Pulsatile GnRH Research Articles

Testosteron und Infertilität

About 15% of men and women in their reproductive years remain childless; in 50% of cases the reasons can be found in the male partner. It is important to define as exactly as possible the reasons for infertility to select those men who can be treated by a causal treatment option and to find the optimum treatment strategy. While FSH is the key hormone for intact spermatogenesis, testosterone as an important modifier of quantitative normal sperm production may sometimes be underestimated. Exogenous substitution with testosterone will suppress spermatogenesis and is thus not indicated in hypogonadal males with hypogonadism and infertility at the same time. In males with normal to elevated gonadotrophins and symptomatic testosterone deficiency, treatment is difficult and evidence-based recommendations are missing. The situation in males presenting with secondary hypogonadotropic hypogonadism exhibiting one of the clearly defined and treatable reasons of male infertility is totally different. To stimulate the gonadal function in secondary hypogonadal patients wishing to father a child, pulsatile GnRH or subcutaneous gonadotrophin treatment are used. They stimulate both testosterone production and spermatogenesis. After the end of successful fertility treatment, substitution is continued by exogenous testosterone treatment with oral, transdermal or intramuscular physiological preparations.

Serum Levels of Tryptophan, 5-Hydroxytryptophan and Serotonin in Patients Affected with Different Forms of Amenorrhea

Tryptophan (Trp) is present in the serum, partly bound to albumine and in the free form. The unbound portion of circulating tryptophan has the property of crossing the hematoencephalic barrier and being converted within the brain into serotonin (5-HT) through the enzymatic processes of hydroxylation and decarboxylation. The serotoninergic system plays an important role in neuroendocrine control of reproductive hormone secretion, and in particular, it may influence GnRH pulsatility, a function essential for reproductive processes. In this study, we analysed serum levels of tryptophan, serotonin and 5-hydroxytryptophan (5-HTP) in women with three different forms of amenorrhea: 16 patients were diagnosed with anorexia nervosa, 60 patients with functional hypothalamic amenorrhea, and 14 patients with hyperprolactinemia. Data were compared with those of a group of 25 healthy women. Serum Trp levels were significantly (P ≤ 0.05) lower in the anorexic (11.64 ± 0.53 μg/ml, mean ± S.E.) than in the control (12.98 ± 0.37 μg/ml) groups. In addition, in the anorexic group a statistical dispersion of Trp values was shown indicating a bimodal data distribution suggesting the existence of two different subgroups of patients. Regarding 5-HTP, an increase of its serum level was observed in all the groups with amenorrhea with the highest value in hyperprolactinemic patients. On the contrary, no statistical differences in serum 5-HT levels among the four analyzed groups were observed.This study shows that women affected by various forms of amenorrhea present an altered metabolism of tryptophan via serotonin and, in particular, markedly high differences are observed between the two subgroups of anorexic patients.

Decreased Expression of A-Kinase Anchoring Protein 150 in GT1 Neurons Decreases Neuron Excitability and Frequency of Intrinsic Gonadotropin-Releasing Hormone Pulses

The frequency of intrinsic pulsatile GnRH secretion from endogenous GnRH neurons and GT1 GnRH cell lines is stimulated by increased intracellular cAMP levels. The downstream molecules comprising the cAMP signaling pathway are organized in microdomains by a family of scaffolding proteins, A-kinase anchoring proteins (AKAPs). These molecules tether protein kinase A, cAMP-specific phosphodiesterases, phosphatases to known substrates. In neurons AKAP150 organizes many of the signaling molecules known to regulate the excitability and intrinsic pulsatile activity of GnRH neurons. AKAP150 was expressed in both the GT1-1 and GT1-7 cells. We determined the role of AKAP150 in coordinating GT1-1 cell excitability and intrinsic GnRH pulsatile secretion by lowering AKAP150 levels with a small interfering RNA (siRNA) adenovirus construct to AKAP150 (Ad-AKAP150-siRNA). Infection with Ad-AKAP150-siRNA specifically decreased AKAP150 mRNA levels by 74% and protein levels by 53% relative to uninfected cells or cells infected with a luciferase control adenovirus siRNA vector. In GT1 cells, spontaneous Ca(2+) oscillations, an index of neuron excitability, are stimulated by increased levels of intracellular cAMP and lowered by decreased levels. The frequency of spontaneous Ca(2+) oscillations in Ad-AKAP150-siRNA-treated GT1-1 cells decreased by 47.2% relative to controls. A dramatic decrease in the number of spontaneous GnRH pulses was also observed after infection with Ad-AKAP150-siRNA. The interpulse interval increased to 143 +/- 20.25 min in Ad-AKAP150-siRNA infected cells from 32.2 +/- 7.3 min in luciferase control adenovirus siRNA vector-infected cells. These data demonstrate an important role of AKAP150 in coordinating signaling events regulating the frequency of intrinsic pulsatile GnRH secretion.

Role of Seminiferous Tubular Development in Determining the FSH versus LH Responsiveness to GnRH in Early Sexual Maturation

Background: The onset of sexual maturation at puberty is a unique developmental period from a neuroendocrine perspective in that it is characterized by enhanced FSH secretion and FSH responsiveness to exogenous GnRH (vs. LH) from the gonadotrope, yet the mechanism of these dynamics remains unclear. This study aimed to elucidate this phenomenon using a human disease model of GnRH deficiency (idiopathic hypogonadotropic hypogonadism, IHH) in which GnRH input can be experimentally controlled. Methods: 25 GnRH-deficient men were selected for study based upon their baseline testicular volumes (TV) and serum inhibin B (I_B) levels to represent a spectrum of pubertal/testicular development. Subjects underwent: (i) a 12-hour overnight neuroendocrine evaluation for hormonal profiling and determination of endogenous LH secretion pattern, and (ii) a 7-day exposure to a physiologic regimen of exogenous pulsatile GnRH (25 ng/kg every 2 h). Daily measurements of serum testosterone (T) and I_B levels were made and a 2-hour window of frequent blood sampling was monitored to measure LH and FSH following a single i.v. GnRH bolus (25 ng/kg). All subjects were screened for known loci underlying GnRH deficiency and the response to GnRH was tracked according to genotype. Results: Among the entire cohort, no changes were noted in serum T or I_B during the 7 days, thus keeping gonadal feedback relatively constant. However, serum LH and FSH levels increased significantly (p < 0.0001) in the entire cohort. When analyzed by degree of pubertal/testicular development, men with no evidence of prior spontaneous pubertal development (TV ≤3 ml, Group I) showed sharp increases in serum FSH compared to men with some prior evidence of partial puberty (TV >3 ml, Group II, p < 0.0001). Group I exhibited a decreased LH response to GnRH on day 2 compared to day 1 (p < 0.01), which did not recover until day 5 (1–4 vs. 5–7 days, p < 0.0001). Group II displayed robust and equivalent LH responses to GnRH throughout the 7-day study. Genetic studies identified 8 mutations in 4 different loci (DAX1, KAL1, GNRHR, and FGFR1) in this cohort. Conclusions: GnRH-deficient men undergoing GnRH-induced sexual maturation display an inverse relationship between FSH responsiveness to GnRH and baseline testicular size and I_B levels. This observation implies that increasing seminiferous tubule maturity represents the major constraint on FSH responsiveness to GnRH in early puberty. In contrast, LH responsiveness to GnRH correlates directly with duration of GnRH exposure. Attenuated pituitary gonadotropin responses were noted in subjects harboring DAX1 mutations, consistent with known pituitary defects.

Regulation of Lhb and Egr1 gene expression by GNRH pulses in rat pituitaries is both c-Jun N-terminal kinase (JNK)- and extracellular signal-regulated kinase (ERK)-dependent.

Pulsatile GNRH regulates the gonadotropin subunit genes in a differential manner, with faster frequencies favoring Lhb gene expression and slower frequencies favoring Fshb. Early growth response 1 (EGR1) is critical for Lhb gene transcription. We examined GNRH regulation of EGR1 and its two corepressors, Ngfi-A-binding proteins 1 and 2 (NAB1 and NAB2), both in vivo and in cultured rat pituitary cells. In rats, fast GNRH pulses (every 30 min) stably induced Egr1 primary transcript (PT) and mRNA 2-fold (P < 0.05) for 1-24 h. In contrast, slow GNRH pulses (every 240 min) increased Egr1 PT at 24 h (6-fold; P < 0.05) but increased Egr1 mRNA 4- to 5-fold between 4 and 24 h. Both GNRH pulse frequencies increased EGR1 protein 3- to 4-fold. In cultured rat pituitary cells, GNRH pulses (every 60 min) increased Egr1 (PT, 2.5- to 3-fold; mRNA, 1.5- to 2-fold; P < 0.05). GNRH pulses had little effect on Nab1/2 PT/mRNAs either in vivo or in vitro. We also examined specific intracellular signaling cascades activated by GNRH. Inhibitors of mitogen-activated protein kinase 8/9 (MAPK8/9 [also known as JNK]; SP600125) and MAP Kinase Kinase 1 (MAP2K1 [also known as MEK1]; PD98059) either blunted or totally suppressed the GNRH induction of Lhb PT and Egr1 PT/mRNA, whereas the MAPK14 (also known as p38) inhibitor SB203580 did not. In summary, pulsatile GNRH stimulates Egr1 gene expression and protein in vivo but not in a frequency-dependent manner. Additionally, GNRH-induced Egr1 gene expression is mediated by MAPK8/9 and MAPK1/3, and both are critical for Lhb gene transcription.

Changes in anterior pituitary response in patients with idiopathic hypothalamic hypogonadism caused by pulsatile GnRH therapy and testosterone replacement.

This study evaluated in male patients with idiopathic hypothalamic hypogonadism the effect of pulsatile GnRH therapy or testosterone replacement on the response of all anterior pituitary hormones to adequate dynamic stimuli. In nine patients with idiopathic hypothalamic hypogonadism--mean age 21 +/- 1 (mean +/- SE)--a combined pituitary stimulation (CPS) with 200 micrograms TRH, 100 micrograms GnRH, 100 micrograms CRH and 100 micrograms GRH and an insulin tolerance-test (ITT) with 0.1 U insulin/kg body weight were performed. Both tests were repeated during pulsatile GnRH therapy and thereafter on testosterone replacement. Hormone levels were measured by immunometric assays. For statistical analysis the area under the curve (AUC) was used as a measure for hormone response. Testosterone levels did not differ significantly during GnRH therapy (16.6 +/- 2.1 nmol/L) and testosterone replacement (18.5 +/- 1.7 nmol/L). No significant differences were observed before and during the two treatment modalities for TSH and ACTH. PRL increase was significantly higher during GnRH therapy (AUC: 73580 +/- 8940) compared to the rise before treatment (AUC: 36161 +/- 5853; p < 0.01) and on testosterone replacement (AUC: 49995 +/- 6158; p < 0.01). The GH response to CPS and ITT was higher under testosterone replacement (AUC: 1826 +/- 353 and 1423 +/- 125) compared with the pretreatment situation (AUC: 727 +/- 115; p < 0.05 and 541 +/- 110; p < 0.01) and also more pronounced than under GnRH therapy (AUC: 1148 +/- 180 and 798 +/- 129; p < 0.05). FSH and LH after CPS rose significantly more during GnRH therapy (AUC: 864 +/- 122 and 2215 +/- 219) than before (AUC: 418 +/- 61 and 1424 +/- 277; p < 0.01) and on testosterone treatment (342 +/- 83 and 1153 +/- 323; p < 0.05). These results show that GnRH exerts a stimulatory effect on PRL secretion and may modulate GH secretion independently from sex steroids.

Involvement of gamma amino butyric acid (GABA) in the postnatal function of the GnRH pulse generator as determined on the basis of GnRH and GnRH-receptor gene expression in the hypothalamus and the pituitary

In many species the GnRH pulse generator functions early postnatally to become arrested during infancy. In rats highly variable LH levels in 15-day-old animals are suggestive that LH is being released by the pituitary in pulses whereas between day 20 after birth and puberty LH levels are low indicating that the GnRH pulse generator is arrested. In the present study we show on the basis of consecutively withdrawn blood samples in 15-day-old animals that LH pulses are indeed present at that age. The proper function of GnRH receptors in the pituitary is crucially dependent on pulsatile GnRH release from the hypothalamus. In addition, GnRH receptors have been demonstrated in the medial preoptic area and in the mediobasal hypothalamus of adult rats. In 15-day-old animals the functional GnRH pulse generator results in upregulated GnRH receptor gene expression as demonstrated by quantitative RT-PCR. It is not known what neural mechanisms are involved in turning the GnRH pulse generator off during infancy and a GABAergic brake has been discussed. Indeed, when 30-day-old animals were injected with the GABA-A receptor blocking drug bicuculline, this resulted in increased serum LH levels indicating that a tonic GABAergic inhibition is indeed operative at this age.

Isolated Familial Hypogonadotropic Hypogonadism and aGNRH1Mutation

We investigated whether mutations in the gene encoding gonadotropin-releasing hormone 1 (GNRH1) might be responsible for idiopathic hypogonadotropic hypogonadism (IHH) in humans. We identified a homozygous GNRH1 frameshift mutation, an insertion of an adenine at nucleotide position 18 (c.18-19insA), in the sequence encoding the N-terminal region of the signal peptide-containing protein precursor of gonadotropin-releasing hormone (prepro-GnRH) in a teenage brother and sister, who had normosmic IHH. Their unaffected parents and a sibling who was tested were heterozygous. This mutation results in an aberrant peptide lacking the conserved GnRH decapeptide sequence, as shown by the absence of immunoreactive GnRH when expressed in vitro. This isolated autosomal recessive GnRH deficiency, reversed by pulsatile GnRH administration, shows the pivotal role of GnRH in human reproduction.

Lutropin alpha, recombinant human luteinizing hormone, for the stimulation of follicular development in profoundly LH-deficient hypogonadotropic hypogonadal women: a review

Lutropin alpha, recombinant human luteinizing hormone, for the stimulation of follicular development in profoundly LH-deficient hypogonadotropic hypogonadal women: a review Bernd Th Krause1, Ralf Ohlinger2, Annette Haase31Center for Endocrinology and Reproductive Medicine, MVZ Uhlandstr, Berlin, Germany; 2Ernst-Moritz-Arndt-University, Department of Gynecology and Obstetrics, Greifswald, Germany; 3Uhlandstr. 162, 10719 BerlinAbstract: Hypogonadotropic hypogonadism is defined as a medical condition with low or undetectable gonadotropin secretion, associated with a complete arrest of follicular growth and very low estradiol. The main cause can be traced back to an irregular or absent hypothalamic GnRH secretion, whereas only a minority suffers from a pituitary disorder. The choice of treatment to reverse this situation is a pulsatile GnRH application or a direct ovarian stimulation using gonadotropin injections. The goal is to achieve a proper ovarian function in these cases for a short time to allow ovulation and chance of pregnancy. Since the pulsatile GnRH treatment lost its former importance, several gonadotropins are in use to stimulate follicular growth, such as urine-derived human menopausal gonadotropin, highly purified follicle stimulating hormone (FSH) or recombinant FSH, all with different success. The introduction of recombinant luteinizing hormone (LH) and FSH provided an opportunity to investigate the distinct influences of LH and FSH alone and in combination on follicular growth in monofollicular ovulation induction cycles, and additionally on oocyte maturation, fertilization competence of the oocyte and embryo quality in downregulated IVF patients. Whereas FSH was known to be indispensable for normal follicular growth, the role of LH remained questionable. Downregulated IVF patients with this short-term gonadotropin depletion displayed no advance in stimulation success with the use of recombinant LH. Patients with hypogonadotropic hypogonadism undergoing monofollicular stimulation for ovulation induction showed clearly a specific role and need for both hormones in normal follicular growth. Therefore, a combined stimulation with FSH and LH seems to be the best treatment choice. In the first half of the stimulation cycle the FSH dosage should exceed that of LH by 2:1, with an inverse ratio for the second half.Keywords: hypogonadotropic hypogonadism, ovarian stimulation, ovulation induction, recombinantluteinizing hormone

Lutropin alpha, recombinant human luteinizing hormone, for the stimulation of follicular development in profoundly LH-deficient hypogonadotropic hypogonadal women: a review.

Hypogonadotropic hypogonadism is defined as a medical condition with low or undetectable gonadotropin secretion, associated with a complete arrest of follicular growth and very low estradiol. The main cause can be traced back to an irregular or absent hypothalamic GnRH secretion, whereas only a minority suffers from a pituitary disorder. The choice of treatment to reverse this situation is a pulsatile GnRH application or a direct ovarian stimulation using gonadotropin injections. The goal is to achieve a proper ovarian function in these cases for a short time to allow ovulation and chance of pregnancy. Since the pulsatile GnRH treatment lost its former importance, several gonadotropins are in use to stimulate follicular growth, such as urine-derived human menopausal gonadotropin, highly purified follicle stimulating hormone (FSH) or recombinant FSH, all with different success. The introduction of recombinant luteinizing hormone (LH) and FSH provided an opportunity to investigate the distinct influences of LH and FSH alone and in combination on follicular growth in monofollicular ovulation induction cycles, and additionally on oocyte maturation, fertilization competence of the oocyte and embryo quality in downregulated IVF patients. Whereas FSH was known to be indispensable for normal follicular growth, the role of LH remained questionable. Downregulated IVF patients with this short-term gonadotropin depletion displayed no advance in stimulation success with the use of recombinant LH. Patients with hypogonadotropic hypogonadism undergoing monofollicular stimulation for ovulation induction showed clearly a specific role and need for both hormones in normal follicular growth. Therefore, a combined stimulation with FSH and LH seems to be the best treatment choice. In the first half of the stimulation cycle the FSH dosage should exceed that of LH by 2:1, with an inverse ratio for the second half.

Expression of a Gonadotropin-Releasing Hormone Receptor-Simian Virus 40 T-Antigen Transgene Has Sex-Specific Effects on the Reproductive Axis

The GnRH receptor (GnRHR) responds to pulsatile GnRH signals to coordinate pituitary gonadotropin synthesis and secretion. Previously, a 1.2-kb fragment of the 5'-flanking region isolated from the mouse GnRHR gene was shown to target expression to pituitary gonadotropes in vivo. The 1.2-kb gene promoter fused to the simian virus 40 large T antigen (TAg) was used to generate transgenic mice that form gonadotrope-derived pituitary tumors at 4-5 months of age. Transgenic female mice have hypogonadotropic hypogonadism, infantile gonads, and are infertile throughout their life span, whereas males remain reproductively intact until their tumors become large. We hypothesized that the targeted TAg expression causes a sex-specific disruption of the reproductive axis at the level of the pituitary gland. To test this hypothesis, we characterized the pituitary gonadotropin beta-subunit and TAg expression patterns, and measured plasma gonadotropin and gonadal steroid levels in female and male mice before and after pituitary tumor development. TAg expression was observed in transgenic females and males 15 d of age, before tumor development. Interestingly, and in contrast to the transgenic males, pituitary LH beta and FSH beta subunit protein levels, and plasma LH and FSH levels, were reduced in transgenic females. Reproductive organs in transgenic female mice remained underdeveloped but were normal in transgenic males. We conclude that the expression of the TAg transgene driven by the GnRHR gene promoter results in female-specific infertility due to disruption of gonadotropin production and secretion even before tumor development.

Role of Estradiol in Cortisol-Induced Reduction of Luteinizing Hormone Pulse Frequency

Precise control of pulsatile GnRH and LH release is imperative to ovarian cyclicity but is vulnerable to environmental perturbations, like stress. In sheep, a sustained (29 h) increase in plasma cortisol to a level observed during stress profoundly reduces GnRH pulse frequency in ovariectomized ewes treated with ovarian steroids, whereas shorter infusion (6 h) is ineffective in the absence of ovarian hormones. This study first determined whether the ovarian steroid milieu or duration of exposure is the relevant factor in determining whether cortisol reduces LH pulse frequency. Prolonged (29 h) cortisol infusion did not lower LH pulse frequency in ovariectomized ewes deprived of ovarian hormones, but it did so in ovariectomized ewes treated with estradiol and progesterone to create an artificial estrous cycle, implicating ovarian steroids as the critical factor. Importantly, this effect of cortisol was more pronounced after the simulated preovulatory estradiol rise of the artificial follicular phase. The second experiment examined which component of the ovarian steroid milieu enables cortisol to reduce LH pulse frequency in the artificial follicular phase: prior exposure to progesterone in the luteal phase, low early follicular phase estradiol levels, or the preovulatory estradiol rise. Basal estradiol enabled cortisol to decrease LH pulse frequency, but the response was potentiated by the estradiol rise. These findings lead to the conclusion that ovarian steroids, particularly estradiol, enable cortisol to inhibit LH pulse frequency. Moreover, the results provide new insight into the means by which gonadal steroids, and possibly reproductive status, modulate neuroendocrine responses to stress.

GnRH Pulsatility, the Pituitary Response and Reproductive Dysfunction

GnRH plays an essential role in neuroendocrine control of reproductive function. In mammals, the pattern of gonadotropin secretion includes both pulse and surge phases, which are regulated independently. The pulsatile release of GnRH and LH plays an important role in the development of sexual function and in the normal regulation of the menstrual cycle. The importance of GnRH pulsatility was established in a series of classic studies. Fertility is impaired when GnRH pulsatility is inhibited by chronic malnutrition, excessive caloric expenditure, or aging. A number of reproductive disorders in women with including hypogonadotropic hypogonadism, hypothlamic amenorrhea, hyperprolactinemia and polycystic ovary syndrome (PCOS) are also associated with disruption of the normal pulsatile GnRH secretion. Despite these findings, the molecular mechanisms of this pulsatile GnRH regulation are not well understood. Here, we review recent studies about GnRH pulsatility, signaling and transcriptional response, and its implications for disease.

Proteasome Regulation of Dynamic Transcription Factor Occupancy on the GnRH-Stimulated Luteinizing Hormone β-Subunit Promoter

GnRH is the main modulator of LH secretion and transcription of the LH subunit genes in pituitary gonadotropes. The LHbeta gene is preferentially transcribed during pulsatile GnRH stimuli of one pulse/30 min and is thus carefully controlled by specific signaling pathways and transcription factors. We now show that GnRH-stimulated LHbeta transcription is also influenced by the ubiquitin-proteasome system. GnRH-stimulated activity of an LHbeta reporter gene was prevented by proteasome inhibitors MG-132 and lactacystin. Inhibition was not rescued by overexpression of two key transcription factors for LHbeta, early growth response-1 (Egr-1) and steroidogenic factor-1 (SF-1). Increased endogenous LHbeta transcription after GnRH treatment was also prevented by MG-132, as measured by primary transcript assays. To investigate possible mechanisms of LHbeta transcriptional inhibition by proteasome blockade, we employed chromatin immunoprecipitation to measure LHbeta promoter occupancy by transcription factors. Without GnRH, binding was low and unorganized. With GnRH, Egr-1 and SF-1 associations were stimulated, cyclic, and coincidental, with a period of approximately 30 min. MG-132 disrupted GnRH-induced Egr-1 and SF-1 binding and prevented phosphorylated RNA polymerase II association with the LHbeta promoter. Egr-1, but not SF-1, protein was induced by GnRH and accumulated with MG-132. Egr-1 and SF-1 were ubiquitinated in gonadotropes and ubiquitinated forms of these factors associated with the LHbeta promoter, suggesting their degradation may be key for LHbeta proteasome-dependent transcription. Together, these results demonstrate that degradation via the proteasome is vital to GnRH-stimulated LHbeta expression, and this occurs in part by allowing proper transcription factor associations with the LHbeta promoter.

Psychosocial Stress Inhibits Amplitude of Gonadotropin-Releasing Hormone Pulses Independent of Cortisol Action on the Type II Glucocorticoid Receptor

Our laboratory has developed a paradigm of psychosocial stress (sequential layering of isolation, blindfold, and predator cues) that robustly elevates cortisol secretion and decreases LH pulse amplitude in ovariectomized ewes. This decrease in LH pulse amplitude is due, at least in part, to a reduction in pituitary responsiveness to GnRH, caused by cortisol acting via the type II glucocorticoid receptor (GR). The first experiment of the current study aimed to determine whether this layered psychosocial stress also inhibits pulsatile GnRH release into pituitary portal blood. The stress paradigm significantly reduced GnRH pulse amplitude compared with nonstressed ovariectomized ewes. The second experiment tested if this stress-induced decrease in GnRH pulse amplitude is mediated by cortisol action on the type II GR. Ovariectomized ewes were allocated to three groups: nonstress control, stress, and stress plus the type II GR antagonist RU486. The layered psychosocial stress paradigm decreased GnRH and LH pulse amplitude compared with nonstress controls. Importantly, the stress also lowered GnRH pulse amplitude to a comparable extent in ewes in which cortisol action via the type II GR was antagonized. Therefore, we conclude that psychosocial stress reduces the amplitude of GnRH pulses independent of cortisol action on the type II GR. The present findings, combined with our recent observations, suggest that the mechanisms by which psychosocial stress inhibits reproductive neuroendocrine activity at the hypothalamic and pituitary levels are fundamentally different.

Side effects of pulsatile GnRH therapy for induction of ovulation

Summary Pulsatile administration of GnRH by means of a portable pump is an efficient and practical method for the induction of ovulation as a treatment of infertility in hypothalamic amenorrhea. It represents a replacement therapy based on physiological observations. Since the feedback mechanisms between the ovary and pituitary gland remain intact, it is associated with a very low risk of multiple follicular devel -opment and multiple pregnancies. However, the results obtained with this method are critically dependent upon the correct selection of patients as far as the diagnosis of hypothalamic amenorrhea is con -cerned. If this is the case, pulsatile GnRH therapy is safe, associated with a very low risk of multiple pregnancy and, therefore, has to be considered as the therapy of choice in hypothalamic amenorrhea. Financial & competing interests disclosure The authors have no relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript. This includes employment, consultancies, honoraria, stock ownership or options, expert testimony, grants or patents received or pending, or royalties.No writing assistance was utilized in the production of this manuscript.

Regulation of Intracellular Signaling Cascades by GNRH Pulse Frequency in the Rat Pituitary: Roles for CaMK II, ERK, and JNK Activation1

Pulsatile GnRH (GNRH) differentially regulates LH and FSH subunit genes, with faster frequencies favoring Lhb transcription and slower favoring Fshb. Various intracellular pathways mediate the effects of GNRH, including CaMK II (CAMK2), ERK, and JNK. We examined whether activation of these pathways is regulated by GNRH pulse frequency in vivo. GNRH-deficient rats received GNRH pulses (25 ng i.v. every 30 or 240 min for 8 h, vehicle to controls). Pituitaries were collected 5 min after the last pulse, bisected, and one half processed for RNA (to measure beta subunit primary transcripts [PTs]) and the other for protein. Phosphorylated CAMK2 (phospho-CAMK2), ERK (mitogen-activated protein kinase 1/3 [MAPK1/3], also known as p42 ERK2 and p44 ERK1, respectively), and JNK (MAPK8/9, also known as p46 JNK1 and p54 JNK2, respectively) were determined by Western blotting. The 30-min pulses maximally stimulated Lhb PT (8-fold), whereas 240 min was optimal for Fshb PT (3-fold increase). Both GNRH pulse frequencies increased phospho-CAMK2 4-fold. Activation of MAPK1/3 was stimulated by both 30- and 240-min pulses, but phosphorylation of MAPK3 was significantly greater following slower GNRH pulses (240 min: 4-fold, 30 min: 2-fold). MAPK8/9 activation was unchanged by pulsatile GNRH in this paradigm, but as previous results showed that GNRH-induced activation of MAPK8/9 is delayed, 5 min after GNRH may not be optimal to observe MAPK8/9 activation. These data show that CAMK2 is activated by GNRH, but not in a frequency-dependant manner, whereas MAPK3 is maximally stimulated by slow-frequency GNRH pulses. Thus, the ERK response to slow pulse frequency is part of the mechanisms mediating Fhb transcriptional responses to GNRH.

Converse Regulatory Functions of Estrogen Receptor-α and -β Subtypes Expressed in Hypothalamic Gonadotropin-Releasing Hormone Neurons

Estradiol (E(2)) acts as a potent feedback molecule between the ovary and hypothalamic GnRH neurons, and exerts both positive and negative regulatory actions on GnRH synthesis and secretion. However, the extent to which these actions are mediated by estrogen receptors (ERs) expressed in GnRH neurons has been controversial. In this study, Single-cell RT-PCR revealed the expression of both ERalpha and ERbeta isoforms in cultured fetal and adult rat hypothalamic GnRH neurons. Both ERalpha and ERbeta or individual ERs were expressed in 94% of cultured fetal GnRH neurons. In adult female rats at diestrus, 68% of GnRH neurons expressed ERs, followed by 54% in estrus and 19% in proestrus. Expression of individual ERs was found in 24% of adult male GnRH neurons. ERalpha exerted marked G(i)-mediated inhibitory effects on spontaneous action potential (AP) firing, cAMP production, and pulsatile GnRH secretion, indicating its capacity for negative regulation of GnRH neuronal function. In contrast, increased E(2) concentration and ERbeta agonists increase the rate of AP firing, GnRH secretion, and cAMP production, consistent with ERbeta-dependent positive regulation of GnRH secretion. Consonant with the coupling of ERalpha to pertussis toxin-sensitive G(i/o) proteins, E(2) also activates G protein-activated inwardly rectifying potassium channels, decreasing membrane excitability and slowing the firing of spontaneous APs in hypothalamic GnRH neurons. These findings demonstrate that the dual actions of E(2) on GnRH neuronal membrane excitability, cAMP production, and GnRH secretion are mediated by the dose-dependent activation of ERalpha and ERbeta expressed in hypothalamic GnRH neurons.

Kisspeptin and the regulation of the hypothalamic–pituitary–gonadal axis in the rhesus monkey ( Macaca mulatta)

The present article reviews recent studies of monkeys and, in some cases, humans that have been conducted to examine the role of kisspeptin-GPR54 signaling in the regulation of the hypothalamic–pituitary–gonadal axis in higher primates. This area of peptide biology was initiated in 2003 by the discovery that loss of function mutations of GPR54 in man were associated with hypogonadotropic hypogonadism and absent or delayed puberty. Puberty in the monkey, an experimental model commonly used to study this fundamental developmental stage, is first described. This is followed by a review of the role of kisspeptin in the regulation of the postnatal ontogeny of GnRH pulsatility. The roles of kisspeptin in GnRH pulse generation and in the feedback loops governing gonadotropin secretion in primates are then discussed. A brief section on kisspeptin-GPR54 signaling at the pituitary and gonadal levels is also included. The review concludes with a discussion of the phenomenon of GPR54 downregulation by continuous exposure to kisspeptin and its therapeutic implications.

Development of hyperandrogenemia in women receiving pulsatile GnRH therapy

Development of hyperandrogenemia in women receiving pulsatile GnRH therapy