Forms Of Gonadotropin-releasing Hormone Research Articles

Effects of photoperiod on pituitary levels of three forms of GnRH and reproductive hormones in the male European sea bass ( Dicentrarchus labrax, L.) during testicular differentiation and first testicular recrudescence

Photoperiod is one of the main external cues in the control of different stages of fish development. In the present work, the effect of several photoperiods, known to delay spawning in adult fish, was investigated in juvenile male sea bass ( Dicentrarchus labrax) during testicular differentiation and first testicular recrudescence. Fish were exposed to constant long (LO), expanded (EX), constant short photoperiod with long photoperiod in October (SLoct) and constant short photoperiod with long photoperiod in December (SLdec) during two consecutive years. Pituitary levels of three forms of gonadotropin-releasing hormone (GnRH) [sea bream GnRH (sbGnRH), chicken GnRH-II (cGnRH-II), and salmon GnRH (sGnRH)] were analysed and plasma levels of gonadotropin (LH), testosterone (T), and 11-ketotestosterone (11-KT) were determined in the same periods. During both periods, pituitary levels of sbGnRH were higher than cGnRH-II and sGnRH in all groups. The three GnRHs had significantly lower values in November 1995 in groups exposed to artificial photoperiods during the testicular differentiation and growth period compared to the control group. In addition, a significant effect of LO, EX, and SLoct photoperiods was observed in the LH plasma profile, showing a significant delay in the peak of LH compared to the control group. However, no significant effect of the artificial photoperiods on the pituitary content of the three GnRHs has been observed during the first testicular recrudescence. Deeper studies are needed to understand the effect of artificial photoperiods on the endocrine events occurring during both histological testicular differentiation and growth and first testicular maturation in the sea bass.

15α-Hydroxytestosterone induction by GnRH I and GnRH III in Atlantic and Great Lakes sea lamprey ( Petromyzon marinus L.)

The sea lamprey ( Petromyzon marinus L.) represents one of the two most ancient classes of vertebrates and possesses a functional hypothalamus–pituitary–gonadal axis. However, the presence and functionality of androgens in the sea lamprey remain elusive. Recently, 15α-hydroxytestosterone (15α-T) has been found in sea lamprey gonads and blood plasma. In this study we examined changes of circulatory concentrations of 15α-T in response to gonadotropin releasing hormone (GnRH) treatments. Plasma concentrations of 15α-T in sea lamprey increased 2–5 times for all GnRH-injected sea lamprey compared to controls ( P<0.0001). However, there were no differences among responses: (1) to the two forms of GnRH (lamprey GnRH I or lamprey GnRH III), (2) to the doses delivered (50, 100, or 200 μg/kg), or (3) between post-injection sample intervals (8 or 24 h). Between lampreys from the Atlantic Ocean and Great Lakes sites, two of seven GnRH form and dosage comparisons showed between-site differences, but were not believed to represent an overall between-site difference. These are the first data to show a response of a C19 steroid to GnRH stimulation in sea lamprey.

Identification and characterization of a reptilian GnRH receptor from the leopard gecko

Gonadotropin-releasing hormone (GnRH) plays a pivotal role in the regulation of reproductive functions through interactions with its specific receptor. We describe the first molecular cloning and characterization of a full-length GnRH receptor (GnRHR) from the leopard gecko Eublepharis macularius. It has a distinct genomic structure consisting of five exons and four introns, compared with all the other reported GnRHR genes. A native GnRH form, cGnRH-II, stimulated inositol phosphate (IP) production in COS-7 cells transiently transfected with the GnRHR, in a dose dependent manner. The mRNA was expressed in all the tissues and organs examined. Molecular phylogenetic analysis revealed that the cloned GnRHR belongs to the type 2/nonmammalian I GnRHR. Low-expression levels were observed from the pituitary glands of reproductively active leopard geckos, indicating the possibility that there is at least one more type of GnRHR highly expressed in the pituitary gland for the gonadotropin secretion in this reptile.

Structure, expression, and cluster organization of genes encoding gonadotropin-releasing hormone receptors found in the neural complex of the ascidian Ciona intestinalis

Gonadotropin-releasing hormone (GnRH) controls reproductive functions in vertebrates. In the present study, two distinct homologues (Ci-GnRHR1 and Ci-GnRHR2) of the vertebrate GnRH receptor (GnRHR) were identified by cDNA cloning from the neural complex of the ascidian Ciona intestinalis. The Ci-GnRHR1 and Ci-GnRHR2 genes are closely linked to each other and form a cluster with another GnRHR-like gene in the genome. Ci-GnRHR1 and Ci-GnRHR2 are more closely related to vertebrate GnRHRs than to the GnRHR-related protein of Drosophila. The phylogenetic analysis indicates that vertebrates and urochordates independently acquired multiple GnRHRs by gene duplications that occurred during the evolution of each lineage. A voltage clamp of Xenopus oocytes injected with synthetic Ci-GnRHR1 mRNA revealed inward currents in response to an ascidian form of GnRH, suggesting that Ci-GnRHR1 is a bona-fide GnRHR. Expression patterns of Ci-GnRHR1 and Ci-GnRHR2 suggest that a GnRH signaling system is involved in regulation of neuronal and reproductive processes as well as in other physiological functions in ascidians.

Gonadotropin-releasing hormones modulate electrical activity of vasotocin and isotocin neurons in the brain of rainbow trout

Gonadotropin-releasing hormone (GnRH) is widely distributed in the vertebrate brains; however, its significance in the brain function is poorly understood. Both GnRH and vasopressin-family hormones are involved in control of reproductive behavior. Anatomical evidence indicated the possible action of GnRH on classical neurosecretory neurons. In the present study, we examined whether GnRH modulates electrical activity of vasotocin (VT) and isotocin (IT) neurons in the brain of rainbow trout (Oncorhynchus mykiss). Two forms of GnRH, salmon GnRH and chicken GnRH II, are present in the rainbow trout brain, and their fibers are localized in the close vicinity of VT and IT neurons. Applications of both GnRH forms elevated the frequency of cell-type-specific synchronous Ca2+ pulses in VT and IT neurons that are blocked by a GnRH-receptor antagonist. Our results showed facilitatory actions of GnRHs on VT and IT neurons, suggesting that GnRH neurons modulate classical neurosecretory neurons to control reproductive behavior.

Gonadotropin-releasing hormones modulate electrical activity of vasotocin and isotocin neurons in the brain of rainbow trout

Gonadotropin-releasing hormone (GnRH) is widely distributed in the vertebrate brains; however, its significance in the brain function is poorly understood. Both GnRH and vasopressin-family hormones are involved in control of reproductive behavior. Anatomical evidence indicated the possible action of GnRH on classical neurosecretory neurons. In the present study, we examined whether GnRH modulates electrical activity of vasotocin (VT) and isotocin (IT) neurons in the brain of rainbow trout ( Oncorhynchus mykiss). Two forms of GnRH, salmon GnRH and chicken GnRH II, are present in the rainbow trout brain, and their fibers are localized in the close vicinity of VT and IT neurons. Applications of both GnRH forms elevated the frequency of cell-type-specific synchronous Ca 2+ pulses in VT and IT neurons that are blocked by a GnRH-receptor antagonist. Our results showed facilitatory actions of GnRHs on VT and IT neurons, suggesting that GnRH neurons modulate classical neurosecretory neurons to control reproductive behavior.

Relative potency of the forms of GnRH and their analogs on LH release in sea bass

The in vivo and in vitro potency of native and modified forms of gonadotropin releasing hormone (GnRH) to release luteotropic hormone (LH) was studied in sea bass Dicentrarchus labrax in particular the hypothalamic fish‐specific sea bream GnRH form (sbGnRH) and the general mesoencephalic form chicken GnRH‐II (cGnRH‐II). The potencies of the natives and their analogs (GnRHas) were referred to that of [D‐Ala6, Pro9Net]‐mGnRHa (LHRHa) at equivalent doses. Analogs of the native peptides [D‐Arg6, Pro9Net]‐cGnRH‐II, [D‐Ala6, Pro9Net]‐cGnRH‐II, [D‐Trp6, Pro9Net]‐sbGnRH and [D‐Ala6, Pro9Net]‐sbGnRH were effective in inducing in vivo LH release (at 15 µg kg−1 body mass), exhibiting longer lasting activity than their corresponding native forms. Injection of sbGnRH and cGnRH‐II provoked a small but significant peak of circulating LH at 1·5 h after treatment (a.t.) decreasing down to basal levels at 4 h a.t. [D‐Arg6, Pro9Net]‐cGnRH‐II, [D‐Ala6, Pro9Net]‐cGnRH‐II and [D‐Ala6, Pro9Net]‐mGnRHa evoked a higher and a more sustained elevation of LH, peaking at 12 h a.t. and returning to basal levels between 48 and 72 h a.t. [D‐Trp6, Pro9Net]‐sbGnRH and [D‐Ala6, Pro9Net]‐sbGnRH also induced a significant surge of LH in plasma at 4 h a.t. turning to the basal levels at 24 h a.t. These rises, however, were of less amplitude and duration than the observed after treatment with cGnRH‐II analogs and [D‐Ala6, Pro9Net]‐mGnRHa. The in vitro stimulation of dispersed pituitary cells with the different native and modified forms of GnRH resulted in a dose‐dependent increase in the quantity of LH released at 24 h a.t. [D‐Arg6, Pro9Net]‐cGnRH‐II and [D‐Ala6, Pro9Net]‐cGnRH‐II induced the highest response of LH in vitro release followed by salmon GnRH (sGnRH), [D‐Ala6, Pro9Net]‐mGnRHa and [D‐Trp6, Pro9Net]‐sbGnRH. The lowest activity was exhibited by sbGnRH. Collectively, the in vitro biological activity (compared by their EC50) can be ordered as follows: [D‐Arg6, Pro9Net]‐cGnRH‐II &gt; [D‐Ala6, Pro9Net]‐cGnRH‐II &gt; sGnRH &gt; [D‐Ala6, Pro9Net]‐mGnRHa &gt; [D‐Trp6, Pro9Net]‐sbGnRH &gt; [D‐Ala6, Pro9Net]‐sbGnRH &gt; cGnRH‐II &gt; sbGnRH.

Molecular characterization of the GnRH system in zebrafish ( Danio rerio): cloning of chicken GnRH-II, adult brain expression patterns and pituitary content of salmon GnRH and chicken GnRH-II

The zebrafish has proven to be a model system with unparalleled utility in vertebrate genetic and developmental studies. Substantially less attention has been paid to the potential role that zebrafish can play in answering important questions of vertebrate reproductive endocrinology. As an initial step towards exploiting the advantages that the zebrafish model offers, we have characterized their gonadotropin-releasing hormone (GnRH) system at the molecular level. GnRHs comprise a family of highly conserved decapeptide neurohormones widely recognized to orchestrate the hormonal control of reproduction in all vertebrates. We have isolated the gene and cDNA encoding chicken GnRH-II (cGnRH-II) from zebrafish, as well as several kilobases of upstream promoter sequence for this gene. As the gene encoding salmon GnRH (sGnRH) has been previously isolated ( Torgersen et al., 2002), this is the second GnRH gene isolated from zebrafish to date. We have localized expression of these two genes in the brains of reproductively mature zebrafish using in situ hybridization. sGnRH is localized to the olfactory bulb-terminal nerve region (OB-TN), the ventral telencephalon-preoptic area (VT-POA) and, as we report here for the first time in any teleost species, the hindbrain. cGnRH-II is expressed exclusively in the midbrain, as has been found in all other jawed vertebrate species examined. Finally, the levels of both GnRH peptides in pituitaries of reproductively mature zebrafish were quantified using specific ELISAs. sGnRH pituitary peptide levels were shown to be 3- to 4-fold higher than cGnRH-II pituitary peptide. The cumulative results of these experiments allow us to conclude that zebrafish express just two forms of GnRH in a site-specific manner within the brain, and that sGnRH is the hypophysiotropic GnRH form. This work lays the foundation for further research into the control of reproduction in zebrafish, such as the functional significance of multiple GnRHs in vertebrates, and the molecular mechanisms controlling tissue-specific GnRH expression.

Multi-factorial role of GnRH-I and GnRH-II in the human ovary

Normal ovarian functions are regulated by a wide variety of endocrine hormones, local paracrine and autocrine factors, which functionally interact with each other in a highly coordinated fashion. Recent findings have demonstrated that both forms of gonadotropin-releasing hormone (GnRH-I and GnRH-II) are expressed in various compartments of the human ovary including the granulosa–luteal cells, ovarian surface epithelial cells and ovarian tumors, and their expressions have been shown to be tightly regulated by gonadal steroids and gonadotropins. Functionally, these neuropeptides exert diverse biological effects in the ovary via binding to their cognate receptors, supporting the notion that these peptides act as paracrine and autocrine factors in modulating local ovarian functions. In this review, we will summarize recent literatures regarding the regulation of GnRH-I and GnRH-II gene expressions in the human ovary, and discuss the possible signal transduction mechanisms by which these hormones exert their actions in the gonad. Recent cloning of the second form of the GnRH receptor (GnRH-II receptor) in primates and other vertebrates demonstrated that it was structurally, and thus, functionally distinct from the GnRH-I receptor. Cell proliferation studies showed that GnRH-II inhibited the growth of human ovarian cancer cells that express GnRH-II but not GnRH-I receptor, indicating that the GnRH-II binding sites are functional in these cells. However, it remains unknown if GnRH-II receptor is expressed as a full-length, properly processed and functional gene transcript in humans, and its potential physiological roles such as differential regulation of gonadotropin secretion, neuroendocrine modulation and female sexual behavior await further investigation.

Differential regulation of gonadotropin-releasing hormone (GnRH)I and GnRHII messenger ribonucleic acid by gonadal steroids in human granulosa luteal cells.

In humans, reproduction was generally believed to be controlled by only one form of GnRH (called mammalian GnRH or GnRHI). However, recently, a second form of GnRH, analogous to chicken GnRHII, was discovered in several tissues, including the human ovary. The regulation and function of GnRHI in the hypothalamus has been well studied. However, the function and regulation of GnRHI, and particularly GnRHII in the ovary, is less well understood. Because gonadal sex steroids are one of the main regulators of reproduction, we investigated, in the present study, the regulation of GnRHI and GnRHII mRNA expression by 17beta-estradiol (E2) and RU486 (a progesterone antagonist) in human granulosa luteal cells (hGLCs). The levels of the mRNA transcripts encoding the two GnRH forms were examined using semiquantitative RT-PCR followed by Southern blot analysis. With time in culture, GnRHI and GnRHII mRNA levels significantly increased, by 120% and 210%, at d 8 and d 1, respectively. The levels remained elevated until the termination of these experiments at d 10. A 24-h treatment of hGLCs with E2 (10(-9) to 10(-7) M) resulted in a dose-dependent decrease and increase in mRNA expression of GnRHI and GnRHII, respectively. E2 (10(-9) M) significantly decreased GnRHI mRNA levels (by 55%) and increased GnRHII mRNA levels (by 294%). Time-course studies demonstrated that E2 (10(-9) M) significantly decreased GnRHI mRNA levels in a time-dependent manner, with maximal inhibition of 77% at 48 h. In contrast, GnRHII mRNA levels significantly increased in a time-dependent fashion, reaching a maximum level of 280% at 24 h. Cotreatment of hGLCs with E2 and tamoxifen (an E2 antagonist) reversed the inhibitory and stimulatory effects of E2 on the mRNA expression of GnRHI and GnRHII, respectively. Time- and dose-dependent treatment with RU486 did not affect GnRHI mRNA levels in hGLCs. In contrast, RU486 treatment significantly increased GnRHII mRNA levels in hGLCs in a time- and dose-dependent fashion, with a maximum increase being observed at 24 h (with 10(-5)M RU486). In summary, the present study demonstrated that the expression of GnRHI and GnRHII at the transcriptional level is differently regulated by E2 and P4 in hGLCs.

GnRH systems in masu salmon and barfin flounder

Multiple forms of the gonadotropin-releasing hormone (GnRH) exist in teleost fish. A salmonid fish, masu salmon Oncorhynchus masou has salmon GnRH (sGnRH) and chicken GnRH-II (cGnRH-II). sGnRH neurons were scattered from the olfactory nerve through the ventral telencephalon (VT) and the preoptic area (POA). sGnRH but not cGnRH-II was detected in the pituitary. sGnRH mRNA levels in the VT and the POA increased during gonadal maturation, suggesting that sGnRH neurons in these areas are involved in gonadal maturation. sGnRH neurons were first detected in a cluster near the olfactory epithelium 40 days after fertilization. sGnRH neurons were not detected in the brain by the olfactory epithelia lesion, suggesting that sGnRH neurons are derived from the olfactory epithelium. A pleuronectiform fish, barfin flounder Verasper moseri has sGnRH, cGnRH-II and seabream GnRH (sbGnRH). sGnRH and cGnRH-II-immunoreactive fibers were observed throughout the brain, but not in the pituitary. sbGnRH neurons were located in the POA and sent fibers to the pituitary, indicating that sbGnRH is involved in GTH secretion. Judging from the location of neuronal somata and their projections, it is indicated that three GnRH systems exist in the barfin flounder; the TN-, the MT- and the POA-GnRH system. However, in masu salmon, clear anatomical identification of the TN- and the POA-GnRH system is difficult, because the GnRH neurons located in the ventral forebrain are consecutive and the GnRH form produced in these neurons is the same (sGnRH). Thus, it is suggested in masu salmon that sGnRH neurons are derived from the olfactory epithelium, migrate into the brain and play different roles according to the location in the brain.

Localization of gonadotropin-releasing hormone in the central nervous system and a peripheral chemosensory organ of Aplysia californica

Gonadotropin-releasing hormone (GnRH) is a neurohormone crucial for the regulation of reproductive and neural functions in vertebrates. Recent discoveries of GnRH immunoreactivity (IR) in a number of invertebrates raised the possibility that GnRH may be an ancient molecule that had arisen before the emergence of Phylum Chordata. We previously demonstrated the presence of a GnRH IR similar to the mammalian (m) and tunicate I (tI) forms of GnRH in the hemolymph and ovotestis of an opisthobranch mollusk, Aplysia californica; however, the presence of GnRH in the central nervous system (CNS) of A. californica could not be detected with the available antisera against various forms of chordate GnRH. In the present study, we performed immunohistochemistry (IHC) to localize the presence of GnRH in the CNS and a peripheral chemosensory organ, the osphradium, of A. californica. A newly generated antiserum against tI-GnRH revealed the strong expression of GnRH IR in neurons of all CNS ganglia. A notable asymmetry in immunostaining was detected in the left and right abdominal hemiganglia. The CNS is rich in tI-GnRH immunoreactive neurons but lacks mGnRH IR, whereas the osphradium contains abundant mGnRH immunoreactive neurons but lacks tI-GnRH IR. The extract of CNS failed to stimulate the release of LH from mouse pituitary, demonstrating that the A. californica GnRH IR is structurally different from what is required to bind and activate mammalian GnRH receptor. Together, these results indicate the presence of at least two distinct GnRH systems in A. californica. The presence of GnRH in the osphradium is consistent with the long-standing anatomical relationship between GnRH and the chemosensory system observed in vertebrates.

Social regulation of gonadotropin-releasing hormone.

Behavioral interactions among social animals can regulate both reproductive behavior and fertility. A prime example of socially regulated reproduction occurs in the cichlid fish Haplochromis burtoni, in which interactions between males dynamically regulate gonadal function throughout life. This plasticity is mediated by the brain, where neurons that contain the key reproductive regulatory peptide gonadotropin-releasing hormone (GnRH) change size reversibly depending on male social status. To understand how behavior controls the brain, we manipulated the social system of these fish, quantified their behavior and then assessed neural and physiological changes in the reproductive and stress axes. GnRH gene expression was assessed using molecular probes specific for the three GnRH forms in the brain of H. burtoni. We found that perception of social opportunity to increase status by a male leads to heightened aggressiveness, to increased expression of only one of the three GnRH forms and to increases in size of GnRH-containing neurons and of the gonads. The biological changes characteristic of social ascent happen faster than changes following social descent. Interestingly, behavioral changes show the reverse pattern: aggressive behaviors emerge more slowly in ascending animals than they disappear in descending animals. Although the gonads and GnRH neurons undergo similar changes in female H. burtoni, regulation occurs via endogenous rather than exogenous social signals. Our data show that recognition of social signals by males alters stress levels, which may contribute to the alteration in GnRH gene expression in particular neurons essential for the animal to perform in its new social status.

Effects of gonadotropin-releasing hormone (GnRH)-I and -II on regulating the urokinase-type plasminogen activator (uPA) and plasminogen activator inhibitor (PAI-1) in cultured human extravillous cytotrophoblasts

Objective: Implantation in the human is dependent upon the invasion of embryonic trophoblasts into the decidua of the maternal endometrium. The regulated expression of uPA and PAI-1 in the human trophoblasts have been shown to mediate key events in human placenta invasion and placentation. To date, the factors capable of regulating the expression of uPA and PAI-1 in placenta remain poorly characterized. In the present study, we have examined the ability of Gonadotropin-Releasing Hormone (GnRH I) and a novel form of Gonadotropin-Releasing Hormone (GnRH II), that are expressed in the human placenta, to regulate uPA and PAI-1 levels in primary cultures of human extravillous cytotrophoblasts (EVT).

Spatio-temporal expression of gonadotropin-releasing hormone receptor subtypes in gonadotropes, somatotropes and lactotropes in the cichlid fish.

The description of two or more forms of gonadotropin-releasing hormone (GnRH) in most vertebrates suggests multiple roles for this family of peptide hormones. In order to verify these functions, we analysed the anatomical location, time of initial expression and ontogenic changes in three distinct GnRH receptors (GnRH-Rs) in developing and sexually mature tilapia, using antisera raised against the extracellular loop three of the receptor, which is a determinant in ligand-selectivity and receptor coupling to signalling pathways. In all age groups, including males and females, using in situ hybridization and double-label immunological methods, GnRH-R type IA was colocalized in cells containing luteinizing hormone (LH) beta-subunit in the pituitary. GnRH-R type IB was visualized in prolactin cells and LH cells. The type III GnRH-R was expressed in growth hormone cells. On day 8 after fertilization, GnRH-R type III was first seen in growth hormone cells and, subsequently, on day 15, GnRH-Rs type IA and type IB were first seen in LH and prolactin cells, respectively. On day 25, the receptor occupied area per pituitary and the staining intensity of GnRH-R type IA increased significantly, consistent with the hypothesis that differentiation of GnRH neurones and their inputs to the pituitary coincide precisely with gonadal sex differentiation and steroidogenesis in tilapia. The differential distribution of GnRH-Rs in the pituitary provides the first clear evidence that the three native GnRH variants in tilapia have cognate receptors, each capable of regulating different pituitary endocrine cells.

Molecular cloning of three cDNAs encoding different GnRHs in the brain of barfin flounder

To examine the reproductive endocrinology of a large pleuronectiform fish, barfin flounder, Verasper moseri, a promising candidate for aquaculture and resource enhancement in northern Japan due to its high commercial value, three gonadotropin-releasing hormones (GnRHs) in the brain was identified by isolation of their cDNAs. This species had three molecular forms of GnRH; salmon GnRH (sGnRH), chicken GnRH-II (cGnRH-II), and seabream GnRH (sbGnRH). Each GnRH cDNA encoded a signal peptide (SP), GnRH, and a GnRH-associated peptide (GAP), which was connected to GnRH by a Gly–Lys–Arg sequence. The sGnRH cDNA encoded an SP composed of 23 amino acids and a GAP composed of 54 amino acids. The cGnRH-II cDNA encoded an SP of 23 amino acids and a GAP of 49 amino acids. The sbGnRH cDNA encoded an SP of 26 amino acids and a GAP of 57 amino acids. In situ hybridization showed that the genes for sGnRH, cGnRH-II, and sbGnRH are expressed in the ventromedial olfactory bulbs and the terminal nerve ganglion, the midbrain tegmentum, and the preoptic area, respectively. These results indicate that sbGnRH neurons in the preoptic area are involved in gonadotropin secretion in barfin flounder.

Guinea Pig Gonadotropin-Releasing Hormone: Expression Pattern, Characterization and Biological Activity in Rodents

Gonadotropin-releasing hormone (GnRH) is a decapeptide widely known for its role in regulating vertebrate reproduction by serving as a signal from the hypothalamus to pituitary gonadotropes. The first form of GnRH to be identified was isolated from mammals (mGnRH) and the same form has been reported for all mammals studied, which includes marsupials and placental mammals. Later, another variant, chicken GnRH-II (cGnRH-II) was shown to be expressed together with mGnRH in the brains of all jawed vertebrates, including mammals such as rats, monkeys and humans. Our objective was to characterize a third form of GnRH that was isolated previously as mRNA from guinea pigs (gpGnRH), but has not been reported for any other mammal to date. Furthermore, the gonadotropic activity of gpGnRH has not been fully characterized. Our results, using chromatographical and immunological methods, show for the first time that gpGnRH is expressed together with mGnRH in some rodents (wild guinea pig and capybara), but not in others (mouse and hamster). Also, the gonadotropic activity of gpGnRH and mGnRH was tested in two different rat cell culture systems. Although there have been reports that the salmon(s) form of GnRH is present in mammals, we did not detect sGnRH in capybara, wild guinea pigs, hamsters, rats or mice. Taken together with previous reports, the present results support the idea that the expression of multiple GnRH variants in a single species is a common pattern in most vertebrate groups.

Expression of prepro-GnRH and GnRH receptor messengers in rainbow trout ovary depends on the stage of ovarian follicular development.

Gonadotropin-Releasing Hormones (GnRHs) are decapeptides well known to regulate the reproductive cycle. They are expressed not only in the brain, but also in other tissues including the gonads. It is believed that they may be involved in the endocrine and paracrine regulation of the reproductive cycle. To date, two forms of GnRH have been identified in salmonids: salmon (sGnRH) and chicken II (cGnRH-II). In the present study, the temporal expression of sGnRH-1, sGnRH-2, cGnRH-II, and rtGnRH receptor genes was studied in rainbow trout ovary during the reproductive cycle according to the stages of follicular development. Using RT-PCR coupled with Southern-blot hybridization, sGnRH-1, sGnRH-2, cGnRH-II, and rtGnRH-R transcripts were detected in morphologically nondifferentiated ovaries as early as 55-65 days post-fertilization and throughout all stages of vitellogenesis. Using Northern blot analysis, cGnRH-II mRNA was detected only in immature previtellogenic ovary, whereas sGnRH mRNA was detected also during early and mid-exogenous vitellogenesis. No sGnRH mRNA was detected at the end of vitellogenesis. In maturing pre-ovulated ovary, sGnRH transiently reappeared before germinal vesicle breakdown (GVBD) and decreased thereafter. A few days after ovulation, a strong sGnRH mRNA expression was found in ovarian tissue as the eggs were kept in the body cavity of females. However, in females stripped just after ovulation, sGnRH mRNA levels remained low in ovary during several weeks. Fully spliced sGnRH-1 and sGnRH-2 messengers were mostly expressed during the reproductive cycle; however different sGnRH-1 and sGnRH-2 splicing variants containing intronic sequences were also detected. Some of these messengers may encode prepro-GnRH precursors with truncated GnRH-associated peptides. The stage-dependent expression and different cell localization of sGnRH, cGnRH-II, and rtGnRH-R transcripts suggest that GnRH-like peptides may have different roles in the paracrine regulation of ovarian follicular development.

Evolutionary development of three gonadotropin-releasing hormone (GnRH) systems in vertebrates

Gonadotropin-releasing hormone (GnRH) is the neuropeptide that links the brain to the reproductive system. Most vertebrate species express two forms of GnRH, which differ in amino acid sequence, localization, distribution, and embryological origin. The GnRH system in the ventral forebrain produces a species-specific GnRH form and projects toward the gonadotropic cell in the pituitary. The GnRH neurons of this system originate from the olfactory placode and migrate into the brain during early development. The other GnRH system is localized in a nucleus in the midbrain, where large cells express chicken-GnRH-II, of which the function is still unclear. In modern teleosts, a third GnRH system is present in the terminal nerve, which contains salmon GnRH. The three GnRH systems appear at different times during fish evolution. Besides the two accepted lineages in GnRH evolution (of conserved chicken GnRH-II in the midbrain and of mammalian GnRH or species-specific GnRH in the hypophysiotropic system), we propose a third lineage: of salmon GnRH in the terminal nerve.

Chimaeric gonadotropin-releasing hormone (GnRH) peptides with improved affinity for the catfish (Clarias gariepinus) GnRH receptor

The gonadotropin-releasing hormone (GnRH) receptor in catfish differs from its mammalian counterparts in showing a very low affinity for the hypothalamic GnRH form [i.e. catfish GnRH (cfGnRH)] and a very high affinity for the highly conserved mesencephalic GnRH, chicken GnRH-II (cGnRH-II). In the present study we investigated the molecular interactions between ligand and receptor involved in determining the ligand selectivity of the catfish GnRH receptor. Studies on the binding characteristics of the catfish GnRH receptor for cfGnRH and cGnRH-II as well as for mammalian GnRH (mGnRH) and synthetic chimaeric GnRHs, differing at positions 5, 7 and 8, revealed that the low affinity of the catfish receptor for cfGnRH can be improved by replacing Leu7 by a tryptophan residue and/or Asn8 by either a tyrosine or an arginine residue. Testing cfGnRH and cGnRH-II as well as mGnRH and the chimaeric GnRHs on Asp304 → Ala, Asp304 → Glu and Asp304 → Asn mutant catfish GnRH receptors revealed that Asp304 of the catfish receptor mediates the recognition of Arg8 in mGnRH, as well as in the chimaeric peptides [Arg8]cfGnRH and [Arg8]cGnRH-II, but seems to be less important for the recognition of Tyr8 in cGnRH-II. On the basis of these results, a three-dimensional model for the binding of [Arg8]cGnRH-II to the catfish GnRH receptor is proposed.