Administration of low dose pulsatile gonadotropin-releasing hormone (GnRH) to GnRH-deficient men regulates free alpha-subunit secretion

Although pharmacological doses of GnRH and TRH stimulate free alpha-subunit (alpha-subunit) secretion from the pituitary, little is known about the pattern and control of alpha-subunit release under physiological circumstances. Euthyroid men with idiopathic hypogonadotropic hypogonadism, a condition of deficient GnRH release, provide a unique opportunity to study alpha-subunit secretion before and during administration of a physiological regimen of GnRH administration. Before GnRH therapy, six euthyroid IHH men with normal endogenous TSH secretion had circulating alpha-subunit levels close to or below assay detection limits, with a mean level less than 0.5 ng/ml. During 12-42 weeks of physiological GnRH replacement, serum alpha-subunit concentrations rose to a mean value of 2.07 +/- 0.3 (+/- SEM) ng/ml (P less than 0.01). After GnRH administration, alpha-subunit was released in a pulsatile pattern following each dose of GnRH and mirrored the secretory pattern of LH. Increases in serum alpha-subunit concentrations during GnRH administration were closely correlated with increases in LH (r = 0.91; P less than 0.01), but not FSH (r = 0.24; P = NS), levels. In addition, a situation in which LH secretion was clearly predominant and FSH levels were barely detectable was created by increasing the frequency of GnRH administration to every 30 min. In this circumstance, free alpha-subunit concentrations increased in conjunction with LH levels in the face of decreased FSH levels. We conclude that replacement of GnRH regulates both the level and pattern of alpha-subunit secretion in GnRH-deficient men, and that there is tight correlation of alpha-subunit with LH, but not with FSH, secretion.     

Preoptic area brain grafts in hypogonadal (hpg) female mice abolish effects of congenital hypothalamic gonadotropin-releasing hormone (GnRH) deficiency

Normal fetal preoptic area (POA), a site of GnRH production, was implanted into the third ventricle of adult female hypogonadal (hpg) mice. When the grafts were successful, the mice (genetically deficient in hypothalamic GnRH) responded with vaginal opening, cornified vaginal cells, ovarian and uterine development, and increased pituitary FSH content and plasma LH concentrations. Similar results were obtained with fetal POA tissue, whether derived from males or females. Two of four hpg mice with POA grafts mated when caged overnight with males. Hpg females that received cortical tissue from fetuses or from 16-day-old pups, or POA tissue from 16-day-old pups, showed none of these changes, remaining similar to untreated hpg females.     

Bio- and immunoactive luteinizing hormone responses to low doses of gonadotropin-releasing hormone (GnRH): dose-response curves in GnRH-deficient men

Previous investigations of the effects of GnRH on pituitary LH responses in normal men required pharmacological doses of GnRH to avoid the confounding effects of endogenous GnRH secretion and employed nonphysiological dose intervals. To examine the role of GnRH in determining both the qualitative and quantitative nature of physiological LH responses, we studied five GnRH-deficient men in whom pituitary and gonadal function had been normalized with GnRH replacement. Both bio- and immunoactive LH responses were evaluated in these men after a wide range of GnRH doses (7.5-250 ng/kg) administered at a physiological frequency (every 2 h), while gonadal steroid levels were within the normal adult male range. In addition, the amplitude and contour of the immunoactive LH pulses were compared to those of 15 normal men to assure that these experiments achieved physiological pituitary responses. The relationship between bio- and immunoactive LH was compared between patients, between doses as the amount of GnRH was increased, and within pulses of LH. As the dose of GnRH was increased, both bio- and immunoactive LH responses increased in a log-linear fashion when assessed by both amplitude (r = 0.96 for bioactive LH and r = 0.98 for immunoactive LH) and area under the curve (r = 0.99 for bioactive LH and r = 0.97 for immunoactive LH). GnRH doses of 7.5 and 25 ng/kg produced LH responses with amplitudes similar to those in normal men. The relationship between bio- and immunoactive LH between patients and between differing doses of GnRH was analyzed by comparing the slopes of lines fit to individual bioactive vs. immunoactive LH plots after each dose of GnRH in each patient. There was a marked variation in the relationship of bio- to immunoactive LH between patients (P less than 0.005). No change was found in the biopotency of LH as the dose of GnRH was increased (P less than 0.10). Finally, no variation of the bioactivity of LH was evident within individual pulses. We conclude that a log-linear relationship exists between doses of GnRH that produce physiological LH pulses and both bio- and immunoactive LH responses; the bioactivity of secreted LH varies markedly between patients; the relative bioactivity of LH in an individual does not change as the dose of GnRH is increased; and no change in bioactivity of LH responses was demonstrated within pulses of LH.     

Effects of increasing the frequency of low doses of gonadotropin-releasing hormone (GnRH) on gonadotropin secretion in GnRH-deficient men

The effects of increasing the frequency of pulsatile GnRH administration on LH and FSH responsiveness were studied in five GnRH-deficient men who had achieved normal sex steroid levels during prior long term GnRH replacement. Intravenous doses of GnRH were employed that had previously been demonstrated to produce LH and FSH levels in each subject similar to those in normal men. Both acute and chronic changes in pituitary responses were studied after progressive increases in GnRH frequency (from every 120 to 60 min, from 60 to 30 min, and from 30 to 15 min) during three 12-h admissions, each separated by 7 days. During the two intervals between the studies GnRH frequency was 60 and 30 min, respectively. Pituitary responses were characterized by determining the mean serum LH and FSH levels, LH pulse amplitudes, and mean LH and FSH levels which were normalized for the frequency of GnRH administration (nLH and nFSH). As the frequency of GnRH stimulation was increased acutely, mean serum LH levels rose progressively, in contrast to both LH pulse amplitude and nLH levels which decreased, while serum testosterone (T) concentrations remained constant. No further evidence of gonadotroph desensitization occurred after chronic GnRH administration at either 60- or 30-min intervals. At higher frequencies of GnRH stimulation, discrete pulses of LH were not always apparent after injections of GnRH, and in two men, marked destabilization of the gonadotroph responses occurred. Even without detectable LH pulses, serum T levels did not decline during administration of GnRH at intervals as rapid as 15 min. In contrast, there was no change in mean FSH concentrations, although nFSH values decreased progressively as the GnRH frequency was increased. nFSH levels fell to a greater degree than nLH after each increase in GnRH frequency. Thus, pituitary gonadotroph responsiveness to a fixed dose of GnRH decreased as the frequency of GnRH stimulation increased. FSH responsiveness decreased to a greater degree than did LH. Gonadotropin secretory responses are destabilized at higher frequencies of GnRH administration. Pulsatile LH stimulation of the testes does not appear necessary to maintain T secretion.     

Pulsatile gonadotropin secretion after discontinuation of long term gonadotropin-releasing hormone (GnRH) administration in a subset of GnRH-deficient men

Normal pituitary and gonadal function can be maintained with long term pulsatile GnRH administration in men with idiopathic hypogonadotropic hypogonadism (IHH), and both pituitary and gonadal priming occur during the process of GnRH-induced sexual maturation. Still, the long term effects of discontinuing GnRH therapy in IHH men have not been examined. Therefore, we evaluated the patterns of gonadotropin and alpha-subunit secretion before and after a prolonged period of pulsatile GnRH administration in 10 IHH men. Before exogenous GnRH stimulation, no patient had any detectable LH pulsations. In 6 of these men, who were typical of most of our IHH patients (group I), no LH pulsations were detectable after cessation of GnRH administration. However, in the other 4 men (group II), LH pulsations were easily detectable despite cessation of exogenous GnRH stimulation, and the amplitude (9.3 +/- 3.5 IU/L) and frequency (13.8 +/- 1.7 pulses/day) of these LH pulses were similar to those in 20 normal men (10.6 +/- 0.7 IU/L and 11.0 +/- 0.7 pulses/day). Three of these 4 men in group II maintained normal serum testosterone levels after discontinuation of GnRH delivery. To determine if there were any characteristics that might be useful in predicting which IHH men could maintain normal pituitary-gonadal function after long term GnRH administration, we evaluated various clinical and hormonal parameters at the time of initial presentation. Mean alpha-subunit levels (P less than 0.01) and alpha-subunit pulse amplitude (P less than 0.02) were significantly higher in the group II than the group I men, suggesting that the group II patients had partial, rather than complete, deficiency of endogenous GnRH secretion. None of the other parameters that were assessed distinguished the two groups. We conclude that gonadotropin and sex steroid levels return to their pretreatment state in the majority of IHH men when long term GnRH administration is discontinued. Normal pituitary-gonadal function can be maintained after discontinuation of long term GnRH administration in a rare subset of IHH men who present with higher levels of alpha-subunit. We hypothesize that these latter IHH men have an incomplete GnRH deficiency and that long term exogenous GnRH administration induces pituitary and gonadal priming, which subsequently enables them to sustain normal pituitary and gonadal function in response to their own enfeebled GnRH secretion.     

Transplanted gonadotropin-releasing hormone neurons promote pulsatile luteinizing hormone secretion in congenitally hypogonadal (hpg) male mice

Congenitally hypogonadal (hpg) male mice are unable to synthesize biologically active gonadotropin-releasing hormone (GnRH). Implantation of normal fetal preoptic area tissue containing GnRH neurons into the third ventricle of adult hpg males significantly elevates pituitary levels of luteinizing hormone (LH) and corrects their hypogonadism. In all responding animals, immunoreactive GnRH neurons within the transplant innervate the median eminence of the host. To assess whether gonadal recovery in hpg hosts results from pulsatile secretion of GnRH from grafted neurons, we compared the pattern of variation in plasma LH levels in 19 hpg graft recipients with testicular growth to that of 10 normal adult mice. All animals were castrated prior to receiving an indwelling catheter in the jugular vein. Sequential blood samples were collected (t = 10 min) and assayed for LH. Pulsatile LH secretion was seen in 11 of 19 hpg hosts and in all control mice. While there was great variability between individual animals, measures of baseline LH, LH pulse amplitude and duration, interpulse interval, and LH pulse frequency revealed no difference between hpg graft recipients and normal castrates in their LH pulse pattern. Immunocytochemical analysis of the brain in hpg hosts suggested no correlation between any parameter of pulse activity and individual differences in GnRH cell number or GnRH fiber outgrowth into the median eminence. Sources of variation in LH secretion among graft recipients, and between hpg hosts and normal mice, are discussed. We suggest that transplanted GnRH neurons are capable of integration into a GnRH 'pulse generator' which can support a near-normal pattern of pulsatile LH secretion, leading to testicular growth and steroid production.     

The interaction of gonadotropin-releasing hormone and estradiol on luteinizing hormone and prolactin gene expression in female hypogonadal (hpg) mice

We have investigated the interaction of estrogen with GnRH on the regulation of LH subunit mRNA in female hypogonadal (hpg) mice receiving constant frequency and amplitude pulsatile GnRH treatment for up to 18 days. The level of cytosolic common alpha mRNA in female hpg mouse pituitaries was 45 +/- 6% of normal female littermate values, and treatment with pulsatile GnRH increased alpha mRNA to 40% above the normal value at 24 h and 2-4 times normal at 7 and 12 days (P less than 0.001); by 18 days levels had returned to those of untreated hpg controls. Concurrent treatment with estradiol (E2) did not affect those changes. However, in ovariectomized hpg mice the 2- to 4-fold rise in alpha mRNA was sustained for 18 days with GnRH treatment. E2 treatment alone for 7 and 12 days doubled alpha mRNA. LH beta mRNA levels in untreated female hpg mice were between 5-10% of normal values. Levels increased significantly (77 +/- 6.4%) 24 h after GnRH treatment and were normal at 7, 12, and 18 days. E2 together with GnRH did not affect the LH beta mRNA increase at 12 days, but reduced it to 45% of normal at 18 days. Ovariectomy did not alter the LH beta mRNA response to GnRH treatment, and E2 treatment alone did not increase LH beta mRNA. Serum LH concentrations were normalized by GnRH treatment at all times and did not increase in ovariectomized animals. LH release was prevented when E2 was combined with GnRH. Pituitary LH content in hpg mice was 20% of normal and increased gradually with GnRH treatment. Neither concurrent treatment with E2 nor ovariectomy affected the GnRH-induced synthesis of LH. PRL mRNA levels were 30-40% of normal littermate values in untreated female hpg mice, and pulsatile GnRH increased these to 70-80% of normal. E2 alone raised PRL mRNA slightly above normal values, although together with GnRH this rise was attenuated by about 40%. Pulsatile GnRH treatment of ovariectomized hpg mice did not increase PRL mRNA. E2 increased pituitary PRL content, and GnRH did not attenuate this aspect of E2 action. Serum PRL levels rose with E2 treatment at 7 and 12 days, and concurrent GnRH treatment prevented the rise at 12 days. We conclude the following: 1) The stimulatory action of pulsatile GnRH on the expression of both common alpha and LH beta mRNA is rapid (less than 24 h).(ABSTRACT TRUNCATED AT 400 WORDS)     

Topical absorption of gonadotropin-releasing hormone (GnRH) in goldfish

The studies reported here show that exogenous GnRH can be absorbed by goldfish from the intraperitoneal (ip) cavity, gill surface, or surrounding water. Mammalian rather than teleost GnRH was applied in order to ensure that GnRH measurement in plasma did not reflect the native form. A radioimmunoassay (RIA) specific to mammalian GnRH was used to measure the concentration of absorbed GnRH; validation for this approach was provided by HPLC and cross-reactivity studies in which mammalian GnRH was shown not to be present in control goldfish brain or pituitary extracts. Plasma concentration of GnRH was highest at the first sampling time, 4 min after administration, for all three routes. For intraperitoneal injection, plasma concentration was halved in 12 min, a period comparable with the half-life in rats. The pituitary content of GnRH also increased rapidly during the first 4 min after ip injection and remained high for 60 min. Absorption of GnRH from the gill was equally effective with water or dimethyl sulfoxide (DMSO) as vehicle.     

Utility of free alpha-subunit as an alternative neuroendocrine marker of gonadotropin-releasing hormone (GnRH) stimulation of the gonadotroph in the human: evidence from normal and GnRH-deficient men.

To examine the hypothesis that the secretion of free alpha-subunit (FAS) can serve as an alternative to LH as a neuroendocrine marker of gonadotroph stimulation by GnRH in euthyroid humans, we have investigated the relationship of pulsatile FAS secretion in euthyroid GnRH-deficient men (n = 10) before and after exogenous GnRH stimulation and in normal men under the influence of endogenous GnRH secretion (n = 18). Before GnRH exposure, the GnRH-deficient men showed a complete absence of both LH and FAS pulses. During the initial 7 days of GnRH exposure, all GnRH-deficient men exhibited pulsatile release of FAS by the third day, whereas the appearance of pulsatile release of LH and FSH was more variable. Long term administration of GnRH led to pulses of LH and FAS that were 100% concordant with a demonstrable dose-response relationship between GnRH and FAS, which was quantitatively similar to but more exuberant than that for LH. All doses of GnRH that produced LH pulses within the normal adult range yielded supraphysiological FAS pulses. Analysis of distribution histograms of interpulse intervals and pulse amplitudes of LH and FAS in both normal and GnRH-deficient subjects demonstrated no significant difference between these glycoproteins in interpulse intervals in either the normal or GnRH-deficient groups or in the pulse amplitudes in the GnRH-deficient subjects. There was, however, a significant difference (P less than 0.01) between the distribution histogram of LH and FAS pulse amplitudes in normal men. We conclude that the pulsatile secretion of FAS in euthyroid men 1) is determined by GnRH secretion, 2) is the initial glycoprotein to be secreted in a pulsatile fashion from the gonadotroph during early GnRH exposure in GnRH-deficient men, 3) demonstrates a dose-response relationship to exogenous GnRH which is more robust than that of LH in GnRH-deficient men receiving GnRH, and 4) can, therefore, serve as a complementary and powerful tool with LH for the study of GnRH neurosecretory dynamics.     

Luteinizing hormone release and gonadotropin-releasing hormone (GnRH) receptor internalization: independent actions of GnRH

Three different approaches are described which provide independent and new evidence that gonadotropin-releasing hormone (GnRH) internalization and GnRH-stimulated LH release are distinct actions of the releasing hormone. 1) Removal of GnRH from medium bathing the pituitary cell cultures resulted in the prompt return of LH release to basal levels. This finding indicated that a continuous supply of externally applied GnRH is required for the stimulation of LH release. 2) Covalent immobilization of D-Lys6-des-Gly10-Pro9-ethylamide GnRH (a GnRH agonist) on agarose beads resulted in a derivative which stimulated LH release with full efficacy. At concentrations of immobilized releasing hormone analog sufficient to evoke gonadotropin release, the quantity of LH release was restricted by the number of beads added. This finding was interpreted as evidence that the attachment of immobilized agonist was stable during the bioassay and indicated that LH release could be stimulated with full efficacy without the requirement for GnRH internalization. 3) Comparative studies using image-intensified microscopy and the cell culture bioassay showed that 100 microM vinblastin markedly inhibited large scale patching and capping of the GnRH receptor (viewed by image-intensified microscopy), but did not alter the EC50 or efficacy of LH release stimulated by GnRH or the agonist described above. These observations indicated that internalization as well as large scale patching and capping of the GnRH receptor are not required for LH release.     

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.     

Molecular polymorphism of native gonadotropin-releasing hormone (GnRH) is restricted to mammalian GnRH and [hydroxyproline9] GnRH in the developing rat brain

Although chicken gonadotropin-releasing hormone (GnRH)-II is thought to occur in most animal species, its presence and that of two other variants (lamprey GnRH-III, salmon GnRH) is questionable in rodents. Here we report on the GnRH peptides present in the hypothalamus and the remaining brain of rat of both sexes during development. No immunoreactivity was detected in the elution zone of either native or hydroxylated forms of the above three variants in any of brain extracts chromatographed. The main peptides detected were mammalian GnRH (mGnRH) and m[hydroxyproline9]GnRH (mHypGnRH). In the hypothalamus, these peptides were associated with their free acid and precursor forms. N-terminal fragments from both native decapeptides (GnRH) and mGnRH (GnRH) were observed only in the hypothalamus. C-terminal fragments were detected in both tissues. The relative proportions of mGnRH and mHypGnRH showed no developmental changes in the remaining brain. The hypothalamic proportions of mHypGnRH were high on day 5, and decreased from day 15 onwards. The [Gly11]-precursor to mHypGnRH molar ratio was twofold lower than with the non-hydroxylated peptides. The mGnRH to GnRH molar ratio increased in males but decreased in females during development. No sex-related differences were observed in the native decapeptide to GnRH molar ratio. It was concluded that (1) chicken GnRH-II is not present in all mammals, (2) mGnRH and mHypGnRH are the main GnRH isoforms present in the rat brain, (3) the processing of [Gly11]-precursor into mHypGnRH occurs at a higher rate than that of mGnRH, and (4) the catabolism does not interfere with the developmental changes undergone by the mGnRH and mHypGnRH brain contents.     

Fanconi anemia A is a nucleocytoplasmic shuttling molecule required for gonadotropin-releasing hormone (GnRH) transduction of the GnRH receptor

GnRH binds its cognate G protein-coupled GnRH receptor (GnRHR) located on pituitary gonadotropes and drives expression of gonadotropin hormones. There are two gonadotropin hormones, comprised of a common alpha- and hormone-specific beta-subunit, which are required for gonadal function. Recently we identified that Fanconi anemia a (Fanca), a DNA damage repair gene, is differentially expressed within the LbetaT2 gonadotrope cell line in response to stimulation with GnRH. FANCA is mutated in more than 60% of cases of Fanconi anemia (FA), a rare genetically heterogeneous autosomal recessive disorder characterized by bone marrow failure, endocrine tissue cancer susceptibility, and infertility. Here we show that induction of FANCA protein is mediated by the GnRHR and that the protein constitutively adopts a nucleocytoplasmic intracellular distribution pattern. Using inhibitors to block nuclear import and export and a GnRHR antagonist, we demonstrated that GnRH induces nuclear accumulation of FANCA and green fluorescent protein (GFP)-FANCA before exporting back to the cytoplasm using the nuclear export receptor CRM1. Using FANCA point mutations that locate GFP-FANCA to the cytoplasm (H1110P) or functionally uncouple GFP-FANCA (Q1128E) from the wild-type nucleocytoplasmic distribution pattern, we demonstrated that wild-type FANCA was required for GnRH-induced activation of gonadotrope cell markers. Cotransfection of H1110P and Q1128E blocked GnRH activation of the alphaGsu and GnRHR but not the beta-subunit gene promoters. We conclude that nucleocytoplasmic shuttling of FANCA is required for GnRH transduction of the alphaGSU and GnRHR gene promoters and propose that FANCA functions as a GnRH-induced signal transducer.     

Hyperprolactinemia inhibits gonadotropin-releasing hormone (GnRH) stimulation of the number of pituitary GnRH receptors

Gonadotropin secretion is diminished in the presence of hyperprolactinemia, and previous studies have shown that PRL can reduce GnRH secretion and impair LH responses to GnRH. To investigate the mechanisms of the inhibitory effects of PRL on the pituitary, we administered intraarterial pulse injections of GnRH (25 ng/pulse every 30 min) to castrate testosterone-implanted male rats placed in restraint cages. Serum PRL, GnRH receptor (GnRH-R), and LH responses to GnRH were measured at intervals over 72 h. In control animals which received saline pulses, serum PRL was transiently elevated to the range of 100-150 ng/ml during the first 24 h, GnRH-R remained stable (approximately 300 fmol/mg protein) and serum LH was low (less than 10 ng/ml) throughout the 72 h. GnRH pulses in castrate testosterone-implanted animals increased GnRH-R to values (approximately 600 fmol/mg) similar to those in castrate controls (no testosterone implant, saline pulses) through 48 h, but GnRH-R declined to baseline values by 72 h in both groups. Serum LH responses to GnRH pulses were only present at 24 h. Administration of bromocriptine throughout the 72 h to immobilized castrate rats or to castrate testosterone-replaced animals treated with GnRH pulses suppressed serum PRL, and GnRH-R concentrations remained elevated through 72 h. Serum LH responses to GnRH pulses were 5- to 20-fold higher in bromocriptine-treated rats, and responses were present throughout the 72 h of the experiment. Delaying the start of bromocriptine treatment until 36 h (after the spontaneous PRL peak) resulted in reduced GnRH-R and LH responses at 72 h. Similarly, administration of ovine PRL (during the first 48 h) to bromocriptine-treated rats produced low GnRH-R concentrations at 72 h. Thus, the transient elevation of PRL seen in immobilized rats can inhibit the GnRH-stimulated increase in GnRH-R and is associated with reduced LH responses to GnRH. These results indicate that PRL has a direct inhibitory effect on the gonadotrope and suggest that impaired GnRH-R responses to GnRH are one of the mechanisms involved in the diminished gonadotropin secretion seen in hyperprolactinemia.     

First intron excision of GnRH pre-mRNA during postnatal development of normal mice and adult hypogonadal mice

Previously, we demonstrated that excision of the GnRH first intron (intron A) was largely attenuated in non-GnRH-producing tissues but accelerated in GnRH neurons. In the present study, we examined the splicing rate of GnRH pre-mRNA in developing normal mice and adult hypogonadal mice. The preoptic area and cerebral cortex were removed from mice at ages 1-7 wk. GnRH pre-mRNA splicing was examined by competitive RT-PCR using a variety of primer sets. The ratio of mature mRNA to intron A-containing RNA species in the preoptic area was lowest in 1- and 2-wk-old mice, significantly augmented in 3-wk-old mice, and further increased until adulthood. In contrast, the ratio of mRNA to intron A-containing RNA in the cerebral cortex was extremely low, drastically decreased in 3-wk-old mice, and remained at low levels until adulthood. These data indicate a preoptic area-specific increase in intron A excision during development. Intron B or C excision in the preoptic area was not significantly changed during development. To elucidate the possible involvement of the exonic splicing enhancers located in GnRH exons 3 and 4 in the developmental increase in intron A excision, we examined the splicing rate of GnRH pre-mRNA in hypogonadal mice whose GnRH exons 3 and 4 were truncated. The intron A excision in the preoptic area of hypogonadal mice was significantly lower than that of normal mice but similar to that in other tissues, such as cerebral cortex and olfactory bulb. To support the functional relevance of intron A-containing RNA species, we examined the translation efficiency of intron A-containing RNA. Insertion of intron A sequence into the upstream portion of the luciferase open reading frame significantly decreased translation efficiency. The present study demonstrates that intron A excision in the preoptic area is developmentally regulated in normal mice but largely attenuated in hypogonadal mice, indicating the functional importance of intron A excision in GnRH pre-mRNA splicing.