The effect of dietary protein restriction on the secretion of LH and FSH in pre-pubertal female lambs.

The effect of restricted dietary protein on the synthesis, storage and release of LH and FSH was studied in pre-pubertal female lambs. The experiment started when the lambs were aged 12 weeks and weighed 26.0+/-1.6 kg. It was conducted for 25 weeks. The lambs were fed isocaloric diets containing either a restricted level of crude protein (8% CP; n=6; treatment R) or an elevated one (18% CP; n=4; treatment E). At 37 weeks of age and before the first oestrous cycle, blood samples were collected over 6 h at 10 min intervals for LH assay. The lambs were slaughtered and their brains recovered and fixed in situ. Immuno-reactive (IR) LH and FSH cells were localised by immunohistochemistry techniques. Messenger RNA analyses used by non-isotope in situ hybridisation with sense and anti-sense riboprobes from beta subunits of LH and FSH cDNA clones. Data were generated using computer analysis to measure the proportion of IR and/or hybridising cells and their optical density for immuno-staining and hybridisation signal. Plasma LH was measured by RIA. The daily live-weight gains were 56.5+/-13.1 g and 97.8+/-14.3 g for R and E lambs, respectively (P<0.05), so that final weights at slaughter were 36.1+/-1.97 kg and 39.1+/-3.44 kg, respectively (P<0.05). The number of cells expressing LH beta mRNA and the optical density of this hybridisation signal was significantly (P<0.001) lower in the R lambs but the number of IR LH positive cells was higher (P<0.001) than for the E lambs. The concentration of LH in the plasma of R sheep was lower (P<0.05) than the E group and this response was associated with a decrease (P<0.05) in LH pulse frequency and amplitude. Dietary protein concentration appeared to have no effect on the IR in FSH cells or on the expression of FSH beta mRNA. In summary, the low protein diet influenced the body weight and weight gain of growing lambs and exerted an inhibitory effect on the synthesis and the release of LH in the pituitary gonadotrophs. No such effect was observed for FSH. It was concluded that the protein concentration of the diet consumed during the growth of female lambs may be an important modulator of processes leading to the pre-pubertal rise in LH.

Isolation and characterization of a rat nitric oxide synthase type I gene promoter that confers expression and regulation in pituitary gonadotrope cells.

Nitric oxide synthase type I (NOS I) is expressed and up-regulated in rat pituitary gonadotrophs. Using rapid amplification of cDNA ends-PCR, 2 major transcripts with 5' ends corresponding to exon 1a but truncated of its first 369 or 384 nucleotides, indicative of two pituitary-specific transcription start sites, were identified. By chromosome walking, we isolated 5'-upstream of this truncated exon termed 1p, a novel -1653/+384-bp genomic region. Transient transfections, using the gonadotrope-derived alpha T3-1 and L beta T2 cell lines and the full-length or 5'-deleted sequences fused to a luciferase reporter gene, demonstrated that cell-specific positive and negative regions were present especially within the -246/-73 region, whereas the +12/+384 region was crucial for transcription. Moreover, in L beta T2 cells, the luciferase activity was increased by GnRH, with the full-length sequence being the most efficient and the -73/+60 region corresponding to the essential zone. The latter region was also crucial for cholera toxin-induced activation. Interestingly, GnRH and cAMP effects were not additive, implying a convergent step in the transduction cascade. These data provide evidence for the presence of several elements controlling NOS I expression in gonadotrophs and demonstrate that GnRH, the prime regulator of gonadotrope function, and cAMP may induce the transcription of NOS I in these cells.

Critical implication of transmembrane Phe310, possibly in conjunction with Trp279, in the rat gonadotropin-releasing hormone receptor activation.

The GnRH-R belongs to the superfamily of heptahelical GPCRs. A three-dimensional model of GnRH binding to its receptor predicted that Trp3 was the most deeply buried residue, potentially allowing it to interact with both Trp279, a highly conserved residue in the TMH 6 of GPCRs, and Phe310, present essentially in TMH 7 of GnRH-Rs. Replacement of Phe310 with Leu, the most common positional residue in GPCRs, induced a slightly decreased Bmax (1.6-fold) and affinity (3.8-fold); in addition, IP production was completely abolished. Similarly, replacement of Trp279 with Ser depressed the Bmax by 5.2-fold, the affinity by 2.3-fold, and totally abrogated IP production. The effect of the double mutation was not additive on binding, since the Bmax was reduced to the level of the Phe310Leu mutant, although the Kd was restored to a value not significantly different from that of the wild-type. The double mutant was also unable to induce IP production. Unexpectedly, no influence of any single or double substitution was noted on receptor internalization. These data provide evidence for the crucial role of Phe310, possibly in conjunction with Trp279, on GnRH transduction and suggest that the conformation for phospholipase C activation may not be required for GnRH-R internalization.

Pituitary adenylate cyclase-activating polypeptide and cyclic adenosine 3',5'-monophosphate stimulate the promoter activity of the rat gonadotropin-releasing hormone receptor gene via a bipartite response element in gonadotrope-derived cells.

Specific type I receptors for pituitary adenylate cyclase-activating polypeptide (PACAP) are present in gonadotrope cells of the anterior pituitary gland. By transient transfection of mouse gonadotrope-derived alphaT3-1 cells, which are direct targets for PACAP and express gonadotropin-releasing hormone receptor (GnRH-R), a marker of the gonadotrope lineage, we provide the first evidence that PACAP stimulates rat GnRH-R gene promoter activity. The EC(50) of this stimulation is compatible with a mediation via activation of the cyclic AMP-dependent signaling pathway and, consistently, co-transfection of an expression vector expressing the protein kinase A inhibitor causes reduction in PACAP as well as cholera toxin-stimulated promoter activity. Deletion and mutational analyses indicate that PACAP activation necessitates a bipartite response element that consists of a first region (-272/-237) termed PACAP response element (PARE) I that includes a steroidogenic factor-1 (SF-1)-binding site and a second region (-136/-101) referred to as PARE II that contains an imperfect cyclic AMP response element. Gel shift experiments indicate the specific binding of the SF-1 and a potential SF-1-interacting factor to PARE I while a protein immunologically related to the cyclic AMP response element-binding protein interacts with PARE II. These findings suggest that PACAP might regulate the GnRH-R gene at the transcriptional level, providing novel insights into the regulation of pituitary-specific genes by hypothalamic hypophysiotropic signals.

Proximal cis-acting elements, including steroidogenic factor 1, mediate the efficiency of a distal enhancer in the promoter of the rat gonadotropin-releasing hormone receptor gene.

The gonadotrope-specific and regulated expression of the GnRH receptor (GnRH-R) gene is dependent on multiple transcription factors that interact with the noncanonical GnRH-R activating sequence (GRAS), the activator protein-1 (AP-1) element, and the steroidogenic factor-1 (SF-1) binding site. However, these three elements are not sufficient to mediate the complete cell-specific expression of the rat GnRH-R gene. In the present study, we demonstrate, by transient transfection in gonadotrope-derived alphaT3-1 and LssT2 cell lines, the existence of a distal enhancer [GnRH-R- specific enhancer (GnSE)] that is highly active in the context of the GnRH-R gene promoter. We show that the GnSE activity (-1,135/-753) is mediated through a functional interaction with a proximal region (-275/-226) that includes the SF-1 response element. Regions of similar length containing either the AP-1 or GRAS elements are less active or inactive. Transfection assays using an artificial promoter containing two SF-1 elements fused to a minimal PRL promoter indicate that SF-1 is crucial in this interaction. In addition, by altering the promoter with deletion and block- replacement mutations, we have identified the active elements of GnSE within two distinct sequences at positions -983/-962 and -871/-862. Sequence analysis and electrophoretic mobility shift experiments suggest that GnSE response elements interact, in these two regions, with GATA- and LIM-related factors, respectively. Altogether, these data establish the importance of the GnSE in the GnRH-R gene expression and reveal a novel role for SF-1 as a mediator of enhancer activity, a mechanism that might regulate other SF-1 target genes.

Modulation of luteinizing hormone subunit gene expression by intracerebroventricular microinjection of gonadotropin-releasing hormone or beta-endorphin in female rats.

The effects of gonadotropin-releasing hormone (GnRH), beta-endorphin and its antagonist naloxone on the expression of luteinizing hormone (LH) subunit genes and LH secretion were examined in ovariectomized and/or cycling female rats through their direct microinjection into the third cerebral ventricle, in the proximity of the hypothalamus-pituitary complex. GnRH (1 nM) induced a significant augmentation of the pituitary content of alpha mRNA when administered 15, 30 or 60 min intervals over 5 h to ovariectomized rats whereas only the 30 and 60 min intervals were effective in increasing LHbeta mRNA, and the 60 min intervals for LH release. This was in agreement with the established concept of a pulse-dependent regulation of gonadotropin synthesis and release. Hourly pulses of GnRH also increased alpha and LHbeta mRNA levels when microinjected in female cycling rats during proestrus or diestrus II. Using this model we observed a marked negative influence of hourly intracerebral microinjections of beta-endorphin on LH mRNA content and LH release in ovariectomized rats while naloxone had no effect. This suggests that endogenous beta-endorphin was unable to exert its negative action on beta-endorphin receptors that were present and responded to the ligand. The present approach would be valuable for the exploration of the mechanisms of action of beta-endorphin or other substances on the functions of the gonadotrophs.

A new compound heterozygous mutation of the gonadotropin-releasing hormone receptor (L314X, Q106R) in a woman with complete hypogonadotropic hypogonadism: chronic estrogen administration amplifies the gonadotropin defect.

We describe a woman with complete hypogonadotropic hypogonadism and a new compound heterozygous mutation of the GnRH receptor (GnRHR) gene. A null mutation L314X leading to a partial deletion of the seventh transmembrane domain of the GnRHR is associated with a Q106R mutation previously described. L314X mutant receptor shows neither measurable binding nor inositol phosphate production when transfected in CHO-K1 cells compared to the wild-type receptor. The disease is transmitted as an autosomal recessive trait, as shown by pedigree analysis. Heterozygous patients with GnRHR mutations had normal pubertal development and fertility. The present study shows an absence of LH and FSH response to pulsatile GnRH administration (20 microg/pulse, sc, every 90 min). However, GnRH triggered free alpha-subunit (FAS) pulses of small amplitude, demonstrating partial resistance to pharmacological doses of GnRH. FSH, LH, and FAS concentrations were evaluated under chronic estrogen treatment and repeat administration of GnRH. Not only were plasma FSH, LH, and FAS concentrations decreased, but FAS responsiveness was reduced. This new case emphasizes the implication of the GnRH receptor mutations in the etiology of idiopathic hypogonadotropic hypogonadism. We also have evidence for a direct negative estrogen effect on gonadotropin secretion at the pituitary level, dependent on the GnRHR signaling pathway.

Functional importance of transmembrane helix 6 Trp(279) and exoloop 3 Val(299) of rat gonadotropin-releasing hormone receptor.

Previous studies have established that the interaction of gonadotropin-releasing hormone (GnRH) with its receptor (GnRHR) would require partial entry of the N- and C-terminal regions of ligand into the transmembrane core. The functional significance of the conserved aromatic residue Trp(279) present in the transmembrane helix 6, and Val(299) located in exoloop 3 of the rat GnRHR was investigated by mutagenesis followed by expression in Chinese hamster ovary-K1 cells. Compared with wild-type, substitution of Trp(279) with Ser or Arg resulted in a marked reduction or total abolition, respectively, of ligand binding and, in both cases, abrogation of GnRH-induced inositol phosphate production. A total absence of functionality was observed when Val(299) was simply replaced with Ala. Mention should be made that an expression of all mutated and wild-type receptor proteins was observed. Interestingly, the double mutant [Trp(279)Arg/Val(299)Ala]GnRHR restored B(max) to wild type (504 +/- 43 versus 541 +/- 41 fmol/mg protein), but with a diminished affinity (4.95 +/- 1.05 versus 0.94 +/- 0.35 nM), and GnRH failed to induce inositol phosphate. No influence of the mutations was seen on internalization of the receptor. The three-dimensional model of GnRH binding to the rat GnRHR was built predicting that Trp(279) is buried at 20 A in the transmembrane core of the receptor, directly in contact with Trp(3) of GnRH. In contrast, Val(299) is located in a region that cannot be precisely defined at the extracellular end of transmembrane helix 7. Although models cannot provide any clue concerning the observed interactivity between the two distal residues, altogether these data reveal the functional importance of both GnRHR Trp(279) and Val(299) and suggest that Trp(279), interacting with GnRH Trp(3), represents the bottom of the binding pocket.

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.

Tissue-specific pattern of variant transcripts of the human gonadotropin-releasing hormone receptor gene.

The expression pattern of the GnRH receptor was investigated in a variety of normal and neoplastic human tissues by RT-PCR-Southern blotting. In addition to the full-length cDNA (sb1), we identified two other transcripts: the first (sb2) was characterized by a 128 bp deletion as previously described; the second was an unexpected finding composed of a shorter cDNA (sb3), the sequence of which revealed a 220 bp deletion corresponding in size to exon 2. These three transcripts were found in normal pituitary and pituitary adenomas, and in granulosa tumors, but not in testis, where sb2 was lacking. Only sb1 was expressed in normal, fibrocystic and malignant breast tissue. No transcript with a full-length region was found in endometrium, intestine or lymphocytes. This is the first report that shows that splicing of the gonadotropin-releasing hormone receptor gene is tissue dependent. We also determined the intron-exon nucleotide sequence of the gene and identified an MaeIII polymorphic site in exon 1 created by a silent C453T transition found in 10% of unrelated French whites.

Resistance of hypogonadic patients with mutated GnRH receptor genes to pulsatile GnRH administration.

We have studied a kindred with three siblings with isolated hypogonadotropic hypogonadism caused by compound heterozygote mutations in the GnRH receptor gene. The disorder was transmitted as an autosomal recessive trait. The R262Q mutation in intracellular loop 3 of the receptor was associated with a mutation in the third transmembrane domain of the receptor, A129D, that has never been described before. This A129D mutation results in a complete loss of function, indicated by the lack of inositol triphosphate (TP3) 3 production by transfected Chinese hamster ovary (CHO) cells after GnRH stimulation. The two brothers had microphallus and bilateral cryptorchidism and were referred for lack of puberty, whereas their sister had primary amenorrhea and a complete lack of puberty. Their basal gonadotropin concentrations were below the reference range, and their endogenous LH secretory patterns were abnormal, with a low-normal frequency of small pulses or no apparent LH pulse. Pulsatile GnRH administration (10 microg/pulse every 90 min for 40 h) resulted in increased mean LH without any significant changes in testosterone levels in the two brothers, whereas the LH secretory profile of their sister remained apulsatile. Larger pulses of exogenous GnRH (20 microg every 90 min for 24 h) caused the sister to produce recognizable low amplitude LH pulses. The concentrations of free alpha-subunit significantly increased in all patients during the pulsatile GnRH administration. Thus, these hypogonadal patients are partially resistant to pulsatile GnRH administration, suggesting that they should be treated with gonadotropins to induce spermatogenesis or ovulation rather than with pulsatile GnRH.

Mutations of the GnRH receptor gene: a new cause of autosomal-recessive hypogonadotropic hypogonadism.

Mutations in a few genes have been identified in hypogonadotropic hypogonadism (HH): the gene KAL-1 is involved in X-linked Kallmann syndrome associated with anosmia and mutations in transcription factors, namely, DAX-1 and Prop-1 were also evidenced when associated with other pituitary or endocrine defects. Recently, compound heterozygote mutations in the GnRH receptor gene were described both in males and females and hormonal resistance was confirmed in vitro. There is a wide spectrum of phenotype, ranging from complete HH with lack of pubertal development and cryptorchidism to partial hypogonadism with an arrest of pubertal development. In complete GnRH resistance, endogenous LH secretory patterns were abnormal, either apulsatile or characterized by a low-normal pulse frequency with small pulses or erratic pulses of low amplitude. In patients with partial resistance, basal LH plasma concentration was low, but FSH level was in the normal range. LH pulse analysis revealed normal frequency with decreased amplitude. Mutations are distributed along the coding sequence, as reported for other GPCRs. However, two hot-spots, Q106R and the R262Q, were observed, regardless of the geographic origin of the patients. In most cases, patients responded to GnRH administration, making the GnRH test inappropriate for screening GnRH resistance in IHH.

Multiple elements in the distal part of the 1.2 kb 5'-flanking region of the rat GnRH receptor gene regulate gonadotrope-specific expression conferred by proximal domain.

Several lines of evidence indicate that the number of GnRH receptors (GnRH-R) and therefore, gonadotrope responsiveness to GnRH, is highly dependent upon the level of GnRH-R mRNA. To explore this aspect of regulation, we have isolated a 3.3 kb fragment encompassing the promoter region of the rat GnRH-R gene. Primer extension and RNase protection assays allowed the identification of five major transcriptional start sites located within the 110 bp region upstream of the translation start codon. Transfection experiments using the CAT reporter gene demonstrated that the 1261 bp 5' flanking region is required to direct high efficient expression in the gonadotrope-derived alphaT3-1 cell line thus contrasting with mouse in which the only 500 bp proximal sequence appeared to be sufficient. Another difference between rat and mouse was apparent in the 183 bp region of the rat promoter which induced a 3-fold stimulation of thymidine kinase promoter activity in both alphaT3-1 and CHO cells. Subsequent deletion analysis of the region residing between -1261 and -519 revealed the presence of multiple regulatory domains that contributed to the cell-specific activity. However, despite this efficiency in the context of the wild-type promoter, they failed to induce the activity of the minimal thymidine kinase (TK) promoter in the absence of the proximal 600 bp promoter region. Accordingly, a composite TK promoter containing the entire 1.2 kb promoter induced a 10-fold increase in the activity of the TK promoter in alphaT3-1 cells. Taken together these data suggest that distal regulatory regions are critical and require cooperation with proximal specific-promoter elements for activating basal R-GnRH gene expression in gonadotrope cells.

The GnRH receptor gene is preferentially expressed in functioning gonadotroph adenomas and displays a Mae III polymorphism site.

OBJECTIVE: Given the central role of the GnRH receptor (GnRHR) in the regulation of the gonadotrophin secretion, it might be implicated directly or indirectly in the pathogenesis of gonadotroph tumours. DESIGN: We determined if GnRHR mRNA was expressed in gonadotroph tumours using RT-PCR and analysed the GnRHR gene for the presence of mutations in its coding region, using direct sequencing of PCR products. Results were analysed according to the pattern of expression of alpha, beta-FSH and beta-LH subunit (SU) genes. SUBJECTS: RNA was extracted from 20 gonadotroph tumours identified by immunohistochemistry (> 10% of stained cells): 9 adenomas were functioning (high serum gonadotrophin levels), 3 were associated with high alpha-SU levels and 8 were nonfunctioning. Genomic DNA was extracted from 64 normal subjects. RESULTS: We found GnRHR mRNA in 12 tumours (60%): 8/9 functioning (88%), 1/3 alpha-secreting (33%) and 3/8 nonfunctioning (37.5%) gonadotroph adenomas. There was a significant association between GnRHR expression and immunostaining for beta-FSH (P = 0.014). The nucleotide sequence of the amplified products was identical to that of human pituitary except for the presence, in 3 functioning adenomas, of a silent C to T transition at nucleotide 453 encoding for the serine residue situated in the second intracellular loop at position 151. Heterozygosity provided evidence that both alleles were transcribed in these tumours. This substitution creates a Mae III restriction site. Genomic DNA from normal subjects were then tested for the presence of this new polymorphism. The frequency of the heterozygosity (18.7%) was not significantly different from that found in gonadotroph tumours (25%) and this new Mae III polymorphism site cannot be used as a tumoural marker. CONCLUSION: The GnRHR gene is preferentially expressed in functioning rather than in nonfunctioning gonadotroph adenomas, but no mutations altering the coding region of the gene were found to further substantiate its role in the pathogenesis of gonadotroph tumours.

GnRH-dependent up-regulation of nitric oxide synthase I level in pituitary gonadotrophs mediates cGMP elevation during rat proestrus.

We have recently provided evidence that the concentration and activity of the enzyme nitric oxide synthase type I (NOS I) was stimulated in gonadotrophs by GnRH, suggesting a role for the NOS I/NO pathway in the GnRH control of cell functions. To further establish the GnRH regulation of pituitary NOS I under physiological circumstances, we have examined the expression of NOS I during the 4-day estrus cycle in rats. Western blot analysis demonstrated that NOS I was present in the anterior pituitary at low levels during diestrus I (DI) and diestrus II, then was subject to a significant increase on the afternoon of proestrus to reach a maximal 3-fold increase between 19:00 and 20:00 h, after which NOS I decreased to return, during estrus, to levels within the range detected in diestrus. No such variation was apparent in the posterior pituitary lobe. NADPH-diaphorase histochemistry combined with immuno-identification of the cells revealed that active NOS I was expressed in both the gonadotrophs and the folliculo-stellate cells throughout the whole cycle but only the gonadotrophs showed an up-regulation during proestrus. A high temporal correlation was observed in the profiles of NOS I, pituitary cGMP and serum luteinizing hormone (LH) suggesting an implication of GnRH. Consistently, the administration of a potent GnRH antagonist to rats totally abolished the rise in pituitary NOS I and cGMP, in addition to suppressing, as expected, the surge in the secretion of LH. A role of NOS I as a mediator in the GnRH-induced augmentation in cGMP was further established in vitro by incubating anterior pituitaries in the presence of the NOS inhibitor, L-NMMA (1 mM). Altogether, these data demonstrate that the level and activity of NOS I is up-regulated in gonadotrophs during proestrus, in a manner consistent with a major implication of GnRH over a period during which its release from the hypothalamus, as well as gonadotroph responsiveness, are at maximum. This effect is accompanied by a NOS/NO-mediated rise in cGMP. In the absence of obvious effect on gonadotropin release, the roles of NO and cGMP in the regulation of gonadotroph functions, especially during proestrus, remain to be established.

Fetal expression of GnRH and GnRH receptor genes in rat testis and ovary.

The identification of gonadal gonadotropin-releasing hormone receptors (GnRH-R) and evidence of direct inhibitory effects of GnRH agonists upon steroidogenesis in adult rat gonads, lend credence to a putative intragonadal role of a locally secreted GnRH or GnRH-like peptide. Using reverse transcription-polymerase chain reaction followed by Southern blot hybridization and sequencing, we identified, both in the ovary and in the testis of fetal and adult rats, a fully processed GnRH messenger RNA (mRNA), the sequence of which, in adult testis, was identical to that found in the hypothalamus. We also detected in the testis, but not in the ovary, a transcript containing the first intron. The ontogeny of GnRH and GnRH-R gene expression was studied in rat gonads from 14.5 to 21.5 days post-coitum (dpc), using dot blot hybridization of total RNA. During this period, the levels of cyclophilin mRNA normalized to total RNA remained unchanged. Thus, we used cyclophilin as an internal standard. GnRH mRNA was detected in the ovary at 18.5 dpc, four days later than in the testis, and similar levels were found in both sexes at birth. GnRH-R mRNA was present at 14.5 dpc in the testis and at 15.5 dpc in the ovary, with the levels at 21.5 dpc being 2.4 times higher in the testis than in the ovary. GnRH and GnRH-R mRNA levels increased in both sexes in late fetal development, but this increase appeared two days sooner in the ovary compared with the testis, thus supporting the hypothesis that expression of the GnRH and GnRH-R genes is regulated in a sex-dependent manner during fetal development. In all cases, expression of GnRH and GnRH-R preceded gonadotropin receptors in the gonads and initiation of gonadotropin secretion by the pituitary.

Evidence that gonadotropin-releasing hormone stimulates gene expression and levels of active nitric oxide synthase type I in pituitary gonadotrophs, a process altered by desensitization and, indirectly, by gonadal steroids.

To determine the site and mechanism of action of gonadal steroids on pituitary nitric oxide synthase type I (NOS I), present in both gonadotrophs and folliculo-stellate cells, the effects of castration and steroids were examined in male rats, in the presence of a GnRH antagonist (Antarelix). Western analysis showed a rapid and substantial increase with time, after orchidectomy, of NOS I protein, the concentration doubling in 24 h and reaching a maximal 4- to 5-fold increase after 3-7 days, followed by a progressive decline after 2 weeks. Testosterone or estradiol replacement, or administration of GnRH antagonist, totally abolished the effects of castration, demonstrating a mediation of the steroid effects via GnRH. In noncastrated rats, steroids and the GnRH antagonist also caused a reduction in the levels of NOS I (by 50-60%), consistent with inhibition of endogenous GnRH stimulation. In marked contrast, administration of a potent GnRH agonist (Triptorelin) to intact rats increased the levels of NOS I. A time-course study with a long-lasting formulation showed that rise in NOS I developed rapidly after a lag of approximately 5 h, with a 2-fold increase detectable after 8 h and a maximal 4.5-fold after 48 h. The level declined afterwards in a manner consistent with homologous desensitization that may occur in the continuous presence of GnRH; however, the profile was different and delayed compared with those of gonadotropin release. As observed for NOS I protein, NOS I messenger RNA concentration was increased by castration or GnRH agonist and reduced by steroids or GnRH antagonist. Taken together, these data demonstrate that steroids indirectly regulate NOS I messenger RNA and protein levels, through the hypothalamic modulation of GnRH, which represents the primary regulator of NOS I. No effect of steroids on NOS I was seen in the posterior lobe. NADPH-diaphorase histochemistry coupled to immuno-identification of the cells revealed that the treatments affecting the concentration of NOS I concomitantly altered the activity but exclusively in gonadotrophs and not in folliculo-stellate cells (which do not respond to GnRH), reinforcing the idea that GnRH played a major regulatory role. Expression in gonadotrophs of a GnRH-dependent NOS I and the ensuing production of nitric oxide represents a potentially novel signaling pathway for the neuropeptide in the anterior pituitary, consistent with the previously reported GnRH-induced cGMP production, the role of which remains to be evaluated.

Gonadotropin-releasing hormone and pituitary adenylate cyclase-activating polypeptide affect levels of cyclic adenosine 3',5'-monophosphate-dependent protein kinase A (PKA) subunits in the clonal gonadotrope alphaT3-1 cells: evidence for cross-talk between PKA and protein kinase C pathways.

We have shown previously that protein kinase A (PKA) subunit levels are regulated by activation of PKA or protein kinase C (PKC) in anterior pituitary cells. GnRH also influenced PKA subunit levels, suggesting that hormonal regulation occurs in gonadotrophs, and therefore, we have reexamined this question using the clonal gonadotrope-derived cell line (alphaT3-1 cells). Western blot analysis, using specific immunoaffinity purified immunoglobulins, revealed expression of catalytic (Cat) and regulatory type I (RI) and type II (RII) subunits of PKA in these cells. Activation of adenylyl cyclase (AC) with forskolin, or of PKC with tetradecanoyl phorbol acetate (TPA), caused a rapid (detectable at 0.5-1 h) and concentration-dependent loss of all PKA subunits. Forskolin (10-100 microM) reduced Cat and RI by 60% and RII by 30%, whereas TPA (0.1-1 microM) reduced Cat and RII by 50% and RI by 40%. Simultaneous activation of PKA and PKC caused the expected dose-dependent reductions in Cat, and the effects of forskolin or TPA were nearly additive. RI and RII were reduced similarly by 10 nM TPA, whereas 100 nM TPA tended to prevent the reduction of RI or RII caused by forskolin. GnRH, which activates phosphoinositidase C and not AC in these cells, caused a clear loss of Cat or RII at all concentrations tested and of RI at 0.1 nM. Pituitary adenylate cyclase-activating polypeptide 38, which acts via PVR-1 receptors to stimulate both phosphoinositidase C and AC in these cells, also caused a clear dose-dependent decrease in Cat, RI, and RII, although higher concentrations were needed for the latter effects. Together, the data demonstrate that catalytic and regulatory subunits of PKA are subject to both hormonal and receptor-independent regulation in alphaT3-1 cells, reinforcing the possibility that such effects occur in nonimmortalized gonadotropes. Whereas the effects of PKA activators very likely involve proteolytic degradation of the dissociated PKA holoenzyme, the effects of TPA and GnRH occur in the absence of cAMP elevation by unknown mechanisms. Whatever the mechanisms involved, the data reveal a mechanism for cross-talk between phosphoinositidase C and AC-mediated hormonal signals, in which PKC activation seems to play a pivotal role.

The genes for gonadotropin-releasing hormone and its receptor are expressed in human breast with fibrocystic disease and cancer.

While gonadotropin-releasing hormone (GnRH) or GnRH receptor (GnRHR) have been reported to exist in tissues other than brain and pituitary, there is no report concerning co-expression of GnRH and GnRHR in human breast tissues. To address this question, we have examined whether mRNA for GnRH as well as GnRHR was present in different human breast samples, by employing the reverse transcription-polymerase chain reaction (RT-PCR) protocol followed by Southern blotting of the PCR products. Coexpression of GnRH and GnRHR genes was further confirmed by dot blot hybridization using appropriate [32P]-labeled probes. We thus tested fibrocystic breast (4 cases), invasive ductal carcinomas (6 cases) and 1 adjacent non-neoplastic tissue. GnRHR and GnRH mRNAs were found in all actin-positive samples including malignant as well as nonmalignant tissues. Quantitative determinations of mRNA did not reveal significant differences between malignant and non-malignant breast samples for either GnRH or GnRHR gene expression. Our data show that neither gene was overexpressed in the breast cancer samples compared with normal breast tissue. Since GnRH agonists inhibit breast epithelial cell growth, the presence of GnRHR mRNA suggests that GnRH may specifically affect breast cell growth. Our data thus raise the possibility of an autocrine/paracrine role for GnRH in human mammary gland.