rostral areas. The differential distribution of two GnRH forms suggest that the different GnRH forms may have different physiological functions.     

Rapid separation of gonadotropin-releasing hormone molecular forms by isocratic high-performance liquid chromatography on an ion-exchange column

The purpose of the present work was to develop a chromatographic system for the separation of five molecular forms of the gonadotropin-releasing hormone (GnRH); mammalian GnRH (mGnRH) (LHRH), salmon GnRH (sGnRH), chicken I GnRH (clGnRH), chicken II GnRH (cIIGnRH) and lamprey GnRH I (IGnRH-I). By using an ion-exchange HPLC column and isocratic elution, it was possible to separate properly the five peptides in approximately 20 min. The utility of the system in determining the GnRHs forms present in the brain of two species of vertebrates was examined.     

Multiple molecular forms of gonadotropin-releasing hormone in teleost fish brain

Gonadotropin-releasing hormone (GnRH) immunoreactive peptides in extracts of hake (Merluccius capensis) and tilapia (Tilapia sparrmanii) brain were investigated by high performance liquid chromatography (HPLC) and radioimmunoassay with region-specific antisera. In hake brain, content and concentration of GnRH was higher in the pituitary gland than in the hypothalamic lobes or extrahypothalamic brain. Hake pituitary gland GnRH was purified by six consecutive HPLC systems. The major GnRH molecular form co-eluted with salmon brain GnRH (Trp7, Leu8-GnRH) in four different HPLC systems which were specifically designed to separate the four natural vertebrate GnRHs (mammalian, salmon, chicken I and II). The immunoreactive peak in the final purification step had a retention time identical to that of Trp7, Leu8-GnRH and an UV absorbance (280 nm) peak appropriate for two tryptophan residues in the peptide, as in Trp7, Leu8-GnRH. Six additional less hydrophobic forms of GnRH were detected. Tilapia brain extract contained two major GnRH molecular forms which had identical retention times to chicken GnRH I (Gln8-GnRH) and Trp7, Leu8-GnRH in an HPLC system which separates the natural vertebrate GnRHs. The immunological properties of these two immunoreactive peaks, determined by relative interaction with four region-specific GnRH antisera raised against vertebrate GnRHs, were identical to those of Gln8-GnRH and Trp7, Leu8-GnRH. Additional GnRH molecular forms were also detected. In summary, these findings indicate that a major GnRH molecule in hake pituitary gland is Trp7, Leu8-GnRH, while tilapia brain contains both Trp7, Leu8-GnRH and Gln8-GnRH. Additional GnRH molecular forms were detected in both species.(ABSTRACT TRUNCATED AT 250 WORDS)     

Differential regional distribution and release of two forms of gonadotropin-releasing hormone in the chicken brain

The functional significance of two molecular forms of gonadotropin-releasing hormone (GnRH) in the chicken brain was investigated. The differential distribution of [Gln8]GnRH (chicken GnRH-I, cGnRH-I) and [His5,Trp7,Tyr8]GnRH (chicken GnRH-II, cGnRH-II) was determined using high performance liquid chromatography and radioimmunoassay with region-specific antisera. Potassium-stimulated release of immunoreactive cGnRH-I and cGnRH-II from brain regions was assessed in tissue incubations. cGnRH-I and cGnRH-II varied independently in different brain areas. The concentration of cGnRH-I was highest in the median eminence of the hypothalamus, and a small quantity was also detected in the midbrain and cerebrum. cGnRH-II was more widely distributed throughout the brain, with highest concentrations in areas of the hypothalamus outside the median eminence and in the medulla. Potassium stimulated the release of cGnRH-I from the median eminence 4-fold, while cGnRH-II release was not detectable. Neither cGnRH-I nor cGnRH-II was released from the medulla. These data suggest: 1) cGnRH-I is the prime regulator of gonadotropin release from the pituitary, and 2) cGnRH-II may have a neurotransmitter or neuromodulator role in areas of the brain outside the median eminence.     

Chromatographic and immunological evidence for mammalian GnRH and chicken GnRH II in eel (Anguilla anguilla) brain and pituitary

Gonadotropin-releasing hormone (GnRH) peptides in the brain and pituitary of the European eel (Anguilla anguilla) were investigated by reverse phase high performance liquid chromatography (HPLC) and radioimmunoassay with region-specific antisera. Two GnRH molecular forms were demonstrated in brain and pituitary extracts. One form eluted in the same position as synthetic mammalian GnRH on HPLC and was recognized by antibodies directed against the NH2 and COOH termini of mammalian GnRH as well as by antibodies to the middle region. The second form eluted in the same position as synthetic chicken GnRH II and was recognized by specific antibodies to this molecule. Salmon GnRH and chicken GnRH I were not detected. The occurrence of mammalian GnRH in teleost fish suggests that this molecular form is more ancient than was previously suspected and arose earlier than in primitive tetrapods, or that it has arisen in the eel through random mutation of salmon GnRH. The lack of salmon GnRH in the eel brain indicates that this molecular form is not common to all teleost species. The finding in eel brain of chicken GnRH II, which has previously been described in species of Mammalia, Aves, Reptilia, Amphibia, Osteichthyes, and Chondrichthyes, supports our hypothesis that this widespread structural variant may represent an early evolved and conserved form of GnRH.     

Expression of pejerrey gonadotropin-releasing hormone in three orders of fish

Molecular variants of GnRH were characterized by reverse-phase, high-performance liquid chromatography from brain extracts of fish in three different orders: Synbranchiformes (swamp eel [Synbranchus marmoratus]), Cyprinidontiformes (platyfish [Xiphophorus maculatus] and green swordtail [X. helleri]), and Atheriniformes (Patagonia pejerrey [Odontesthes hatchery]). Also, pituitary gland extracts from the pejerrey O. bonariensis (Atheriniformes) were characterized. Eluted fractions were tested in radioimmunoassays with antisera specific to GnRH, including both antisera that detected only one form of GnRH and those that detected several forms. The results show that brain extracts obtained from all species contained the same three molecular forms of GnRH, which were immunologically and chromatographically undistinguishable from chicken GnRH-II, pejerrey GnRH (pjGnRH), and salmon GnRH. This study supports the hypothesis that expression of these three forms is common in different fish orders and that pjGnRH is the main regulator of pituitary function in these fish.     

Gonadotropin-releasing hormone content in the brain and pituitary of male and female grass rockfish (Sebastes rastrelliger) in relation to seasonal changes in reproductive status.

The involvement of individual molecular forms of GnRH in the regulation of reproductive cyclicity in a viviparous marine teleost, the grass rockfish (Sebastes rastrelliger), was evaluated by relating the brain and pituitary content of the neuropeptide to reproductive status. The presence of sea bream (sb) GnRH, chicken GnRH-II, and salmon GnRH in the brain was confirmed by their elution pattern on HPLC and RIA. In addition, HPLC elution profiles suggest that there may be a fourth form of GnRH. All forms of GnRH were found in male and female brains in all reproductive conditions. However, only sbGnRH could be detected in appreciable amounts in the pituitary. Of the four forms of GnRH found in the rockfish, only sbGnRH fluctuated during the reproductive cycle and large accumulations were detected in the brains and pituitaries of postspawn females and regressed males. The accumulation of sbGnRH at the end of the reproductive cycle is suggested to reflect a decline in GnRH secretion relative to synthesis. The dominance of sbGnRH in the pituitary and its individual fluctuation in relation to seasonal changes in reproductive status suggests that sbGnRH is an important regulator of gonadotropin-mediated reproductive activity in rockfish.     

The identification of gonadotropin-releasing hormone (GnRH) in hypothalamic and extrahypothalamic loci of the human nervous system.

Immunoreactive-like GnRH activity has been identified in 24 of 26 separate loci of the human central nervous system. Tissues, secured from 5 brains at autopsy, were dissected, extracted sequentially with 2N and glacial acetic acid, lyophilized, and eluted in buffered saline for GnRH determinations by specific radioimmunoassay. GnRH concentrations (ng/mg protein) ranged from 8.96 (infundibulum) to 0.001 (cerebellum. middle lobe). Highest extrahypothalamic concentrations of GnRH were found in mamillary body (0.076) and thalamus (0.002). Extrahypothalamic GnRH was identical to synthetic and hypothalamic GnRH by criteria of immunoidentity. No post-mortem GnRH peptidolysis, evaluated experimentally in rats, was evident between 0 and 16 hrs in intact tissues maintained at 4 degrees C. These data suggest that GnRH is distributed throughout regions of the human brain outside the hypothalamus and suggest new, non-endocrine functions for GnRH in the human CNS, analogous the those reported recently for GnRH in experimental animals.     

Effects of gonadotropin-releasing hormone agonists on meiotic maturation of follicle-enclosed oocytes in rabbits

The effects of GnRH agonists on in vitro maturation of rabbit follicle-enclosed oocytes were studied. Rabbit preovulatory follicles were cultured with or without hCG (10(2) ng/ml), buserelin (10(2)-10(5) ng/ml), or leuprolide (10(2)-10(5) ng/ml) for 14 hours in vitro. GnRH agonists induced the resumption of meiosis in the follicle-enclosed oocytes in a dose-dependent manner. The percentage of oocytes achieving GVBD following treatment with 10(5) ng/ml buserelin (87.9 +/- 6.3%) or 10(5) ng/ml leuprolide (86.0 +/- 4.1%) did not differ significantly from hCG-treated control (87.3 +/- 3.8%). Mature oocytes initially were detected within 2 hours of GnRH agonist exposure. Concomitant addition of a GnRH antagonist at 10(4) ng/ml significantly blocked the stimulatory effect of GnRH agonist on oocyte maturation. GnRH agonists significantly stimulated both prostaglandin (PG) E2 (PGE2) and PGF2 alpha production by preovulatory follicles (p less than 0.01), but secreted prostanoid levels did not differ significantly among different concentrations of GnRH agonists. Meiotic maturation of follicle-enclosed oocytes following GnRH agonist exposure began 2 hours earlier than production of PGs. PG production stimulated by GnRH agonists was reduced significantly by indomethacin. However, oocyte maturity in the presence of GnRH agonist plus indomethacin did not differ significantly from that of GnRH agonist alone. GnRH agonistic analogues induce the resumption of meiosis in follicle-enclosed oocytes in rabbits by a mechanism other than PG stimulation.     

Isolation and characterization of glycosylated and non-glycosylated prolactins from alligator and crocodile.

Two molecular forms of prolactin (PRL), glycosylated and non-glycosylated, were isolated from pituitary glands of two reptiles, alligator and crocodile. The reptilian PRLs were extracted under alkaline conditions from the precipitate obtained after pituitaries were first extracted with 0.25 M sucrose, 1 mM NH4HCO3, pH 6.3. Purification was performed by ion exchange chromatography on DE-52, gel filtration on Sephadex G-75 superfine, and reversed phase high performance liquid chromatography. Two forms of both alligator and crocodile PRL, designated PRLI and PRLII, with molecular weights of 26,000 and 24,000 were isolated. Alligator and crocodile PRLI and PRLII were stained specifically in immunoblots with anti-sea turtle PRL and anti-ostrich PRL. Sequence analysis revealed that both forms of alligator and crocodile PRLs consisted of 199 amino acid residues with a glycosylation consensus sequence (Asn-Ala-Ser) at position 60 in alligator and crocodile PRLs with a molecular weight of 26,000 (PRLI). In contrast, Thr was substituted for Asn at position 60 in the PRLs with a molecular weight of 24,000 (PRLII). The sequences of alligator PRLs differed from crocodile PRLs only in position 134: Val for alligator PRLs and Ile for crocodile PRLs. There is a high degree of structural conservation between the reptilian PRLs isolated in this study and avian PRL; each showed 92% sequence identity with chicken PRL and 89% with turkey PRL.     

Dependence of GnRH action on Na+, K+, and Ca2+ in the frog, Rana pipiens, pituitary

The roles of K+, Ca2+, and Na+ ions in the mechanism of gonadotropin releasing hormone (GnRH) action on frog (Rana pipiens) hemipituitaries were studied using an in vitro superfusion system. The effects of elevated K+ alone or in combination with Ca2+-depleted medium, tetrodotoxin (TTX), or with 100 ng/ml GnRH were examined. The involvement of K+ was also studied indirectly through the use of tetraethyl ammonium chloride (TEA). The importance of Ca2+ was established by the loss of responsiveness to GnRH in Ca2+-depleted medium, or in the presence of the Ca2+ competitor CoCl2. The absence of a major dependence of GnRH on Na+ was revealed by the continued gonadotropin secretion after addition of 1 microM TTX to medium containing GnRH or 36.3 mM KCl, or by replacement of NaCL with choline chloride. High (10 X normal) KCl (36.3 mM) stimulated gonadotropin--both LH and FSH--secretion, but the response was more gradual than for GnRH. The inclusion of TEA (to block K+ efflux) in medium with GnRH accentuated the effect of GnRH, and the effects of elevated (36.3 mM) KCl and 100 ng/ml GnRH (a relatively high dose) were additive. Responses to high K+, like GnRH, were abolished by removal of Ca2+ from the medium. Overall, the roles of K+, Ca2+, and Na+ ions in the mechanism of GnRH action are very similar between mammals and frogs; Ca2+ apparently serves a critical function in the mechanism of GnRH action, while Na+ appears not to be involved. K+ can induce gonadotropin secretion, but it is not clear that it plays a direct role in the mediation of the action of GnRH.