Gonadotropin-releasing hormone receptor in the teleost Haplochromis burtoni: structure, location, and function

GnRH acts via GnRH receptors (GnRH-R) in the pituitary to cause the release of gonadotropins that regulate vertebrate reproduction. In the teleost fish, Haplochromis burtoni, reproduction is socially regulated through the hypothalamus-pituitary-gonadal axis, making the pituitary GnRH-R a likely site of action for this control. As a first step toward understanding the role of GnRH-R in the social control of reproduction, we cloned and sequenced candidate GnRH-R complementary DNAs from H. burtoni tissue. We isolated a complementary DNA that predicts a peptide encoding a G protein-coupled receptor that shows highest overall identity to other fish type I GnRH-R (goldfish IA and IB and African catfish). Functional testing of the expressed protein in vitro confirmed high affinity binding of multiple forms of GNRH: Localization of GnRH-R messenger RNA using RT-PCR revealed that it is widely distributed in the brain and retina as well as elsewhere in the body. Taken together, these data suggest that this H. burtoni GnRH receptor probably interacts in vivo with all three forms of GNRH:     

Cloning and expression, pharmacological characterization, and internalization kinetics of the pituitary GnRH receptor in a metatherian species of mammal

Gonadotropin-releasing hormone receptors (GnRH-Rs) expressed in the pituitary of eutherian species of mammal are unique in lacking the cytoplasmic C-terminal tail characteristic of GnRH-Rs of nonmammalian vertebrates and other G protein-coupled receptors. To further investigate evolutionary relationships among vertebrate GnRH-Rs, a full-coding region cDNA of the pituitary GnRH-R was cloned from a metatherian marsupial mammal, the Australian brushtail possum (Trichosurus vulpecula). We have determined the pharmacological characteristics and internalization kinetics of this GnRH-R from an early evolved, metatherian species of mammal and compared it with the corresponding receptors in eutherian species of mammal and nonmammalian vertebrates. The predicted GnRH-R protein from the possum pituitary has high homology with the other mammalian GnRH-Rs (80% identity) and, in common with other mammals, lacks an intracellular C-terminal tail. The ligand selectivity of the possum GnRH-R transfected into COS-1 cells, assessed using inositol phosphate assays and radioreceptor binding assays, was similar to that of the other mammalian GnRH-Rs, and distinct from those of the nonmammalian GnRH-Rs. The pharmacological characteristics of the possum GnRH-R were similar to those of other mammalian GnRH-Rs, for a selection of agonists (including naturally occurring GnRH ligands and superagonists) and antagonists. Receptor-mediated internalization of GnRH agonist by the possum GnRH-R was slightly more rapid than that of the human GnRH-R, while the internalization kinetics of the chicken GnRH-R, in which a cytoplasmic C-terminal tail is present, was considerably more rapid. In terms of the evolution of the GnRH-R in vertebrates, the possum (a metatherian mammal) GnRH-R has a striking resemblance, in both structure and pharmacological characteristics, to GnRH-Rs in eutherian mammals, which are quite distinct from the nonmammalian vertebrate GnRH-Rs, and are unique among G protein-coupled receptors in lacking an intracellular C-terminal tail. The distinct structure of the pituitary GnRH-R in mammalian vertebrates is likely to have important functional consequences in the reproductive physiology of mammals.     

Physiological consequences of social descent: studies in Astatotilapia burtoni

In many species, social interactions regulate reproductive capacity, although the exact mechanisms of such regulation are unclear. Since social stress is often related to reproductive regulation, we measured the physiological signatures of change in reproductive state as they relate to short-term stress and the stress hormone cortisol. We used an African cichlid fish, Astatotilapia burtoni, with two distinct, reversible male phenotypes: dominant (territorial, T) males that are larger, more brightly colored, more aggressive, and reproductively competent and non-dominant males (non-territorial, NT) that are smaller, camouflage colored, and have regressed gonads. Male status, and hence reproductive competence, depends on social experience in this system. Specifically, if a T male is placed among larger male fish, it quickly becomes NT in behavior and coloration, but complete regression of its reproductive axis takes ca. 3 weeks (White et al. 2002). Reproduction in all vertebrates is controlled by the hypothalamic-pituitary-gonadal axis in which the key signaling molecule from the brain to the pituitary is GnRH1. Here, we subjected T males to territory loss, a social manipulation which results in status descent. We measured the effects of this status change in levels of circulating cortisol and testosterone as well as mRNA levels of GnRH1 and GnRH receptor-1 (GnRH-R1) in the brain and pituitary, respectively. Following short-term social suppression (4 h), no change was observed in plasma cortisol level, GnRH1 mRNA expression, GnRH-R1 mRNA expression, or plasma testosterone level. However, following a somewhat longer social suppression (24 h), cortisol and GnRH1 mRNA levels were significantly increased, and testosterone levels were significantly decreased. These results suggest that in the short run, deposed T males essentially mount a neural 'defense' against loss of status.     

Pituitary gonadotropin-releasing hormone (GnRH) receptor responses to GnRH in hypothalamus-lesioned rats: inhibition of responses by hyperprolactinemia and evidence that testosterone and estradiol modulate gonadotropin secretion at postreceptor sites

Increased hypothalamic GnRH secretion appears to influence positively the number of pituitary GnRH receptors (GnRH-R). GnRH-R increase after castration in male rats, and this rise can be prevented by testosterone (T), anti-GnRH sera, or hypothalamic lesions. GnRH also increases serum LH and GnRH-R in hypothalamus-lesioned rats, and these animals injected with exogenous GnRH are, therefore, a good model in which to study the site of steroid feedback at the pituitary level. Adult male and female rats were gonadectomized, and radiofrequency lesions were placed in the hypothalamus. Males received T implants, and females received estradiol implants at the time of surgery. Empty capsules were placed in the control animals. Beginning 3-5 days later, animals in each group were injected every 8 h with vehicle (BSA) or GnRH (0.002-200 micrograms/day) for 2 days. After these GnRH injections, all rats received 6.6 micrograms GnRH, sc, 1 h before decapitation to determine acute LH and FSH responses. GnRH-R were determined by saturation analysis using 125I-D-Ala6-GnRH ethylamide as ligand. In males, GnRH injections increased GnRH-R. T inhibited acute LH and FSH responses to GnRH in all groups, but had little effect on GnRH-R, indicating that T inhibits gonadotropin secretion at a post-GnRH receptor site. In females, the GnRH-R response to GnRH was less marked, and only the 200 micrograms/day dose of GnRH increased GnRH-R, indicating that the positive feedback effects of estradiol at the pituitary level are also exerted at a site distal to the GnRH receptor. There was no positive correlation between the number of GnRH-R and GnRH-stimulated gonadotropin release in males or females. Female rats with hypothalamic lesions had markedly elevated serum PRL levels (greater than 300 ng/ml). Suppression of PRL secretion by bromocryptine resulted in augmented GnRH-R responses to GnRH, and GnRH-R concentrations rose to the same values induced in males. This suggests that hyperprolactinemia inhibits GnRH-R responses to GnRH in females by a direct action on the pituitary gonadotroph.     

Implication of dopaminergic systems on GnRH and GnRHR genes expression in the hypothalamus and GnRH-R gene expression in the anterior pituitary gland of anestrous ewes.

We examined by Real-time PCR how prolonged inhibition of dopaminergic D-2 receptors (DA-2) in the hypothalamus of anestrous ewes by infusion of sulpiride into the third cerebral ventricle affected GnRH and GnRH-R gene expression in discrete parts of this structure and GnRH-R gene expression in the anterior pituitary. Blockaded DA-2 receptors significantly decreased GnRH mRNA levels in the ventromedial hypothalamus but did not evidently affect GnRH mRNA in the preoptic/ anteriorhypothalamicarea. Blockaded DA-2 receptors led to different responses in GnRH-R mRNA in various parts of the hypothalamus; increased GnRH-R mRNA levels in the preoptic/anterior hypothalamic area, and decreased GnRH-R mRNA amounts in the ventromedial hypothalamus stalk/median eminence. An infusion of sulpiride into the III-rd ventricle increased GnRH mRNA levels in the anterior pituitary gland and LH secretion. It is suggested that the increase of GnRH gene expression in the anterior pituitary gland and LH secretion in sulpiride-treated ewes are related with an increase of biosynthesis GnRH with concomitant decreased biosynthesis of GnRH-R protein in the ventromedial hypothalamus/stalk median eminence allowing to an increase of GnRH release.     

The central effect of beta-endorphin and naloxone on the expression of GnRH Gene and GnRH receptor (GnRH-R) gene in the hypothalamus, and on GnRH-R gene in the anterior pituitary gland in follicular phase ewes.

The effect of prolonged intermittent infusion of beta-endorphin or naloxone into the third cerebral ventricle in ewes during the follicular phase of the estrous cycle on the expression of GnRH gene and GnRH-R gene in the hypothalamus and GnRH-R gene in the anterior pituitary gland was examined by Real time-PCR. Activation of micro opioid receptors decreased GnRH mRNA levels in the hypothalamus and led to complex changes in GnRH-R mRNA: an increase of GnRH-R mRNA in the preoptic area, no change in the anterior hypothalamus and decrease in the ventromedial hypothalamus and stalk/median eminence. In beta-endorphin treated ewes the levels of GnRH-R mRNA in the anterior pituitary gland also decreased significantly. These complex changes in the levels of GnRH mRNA and GnRH-R mRNA were reflected in the decrease of LH secretion. Blockade of micro opioid receptors affected neither GnRH mRNA and GnRH-R mRNA nor LH levels secretion. These results indicate that beta-endorphin displays a suppressive effect on the expression of the GnRH gene in the hypothalamus and GnRH-R gene in the anterior pituitary gland, but affects GnRH-R gene expression in a specific manner in the various parts of hypothalamus; altogether these events lead to the decrease in GnRH/LH secretion.     

Identification and characterization of two distinct GnRH receptor subtypes in a teleost, the medaka Oryzias latipes

We report the identification and characterization of two distinct GnRH receptor (GnRH-R) subtypes, designated GnRH-R1 and GnRH-R2, in a model teleost, the medaka Oryzias latipes. These seven-transmembrane receptors of the medaka contain a cytoplasmic C-terminal tail, which has been found in all other nonmammalian GnRH-Rs cloned to date. The GnRH-R1 gene is composed of three exons separated by two introns, whereas the GnRH-R2 gene has an additional intron and therefore consists of four exons and three introns. The GnRH-R1 and GnRH-R2 genes, both of which exist as single-copy genes in the medaka genome, were mapped to linkage groups 3 and 16, respectively. Inositol phosphate assays using COS-7 cells transfected with GnRH-R1 and GnRH-R2 demonstrated that they had remarkably different ligand sensitivities, although both receptors showed highest preference for chicken-II-type GnRH. Phylogenetic analysis showed the presence of three paralogous lineages for vertebrate GnRH-Rs and indicated that neither GnRH-R1 nor GnRH-R2 is the medaka ortholog to mammalian GnRH-Rs that lack a cytoplasmic tail. This, together with an observation that medaka-type GnRH had low affinity for GnRH-R1 and GnRH-R2, suggests that a third GnRH-R may exist in the medaka.     

Four gonadotropin releasing hormone receptor genes in Atlantic cod are differentially expressed in the brain and pituitary during puberty

Gonadotropin releasing hormones (GnRH) are an important part of the brain-pituitary-gonad axis in vertebrates. GnRH binding to its receptors (GnRH-R) stimulates synthesis and release of gonadotropins in the pituitary. GnRH-Rs also mediate other processes in the central nervous system such as reproductive behavior and neuromodulation. As many as five GnRH-R genes have been identified in two teleost fish species, but the function and phylogenetic relationship of these receptors is not fully understood. To gain a better understanding of the functional relationship between multiple GnRH-Rs in an important aquaculture species, the Atlantic cod (Gadus morhua), we identified four GnRH-Rs (gmGnRH-R) by RT-PCR, followed by full-length cloning and sequencing. The deduced amino acid sequences were used for phylogenetic analysis to identify conserved functional motifs and to clarify the relationship of gmGnRH-Rs with other vertebrate GnRH-Rs. The function of GnRH-R variants was investigated by quantitative PCR gene expression analysis in the brain and pituitary of female cod during a full reproductive cycle and in various peripheral tissues in sexually mature fish. Phylogenetic analysis revealed two types of teleost GnRH-Rs: Type I including gmGnRH-R1b and Type II including gmGnRH-R2a, gmGnRH-R2b and gmGnRH-R2c. All four gmGnRH-Rs are expressed in the brain, and gmGnRH-R1b, gmGnRH-R2a and gmGnRH-R2c are expressed in the pituitary. The only GnRH-R differentially expressed in the pituitary during the reproductive cycle is gmGnRH-R2a such that its expression is significantly increased during spawning. These data suggest that gmGnRH-R2a is the most likely candidate to mediate the hypophysiotropic function of GnRH in Atlantic cod.     

Differential gonadotropin-releasing hormone (GnRH) and GnRH receptor messenger ribonucleic acid expression patterns in different tissues of the female rat across the estrous cycle

GnRH, the main regulator of reproduction, is produced in a variety of tissues outside of the hypothalamus, its main site of synthesis and release. We aimed to determine whether GnRH produced in the female rat pituitary and ovaries is involved in the processes leading to ovulation. We studied the expression patterns of GnRH and GnRH receptor (GnRH-R) in the same animals throughout the estrous cycle using real-time PCR. Hypothalamic levels of GnRH mRNA were highest at 1700 h on proestrus, preceding the preovulatory LH surge. No significant changes in the level of hypothalamic GnRH-R mRNA were detected, although fluctuations during the day of proestrus are evident. High pituitary GnRH mRNA was detected during the day of estrus, in the morning of diestrus 1, and at noon on proestrus. Pituitary GnRH-R displayed a similar pattern of expression, except on estrus, when its mRNA levels declined. Ovarian GnRH mRNA levels increased in the morning of diestrus 1 and early afternoon of proestrus. Here, too, GnRH-R displayed a somewhat similar pattern of expression to that of its ligand. To the best of our knowledge, this is the first demonstration of a GnRH expression pattern in the pituitary and ovary of any species. The different timings of the GnRH peaks in the three tissues imply differential tissue-specific regulation. We believe that the GnRH produced in the anterior pituitary and ovary could play a physiological role in the preparation of these organs for the midcycle gonadotropin surge and ovulation, respectively, possibly via local GnRH-gonadotropin axes.     

Homologous ligand induction of pituitary gonadotrophin-releasing hormone receptors in vivo is protein synthesis dependent

This study demonstrates that a single subcutaneous injection of gonadotrophin-releasing hormone (GnRH) (60 ng) to GnRH-deficient (hpg) male mice causes a doubling of pituitary GnRH receptors (GnRH-R). No change in GnRH-R occurs during the time of LH release (15-60 min) or up until 4 h post-GnRH. Between 4 and 12 h there is a progressive increase in GnRH-R, which is still apparent 24 h later. No induction of GnRH-R occurs after the same treatment of intact adult normal mice. The same degree of GnRH-R induction occurs 12 h after a single GnRH injection (60 ng) to orchidectomized hpg male mice, indicating that this effect is mediated by a direct action of GnRH on the pituitary gonadotroph, rather than being secondary to stimulation of some gonadal product. Homologous ligand GnRH-R induction in hpg mouse pituitaries in vivo is prevented by prior treatment with cycloheximide, a non-specific protein synthesis inhibitor. Cycloheximide alone had no effect on GnRH-R in normal male mice but when combined with GnRH caused a 40% depletion of receptors, implying ligand-induced receptor loss without subsequent replenishment. The similarity between the extent, time-course, and dependence on protein synthesis of GnRH induction of its own receptors in vivo and in cultured pituitary cells in vitro indicates that the hpg mouse pituitary behaves like an in vivo pituitary cell culture system in this respect. Similarity of data derived from this in vivo model provides direct support for the view that in vitro studies on the cellular mechanism of GnRH action can be physiologically relevant to the intact animal.     

Pituitary gonadotropin-releasing hormone receptor regulation in the hypogonadotrophic hypogonadal (hpg) mouse

Recent evidence indicates that endogenous GnRH is required for maintenance of its own pituitary receptors (GnRH-R). We have measured GnRH-R in pituitaries of hypogonadotrophic hypogonadal (hpg) mice, in whom hypothalamic GnRH is deficient or absent. The GnRH-R concentration in hpg male mouse pituitaries was 10.6 +/- 1 fmol/pituitary vs. 30.9 +/- 1 fmol/pituitary in normal male littermate pituitaries. Similarly, GnRH-R in female hpg mice (15.2 +/- 1.7 fmol/pituitary) were 30% those of normal random cycling females (51.4 +/- 3.5 fmol/pituitary). There was no difference in receptor affinity (Ka = 1.5-3 C 10(9) M-1) of hpg mouse pituitaries. The pituitary LH content in hpg male and female mice was very similar (range 3.4-4.8 micrograms/pituitary) representing 5% and 19% of normal male (95 +/- 7.2 micrograms/pituitary) and female (18.1 +/- 1.5 micrograms/pituitary) values, respectively. The administration of 50 ng GnRH sc 10 times daily to male hpg mice, increased GnRH-R to 80% of normal values within 3 days. Serum FSH and pituitary FSH content rose to normal male values after 7 days of GnRH injections. However, serum LH remained undetectable and pituitary LH reached only 20% of normal male levels, even after 15 days of GnRH administration. Treatment of hpg male mice with 60 ng GnRH either once daily for 6 days, or 12 times daily for 5 days, increased GnRH-R to 50% of normal male values. Twelve daily injections of GnRH elevated serum FSH to above the normal male range, whereas daily GnRH only doubled untreated hpg levels. Pituitary FSH was stimulated to 50% of normal with 12 daily injections, whereas once daily administration elevated pituitary FSH to 30% of normal values. Both pulsatile regimes depleted pituitary LH. These data demonstrate that: 1) despite absence of bioactive GnRH, GnRH-R values are only reduced to 30% of normal in hpg mouse pituitaries, suggesting that little, if any, endogenous GnRH is required for expression of GnRH receptors. 2) Pituitary GnRH-R number rapidly increase when GnRH is administered to hpg male mice indicating that, as in the rat, GnRH positively regulates its own receptor concentration. 3) The pituitary FSH and LH responses to GnRH treatment in hpg mice depends to a different extent on the frequency and duration of GnRH administration. 4) The hpg mouse provides an ideal animal model for investigating the interaction of defined regiments of exogenous GnRH and gonadal steroids on pituitary GnRH receptor and gonadotroph function.     

Adenosine 3',5'-monophosphate derivatives increase gonadotropin-releasing hormone receptors in cultured pituitary cells

In this study the GnRH receptors (GnRH-R) in cultured rat pituitary cells were examined after treatment with GnRH and cyclic nucleotide derivatives. GnRH at doses of between 10(-11) and 10(-8) M caused GnRH-R increases, 10(-9) M resulting in a 50% stimulation of both GnRH-R and LH release. (Bu)2cAMP increased GnRH-R in a dose dependent manner, with a maximal 2-fold increase at 1 mM, with no effect on LH release in serum-containing medium. The time-course of both the GnRH and (Bu)2cAMP stimulation of GnRH-R was similar, with maximal levels being reached between 6-12 h. There was no difference in the GnRH receptor affinity subsequent to either GnRH or (Bu)2cAMP treatment. GnRH-R increases of 70% were observed when pituitary cells were treated with 1 mM cAMP and 8- bromocAMP , but n-butyric acid, adenosine, and cyclic guanosine monophosphate (all 1 mM) were not effective. Fifty eight millimolar KCl resulted in a 2-fold elevation of GnRH-R. Isobutylmethylxanthine (0.2 mM) did not affect basal receptor levels and slightly enhanced the GnRH-and potassium-stimulated increase of GnRH-R, whereas the increase caused by (Bu)2cAMP was completely prevented. Simultaneous treatment of cultured pituitary cells with either GnRH, KCl, or (Bu)2cAMP and cycloheximide completely prevented GnRH-R increases, while not affecting either basal or GnRH and KCl-stimulated LH secretion. None of the cyclic nucleotides stimulated LH release under the culture conditions employed to examine receptor regulation. However, when incubated in medium not containing serum, both (Bu)2cAMP and cyclic guanosine monophosphate stimulated significant LH release. When the pituitary cells were treated with GnRH in medium without serum, no increase in GnRH-R was measured, although LH release was unaffected. However, absence of serum in the medium did not affect either the K+ or (Bu)2cAMP stimulation of GnRH-R. The GnRH antagonist ( DpGlu1 , D-Phe2, D-Trp3,6) GnRH (1 microM) prevented both GnRH- and (Bu)2cAMP-induced increases in GnRH-R, although not that of 58 mM KCl. The antagonist also inhibited GnRH-stimulated LH secretion, although not that caused by KCl.(ABSTRACT TRUNCATED AT 400 WORDS)     

Pituitary gonadotropin-releasing hormone receptor regulation in mice. I: Males

The regulation of pituitary GnRH receptors (GnRH-R) has been examined in male mice (C3H/HeH/101H F1 hybrid) after castration and testosterone replacement. GnRH-R were quantified in individual mouse pituitaries by equilibration with 125I(D-Ser(tBut)6) des Gly10 GnRH N ethylamide and compared with serum and pituitary LH and FSH concentrations. The equilibrium association constant was 2.7 X 10(9) M-1 for both intact and castrated male mouse pituitary GnRH-R. Six hours after orchidectomy there was a transient 50% reduction in GnRH-R; 13.6 +/- 3.8 fmol/pituitary (castrate) vs. 25.4 +/- 2.5 (intact). A subsequent partial return of binding sites began at 12 h, reaching a peak value of 18.2 +/- 1.5 fmol/pituitary (33% increase vs. 6 h) at 24-h post orchidectomy. This was followed by a gradual decrease in GnRH-R, reaching a plateau by 72 h. The decrease in GnRH-R was associated with a rapid (6-12 h) increase in serum LH and serum FSH. The pituitary GnRH-R concentration remained 45% below intact control values for up to 3 months and was accompanied by a persistent 5-fold rise in serum LH values. Treatment of male mice with testosterone propionate (TP), 25 micrograms/day, completely prevented the GnRH-R fall and the serum and pituitary LH responses to castration, whereas 12.5 micrograms/day TP produced variable results and 5 micrograms/day TP were ineffective. In another strain of mouse (BALB/c white). GnRH-R values also fell by 66% at 7 days post orchidectomy, with no change in the receptor affinity. In mice with androgen resistance from birth due to absence of androgen receptors (Tfm mice), GnRH-R were 14.45 +/- 0.49 vs. 19.8 +/- 1.67 fmol/pituitary in normal male littermates, and serum LH was 472 +/- 78 ng/ml compared with 52.5 +/- 11.7 ng/ml in normals. These findings are qualitatively similar to those in orchidectomized normal adult mice. Thus, in contrast to reports in rats, pituitary GnRH-R content falls after orchidectomy in mice. Possible explanations for this consistent finding include: persistent receptor occupancy by increased endogenous GnRH secretion, endogenous GnRH-induced receptor loss (down-regulation), or a species difference in the pituitary GnRH-R response to removal of negative steroid feedback, unrelated to changes in endogenous GnRH secretion.     

The frequency of gonadotropin-releasing hormone stimulation determines the number of pituitary gonadotropin-releasing hormone receptors

Gonadotropin-releasing hormone (GnRH) induces both synthesis and release of pituitary gonadotropins, but rapid or slow frequencies of stimulation result in reduced LH and FSH secretion. We determined the effects of frequency of GnRH stimulation on pituitary GnRH receptors (GnRH-R). Castrate male rats received testosterone implants (cast + T) to inhibit endogenous GnRH secretion. GnRH pulses were injected by a pump into a carotid cannula and animals received GnRH (25 ng/pulse) at various frequencies for 48 h. In control animals (saline pulses) GnRH-R was 307 +/- 21 fmol/mg protein (+/- SE) in cast + T and 598 +/- 28 in castrates. Maximum GnRH-R was produced by 30-min pulses and was similar to that seen in castrate controls. Faster or slower frequencies resulted in a smaller GnRH-R response and GnRH given every 240 min did not increase GnRH-R over saline controls. Equalization of the total GnRH dose/48 h (6.6 ng/pulse every 7.5 min or 200 ng/pulse every 240 min) did not increase receptors to the maximum concentrations seen after 30-min (25 ng) pulses. Serum LH responses after 48 h of injections were only present after 30-min pulses, and peak FSH values were also seen after this frequency. Serum LH was undetectable in most rats after other GnRH frequencies, even though GnRH-R was increased. These data show that GnRH pulse frequency is an important factor in the regulation of GnRH-R. A reduction of GnRH-R is part of the mechanism of down-regulation of LH secretion by fast or slow GnRH frequencies, but altered frequency also exerts effects on secretory mechanisms at a site distal to the GnRH receptor.     

Desensitization and internalization of human and xenopus gonadotropin-releasing hormone receptors expressed in alphaT4 pituitary cells using recombinant adenovirus

Nonmammalian vertebrates express at least two forms of GnRH and distinct forms of GnRH receptor (GnRH-R) have coevolved with their ligands. Mammalian and nonmammalian GnRH-R have key structural differences (notably the lack of C-terminal tails in mammalian GnRH-R) and comparative studies are beginning to reveal their functional relevance. However, cellular context and receptor density influence G protein-coupled receptor function and may be important variables in such work using heterologous expression systems. Here we report a comparative study using alphaT4 cells (gonadotrope progenitors that lack endogenous GnRH-R) transfected with a mammalian (human) or nonmammalian (Xenopus laevis type I) GnRH-R. Because conventional transfection strategies proved inefficient, recombinant adenovirus expressing these receptors were constructed, enabling controlled and efficient GnRH-R expression. When expressed in alphaT4 cells at physiological density, these GnRH-Rs retain the pharmacology of their endogenous counterparts (as judged by ligand specificity in radioligand binding and inositol phosphate accumulation assays) but do not activate adenylyl cyclase and are not constitutively active. Moreover, the Xenopus GnRH-R rapidly desensitizes and internalizes in these cells, whereas the human GnRH-R does not, and the internalization rates are not dependent upon receptor number. These data extend studies in COS, HEK, and GH3 cells showing that other GnRH-R with C-terminal tails desensitize and internalize rapidly, whereas tail-less mammalian GnRH-R do not. Retention of these distinctions at physiological receptor density in gonadotrope lineage cells, supports the argument that the evolution of nondesensitizing mammalian GnRH-Rs is functionally relevant and related to the development of mammalian reproductive strategies.     

Daily GnRH and GnRH-receptor mRNA expression in the ovariectomized and intact rat

We recently described patterns of GnRH and GnRH receptor (GnRH-R) expression in the hypothalamus, pituitary and ovary throughout the rat estrus cycle. Here, we wished to distinguish between regulatory effects of ovarian factors and underlying circadian rhythmicity. We quantified GnRH and GnRH-R mRNA in the pituitary and hypothalamus of long-term ovariectomized (OVX) rats, at different times of day, using real-time PCR. Furthermore, we expanded our previous study of hypothalamic and pituitary GnRH and GnRH-R expression in intact rats by including more time points throughout the estrus cycle. We found different daily patterns of GnRH and GnRH-R expression in intact versus OVX rats, in both tissues. In the hypothalamus of OVX rats, GnRH mRNA peaked at 12, 16 and 20 h, whereas in the hypothalamus of intact rats we observed somewhat higher GnRH mRNA concentrations at 19 h on every day of the estrus cycle except proestrus, when the peak occurred at 17 h. In this tissue, GnRH-R fluctuated less significantly and peaked at 16 h in OVX rats. During the estrus cycle, we observed higher levels in the afternoon of each day except on estrus. In OVX rats, pituitary GnRH mRNA rose sharply at 9 h, with low levels thereafter. In these animals, pituitary GnRH-R also peaked at 9h followed by a second rise at 22 h. In intact rats pituitary GnRH was high at noon of diestrus-II and on estrus, whereas GnRH-R mRNA was highest in the evening of diestrus-II. This is the first demonstration of daily GnRH and GnRH-R mRNA expression patterns in castrated animals. The observed daily fluctuations hint at underlying tissue-specific circadian rhythms. Ovarian factors probably modulate these rhythms, yielding the observed estrus cycle patterns.     

LH down-regulates gonadotropin-releasing hormone (GnRH) receptor, but not GnRH, mRNA levels in the rat testis.

The demonstration of an inhibitory effect of gonadotropin-releasing hormone (GnRH) agonists upon steroidogenesis in hypophysectomized rats and the presence of mRNA coding for GnRH and GnRH receptors (GnRH-R) in rat gonads suggests that GnRH can act locally in the gonads. To assess this hypothesis, we investigated the effects of GnRH analogs, gonadotropins and testosterone on the levels of both GnRH and GnRH-R mRNA in the rat testis. Using dot blot hybridization, we measured the mRNA levels 2 to 120 h after the administration of the GnRH agonist, triptorelin. We observed an acute reduction of both GnRH and GnRH-R mRNAs 24 h after the injection (about 38% of control). However, the kinetics for testis GnRH-R mRNA were different from those previously found for pituitary GnRH-R mRNA under the same conditions. Initially, the concentrations of serum LH and FSH peaked, then declined, probably due to the desensitization of the gonadotrope cells. In contrast, the GnRH antagonist, antarelix, after 8 h induced a 2.5-fold increase in GnRH-R mRNA, but not in GnRH mRNA, while gonadotropins levels were reduced. Human recombinant FSH had no significant effect on either GnRH or GnRH-R mRNA levels. Inversely, GnRH-R mRNA levels markedly decreased by 21% of that of control 24 h after hCG injection. Finally, 24 h after testosterone injection, a significant increase in GnRH-R mRNA levels (2.3 fold vs control) was found, but a reduction in the concentration of serum LH, probably by negative feedback on the pituitary, was observed. In contrast, GnRH mRNA levels were not significantly altered following testosterone treatment. Since LH receptors, GnRH-R and testosterone synthesis are colocalized in Leydig cells, our data suggest that LH could inhibit the GnRH-R gene expression or decrease the GnRH-R mRNA stability in the testis. However, this does not exclude the possibility that GnRH analogs could also affect the GnRH-R mRNA levels via direct binding to testicular GnRH-R. In contrast, the regulation of GnRH mRNA levels appeared to be independent of gonadotropins. Taken together, our results suggest a regulation of GnRH and GnRH-R mRNA specific for the testis.