Pituitary GnRH Receptor Research Articles

Orally active gnRH-antagonist AEZS-115 inhibits growth of human ovarian and breast cancers in vitro.

Abstract Abstract #3065 Introduction: AEZS-115 is an orally active peptidomimetic antagonist of GnRH, which has been shown to block the pituitary GnRH receptor in nanomolar range. Recently an increasing number of human cancers have been demonstrated to express receptors for GnRH, which mediate growth promoting signals. Accordingly, we tested the antitumor activity of AEZS-115 in in vitro models of human breast and ovarian cancer.
 Material and Methods: Human ovarian SKOV3, Ovcar 3 as well breast cancer cell line MDA-MB 468 were analyzed for GnRH receptor expression by immunocytochemistry. To screen for a putative anti-tumor effect, these cell lines were incubated with increasing concentrations of AEZS-115, peptidic GnRH-antagonist Cetrorelix and GnRH-agonist Triptorelin (1, 10, 100 µm) for 48 hours and the number of viable cells was determined by crystal violet staining as well as by ATP-dependent luminometric assay.
 Results: Cell lines SKOV3, OVCAR3 and MDA-MB 468 expressed GnRH I receptors as demonstrated by immunehistochemistry. Both GnRH-antagonists dose dependently inhibited growth of all three cell lines, while GnRH-agonist Triptorelin showed marginal growth inhibition. At 10 µM AEZS-115 inhibited cell growth by 40-60%, at 100 µM growth inhibition was 60-80%. Inhibition with Cetrorelix at 100 µM ranged from 20-40%, while only minor effects on cell growth were seen at 10 µM. These results obtained by crystal violet staining were confirmed by additional luminometric evaluation of the ATP content after treatment with the respective substances.
 Conclusions: To our knowledge here we report for the first time in vitro anti-cancer activity for a peptidomimetic GnRH antagonist.
 AEZS-115 showed substantial anti-tumor activity in human ovarian and breast cancer cell lines, while GnRH-agonist Triptorelin showed only minor growth inhibition. Surprisingly AEZS-115 was somewhat more potent than peptidic GnRH-antagonist Cetrorelix. Due to the good antitumor-activity and the possible oral administration AEZS-115 should be a promising candidate for in vivo studies. Citation Information: Cancer Res 2009;69(2 Suppl):Abstract nr 3065.

A 1-Year, Open Label, Randomized Phase II Dose Finding Study of Degarelix for the Treatment of Prostate Cancer in North America

Degarelix is a gonadotropin-releasing hormone receptor antagonist (blocker) with rapid onset of action suppressing gonadotropins, testosterone and prostate specific antigen in prostate cancer. In the present open label, randomized study in North America we evaluated the efficacy and safety of a starting dose of 200 mg degarelix followed by monthly injections of 60 or 80 mg during 1 year of prostate cancer treatment. A total of 127 patients (median age 76 years, range 47 to 93) with histologically confirmed prostate cancer were enrolled in the study. Efficacy was assessed by measuring serum testosterone and prostate specific antigen. Median baseline testosterone and prostate specific antigen levels were 4.13 ng/ml (P25-P75 3.03-5.11) and 13.4 ng/ml (P25-P75 6.80-25.7), respectively. The starting dose induced a rapid suppression of testosterone in that 88% of the patients had testosterone levels of 0.5 ng/ml or less 1 month after the injection. For patients who had testosterone levels of 0.5 ng/ml or less after 1 month, 93% and 98% of those receiving maintenance doses of 60 and 80 mg, respectively, had testosterone levels that were consistently 0.5 ng/ml or less at all monthly measurements from 1 month to 1 year. No evidence of testosterone surge was detected. In both groups prostate specific antigen decreased by 96% after 1 year and median time to 90% reduction in prostate specific antigen was 56 days. Six patients (5%) withdrew from the study due to adverse events. Degarelix treatment for 1 year resulted in a fast, profound and sustained suppression of testosterone and prostate specific antigen with no evidence of testosterone surge. Degarelix was well tolerated.

Changes in the GnRH mRNA and GnRH receptor (GnRH-R) mRNA levels in the hypothalamic-anterior pituitary unit of anestrous ewes after infusion of GnRH into the third cerebral ventricle

In the present paper the role of GnRH in the ultrashort loop of the negative feedback action on GnRH secretion was evaluated on the molecular level by the Real-time PCR technique. Specifically, the effect of GnRH infused into the third cerebral ventricle on the expression of GnRH and GnRH receptor (GnRH-R) genes was analyzed in the hypothalamic-pituitary unit of anestrous ewes. GnRH did not significantly affect GnRH mRNA levels in the preoptic/anterior hypothalamic area but drastically increased its level in the ventromedial hypothalamus. In addition, GnRH infusion augmented GnRH-R mRNA level in the entire hypothalamus. In the GnRH-treated animals, anterior pituitary GnRH-R mRNA level and plasma LH concentration were also elevated. The changes in GnRH mRNA and GnRH-R mRNA levels in the hypothalamus in response to treatment with GnRH suggest that GnRH acts differently on the stability of these transcripts. On the basis of presented results it seems that GnRH may affect GnRH and GnRH-R biosynthesis and, consequently, GnRH/LH release.

Pretreatment with gonadotropin-releasing hormone (GnRH) antagonists to prevent the flare-up effect of long-acting GnRH agonists: results of a pilot study

This study evaluated in vivo whether the flare-up effect of GnRH agonists can be suppressed through pretreatment with a GnRH antagonist. The classic flare-up effect caused by 3.8 mg goserelin acetate could not be suppressed through pretreatment using a single dosage of 3.24 mg cetrorelix acetate.

Developmental Programming: Impact of Prenatal Testosterone Excess on Pre- and Postnatal Gonadotropin Regulation in Sheep1

The goal of this study was to explore mechanisms that mediate hypersecretion of LH and progressive loss of cyclicity in female sheep exposed during fetal life to excess testosterone. Our working hypothesis was that prenatal testosterone excess, by its androgenic action, amplifies GnRH-induced LH (but not FSH) secretion and, thus, hypersecretion of LH in adulthood, and that this results from altered developmental gene expression of GnRH and estradiol (E2) receptors, gonadotropin subunits, and paracrine factors that differentially regulate LH and FSH synthesis. We observed that, relative to controls, females exposed during fetal life to excess testosterone, as well as the nor-aromatizable androgen dihydrotestosterone, exhibited enhanced LH but not FSH responses to intermittent delivery of GnRH boluses under conditions in which endogenous LH (GnRH) pulses were suppressed. Luteinizing hormone hypersecretion was more evident in adults than in prepubertal females, and it was associated with development of acyclicity. Measurement of pituitary mRNA concentrations revealed that prenatal testosterone excess induced developmental changes in gene expression of pituitary GnRH and E2 receptors and paracrine modulators of LH and FSH synthesis in a manner consistent with subsequent amplification of LH release. Together, this series of studies suggests that prenatal testosterone excess, by its androgenic action, amplifies GnRH-induced LH response, leading to LH hypersecretion and acyclicity in adulthood, and that this programming involves developmental changes in expression of pituitary genes involved in LH and FSH release.

Peroxisome Proliferator-activated Receptor ??Is Involved in Weaning to Estrus of Primiparous Sows by Regulating the Expression of Hormone Genes in Hypothalamus-pituitary-ovary Axis

The objective of this study was to determine whether peroxisome proliferator-activated receptor y (PPARγ) is involved in the regulation of weaning to estrus of primiparous sows. Twelve sows composed of 6 groups of 2 full-sibs in a similar age (325.2 d), body weight (BW; 152.4 kg) and backfat thickness (BFT; 27.0 mm) at start of lactation, were allocated to accept 31 MJ (restricted group, R-group) or 53 MJ (control group, C-group) DE/d treatment, respectively. The experimental results indicated that the low energy intake resulted in excessive losses of BW and BFT during lactation in R-group sows, which may be related to decrease of serum 15-deoxy-Δ 12,14 -prostaglandin J 2 (15d-PGJ 2 ), a ligand of PPARγ. The obvious peak and the frequency of LH, FSH and estradiol (E 2 ) were only observed in C-group sows. Except for E 2 at d 1 and 2, serum FSH, LH and E 2 concentrations in R-group were lower than those in C-group sows after weaning. However, the serum progesterone (P 4 ) level in R-group sows was always more than that in C-group. The expression abundances of PPARγ and GnRH receptor (GnRH-R) in pituitary, FSH receptor (FSH-R), LH receptor (LH-R), estrogen receptor (ES-R) and aromatase in ovary of anestrous sows were lower than those of estrous sows. Neither the BFT nor the BW was associated with the mRNA abundance of PPARγ in hypothalamus during lactation. Expressions of PPARγ in pituitary and ovary were affected evidently by the BFT changes and only by the loss of BW of sows during and after lactation. Furthermore, PPARγ mRNA level in ovary was significantly related to the expression abundances of GnRH-R, FSH-R, ES-R and aromatase, and GnRH-R was obviously associated with PPARγ expression in pituitary. However, PPARγ expression in hypothalamus likely has no effects on these genes expression and no obvious difference for all sows. Not serum E 2 or P 4 alone but the ratios of E 2 to P 4 and 15d-PGJ 2 to P 4 , and serum FSH and LH were evidently related to PPARγ expression in pituitary and ovary. It is concluded that PPARγ is associated with body conditions, reproduction hormones and their receptor expression, which affected the functions of pituitary and ovary and ultimately the estrus after weaning of primiparous sows.

In vivo characterization of a novel GnRH (gonadotropin-releasing hormone) antagonist, LXT-101, in normal male rats

LXT-101 is a newly developed GnRH (gonadotropin-releasing hormone) analogue. In this study, the in vivo pharmacological profile in intact male rats and binding characters of LXT-101 were illustrated, and regulation of mRNA of hormone receptors related to the pituitary–gonadal axis during and after administration was observed to reveal its molecular mechanism of potent effect and reversibility. After single subcutaneous injections, LXT-101 produced a dose- and time-dependent suppression of serum testosterone level. Multiple administrations and osmotic pump implantation revealed that the time of onset and dose needed to maintain the effect of chemical castration decreased as the frequency of injection increased and gave direct proof that depot formulation could significantly improve the duration of antagonist delivery and pharmacological activities compared to the injectable formulation. And LXT-101 showed excellent character of regulating the pituitary–gonadal axis quickly and reversibly. Competitive binding assay showed that LXT-101 could specifically bind a pituitary GnRH receptor with high affinity. These results indicated that LXT-101 is fit for sustained-release formulation and it might possibly be developed as an ideal candidate for treating sex hormone-sensitive tumors and other disorders.

‘Effective inefficiency’: cellular control of protein trafficking as a mechanism of post-translational regulation

The great writer and polyglot, W Somerset Maugham said, 'I'll give you my opinion of the human race in a nutshell...their heart's in the right place, but their head is a thoroughly inefficient organ.' If his words are applied to trafficking of the human pituitary gonadotropin-releasing hormone receptor, it turns out that he was more right than he knew. Paradoxically, the inefficiency of receptor trafficking to the plasma membrane can bring regulatory advantages to cells. Understanding the mechanism by which cells recognize correctly folded proteins in health and disease opens doors to new therapeutic approaches and provides a more accurate view of mechanisms of normal cell function than is presently available.

Sex Steroids Are Involved in the Regulation of Gonadotropin-Releasing Hormone and Dopamine D2 Receptors in Female Tilapia Pituitary1

Although molecular mechanisms underlying steroid effects on GnRH and dopamine receptors are well documented in mammals, little is known in fish. Herein, we describe the expression of pituitary GnRH and dopamine receptors relative to gonadotropin expression and release. We exposed female tilapia to graded doses of estradiol or 17alpha,20beta-dihydroxy-4-pregnen-3-one (DHP) in vitro, and of estradiol in vivo, and determined mRNA levels of gnrhr1, gnrhr3, drd2, lhb, and fshb by real-time PCR. We also determined gonadotropin levels using specific ELISAs. Exposure to low doses of estradiol caused increased gnrhr3 mRNA levels in vivo and in vitro, probably related to positive feedback on FSH release. Increasing concentrations of estradiol resulted in increased drd2 mRNA levels in vivo and in vitro, inhibition of LH and FSH release, and inhibition of lhb mRNA levels in vivo, possibly related to negative feedback. At high doses of estradiol, FSH release increased in preparation for a new generation of follicles. Exposure to nanomolar doses of DHP resulted in increased drd2 mRNA levels, probably related to negative feedback on LH release. A decrease in drd2 levels at the micromolar range of DHP (concomitant with increased gnrhr3 and fshb mRNA levels) may be related to the recruitment of a new generation of oocytes. Exposure to DHP also resulted in increased lhb mRNA levels toward final oocyte maturation. Salmon GnRH analog (sGnRHa) increased mRNA levels of gnrh1and gnrh3; when combined with DHP, sGnRHa synergistically increased expression of gnrh3 only. These results emphasize the role of sex steroids on positive and negative feedbacks controlling the reproductive cycle.

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

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

Gonadotropin-Releasing Hormone (GnRH) Receptors in Tumors: a New Rationale for the Therapeutical Application of GnRH Analogs in Cancer Patients?

Gonadotropin-Releasing Hormone (GnRH) is the hypothalamic decapeptide which plays a key role in the control of reproductive functions. By binding to specific receptors present on the pituitary gonadotropes, GnRH regulates gonadotropin release and, consequently, steroid hormone secretion from the gonads. When given continuously and at high doses, GnRH agonists suppress the pituitary gonadal axis through the down-regulation and desensitization of its own receptors. Based on this rationale, pituitary GnRH receptors represent the target for the successful utilization of GnRH agonists (that are more stable than the native peptide) for the treatment of hormone-dependent tumors (e.g., prostate, breast, endometrial, ovarian cancers). The observation that GnRH receptors are expressed in steroid-dependent tumors, and that their activation reduces cell proliferation and metastatic behavior of cancer cell lines, both in vitro and in vivo (when inoculated into nude mice), indicates a possible additional and more direct antitumor activity for these compounds. Interestingly, GnRH receptors have been shown to be expressed also in androgen-independent prostate carcinoma, as well as in tumors that are not classically considered hormone-related (e.g., melanoma), suggesting a clinical utility of the administration of GnRH analogs also in these tumors. More recently, GnRH agonists have been proposed as useful carriers to target cytotoxic drugs or toxins to cancer cells displaying the specific GnRH receptors. A second form of GnRH (designated GnRH-II) has been discovered in most vertebrates, including humans. GnRH-II has been suggested to act through a 'putative' cognate type II GnRH receptor, which is distributed in different tissues, both normal and tumoral. In humans, a full-length functional type II GnRH receptor has not been found. Therefore, its functions as well as its possible utility as a molecular target for a GnRH-II based therapy in oncology still has to be clarified. This review will focus on the role of GnRH receptors in the control of tumor growth, progression and dissemination. It will also be discussed whether the presence of these receptors might represent an additional rationale for the clinical utility of GnRH analogs as anticancer drugs.

Differential social regulation of two pituitary gonadotropin-releasing hormone receptors

In many vertebrates, social interactions regulate reproductive capacity by altering the activity of the hypothalamic–pituitary–gonadal (HPG) axis. To better understand the mechanisms underlying social regulation of reproduction, we investigated the relationship between social status and one main component of the HPG axis: expression levels of gonadotropin-releasing hormone receptor (GnRH-R). Social interactions dictate reproductive capacity in the cichlid fish Astatotilapia burtoni. Reproductively active territory holders suppress the HPG axis of non-territorial males through repeated aggressive encounters. To determine whether the expression of GnRH-R is socially regulated, we quantified mRNA levels of two GnRH-R variants in the pituitaries and brains of territorial (T) and non-territorial (NT) A. burtoni males. We found that T males had significantly higher levels of pituitary GnRH-R1 mRNA than NT males. In contrast, GnRH-R2 mRNA levels in the pituitary did not vary with social status. Pituitaries from both T and NT males expressed significantly higher mRNA levels of GnRH-R1 than GnRH-R2. GnRH mRNA levels in the brain correlated positively with GnRH-R1 mRNA levels in the pituitary but did not correlate with pituitary GnRH-R2. Measurements of GnRH-R1 and GnRH-R2 mRNA levels across the whole brain revealed no social status differences. These results show that, in addition to the known effects of social status on other levels of the HPG axis, GnRH receptor in the pituitary is also a target of social regulation.

New Peptidic GnRH Antagonists Offer a Broad Range of Therapeutic Applications

Luteinizing hormone-releasing hormone (GnRH) antagonists competitively block pituitary GnRH receptors, thus leading to an immediate decrease of the circulating levels of follicle stimulating hormone (FSH) and luteinizing hormone (LH) and consequently of sex steroids. They are clinically used when pharmacologically induced hormone suppression is desired, e.g. in sex steroid dependent benign and malignant diseases and for prevention of premature LH surges in assisted reproduction. The article reviews the clinical experience with the well established GnRH antagonists cetrorelix, ganirelix and the approved, but recently withdrawn abarelix. Clinical studies with the new depot formulations of cetrorelix, teverelix, degarelix and D-63153 are summarized. Keywords: GnRH antagonists, cancer, sex-steroid dependent disease, in vitro fertilization, cetrorelix, review

Antagonistes de la GnRH: présent et futur

Antagonists act by competitive inhibition of pituitary GnRH receptors for which they have a high affinity with a dose-dependent activity. The inhibition concerns LH but also FSH by inhibiting their secretion for 24 hours. At this time, only three molecules are available. Cetrorelix is mainly used for in vitro fertilization protocols, but research is currently being carried out in benign prostatic hypertrophy. Ganirelix is only used in medically assisted procreation. Abarelix is used in the United States as a monthly “depot” injection for the treatment of prostate cancer resistant to other therapies. Other molecules including orally active antagonists are in the course of clinical evaluation or in preclinical phases. The development of this therapeutic class is hampered by conflicting industrial interests and also by problems of tolerability in particular because of the side effects due to induced histamine release. The current indications in France are therefore limited to Assisted Reproductive Technology, and recently, prostate cancer pending comparative studies versus agonists. The indications under development are endometriosis, myoma and precocious puberty, fields in which agonists have already been found to be effective. Male contraception seems to have a future however this therapeutic regimen remains very expensive. Finally, in benign prostatic hypertrophy, the antagonists administrated intermittently may be used to rapidly improve urinary flow.

Les résistances à la GnRH et le gène GRP54

Current data make it possible to relate idiopathic hypogonadotrophic hypogonadism to mutations affecting the GnRH I-1 receptor and also to new “loss-of-function” mutations concerning another receptor, GPR54. It now seems that mutations of the pituitary GnRH receptor are not the only explanation of most cases of sporadic isolated hypogonadotrophic hypogonadism, and, on the contrary, there are certain familial forms, where no mutation has been demonstrated, suggesting the potential involvement of other genes. The role of another G protein-coupled glycoprotein membrane receptor, GRP54, already known for its involvement as a metastasis suppressor, has been demonstrated. Bioclinical studies of families affected with the disorder by pheno/genotypic correlation demonstrated that “loss-of-function” mutations affecting the GPR54 gene coding for GPR54 are the cause of hypogonadotrophic hypogonadism. GPR54 therefore appears to be involved at hypothalamic and pituitary level. It is not involved in sexual differentiation, but may modulate GnRH secretion or affect its pituitary response. Further investigations are required to determine the levels of action of this receptor which may provide a new pharmacological target in the future.

Pituitary gonadotropin-releasing hormone (GnRH) receptor: Structure, distribution and regulation of expression

Reproduction in mammals is controlled by interactions between the hypothalamus, anterior pituitary and gonads. Interaction of GnRH with its cognate receptor is essential to regulating reproduction. Characterization of the structure, distribution and expression of GnRH receptors (GnRH-R) has furthered our understanding of the physiological consequences of GnRH stimulation of pituitary gonadotropes. Based on the putative topology of the amino acid sequence of the GnRH-R and point mutation studies, key elements of the GnRH-R have been identified to play a role in ligand recognition and binding, G-protein activation and internalization. Normally, reproductive function is mediated by GnRH-R expressed only on the membranes of pituitary gonadotropes. The density of GnRH-R on gonadotropes determines their ability to respond to GnRH. This density is highest just prior to ovulation and likely is important for complete expression of the pre-ovulatory surge of LH. Therefore, knowledge regarding what regulates the density of GnRH-R is essential to understanding changes in pituitary sensitivity to GnRH and ultimately, to expression of the LH surge. Regulation of GnRH-R gene expression is influenced by a multitude of factors including gonadal steroid hormones, inhibin, activin and perhaps most importantly GnRH itself.

Differential Gonadotropin-Releasing Hormone (GnRH) and GnRH Receptor Messenger Ribonucleic Acid Expression Patterns in Different Tissues of the Female Rat across the Estrous Cycle

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

Cloning and Characterization of a Functional Type II Gonadotropin-Releasing Hormone Receptor with a Lengthy Carboxy-Terminal Tail from an Ancestral Vertebrate, the Sea Lamprey

A full-length transcript encoding a functional type II GnRH receptor was cloned from the pituitary of the sea lamprey, Petromyzon marinus. The current study is the first to identify a pituitary GnRH receptor transcript in an agnathan, which is the oldest vertebrate lineage. The cloned receptor retains the conserved structural features and amino acid motifs of other known GnRH receptors and notably includes a C-terminal intracellular tail of approximately 120 amino acids, the longest C-terminal tail of any vertebrate GnRH receptor identified to date. The lamprey GnRH receptor was shown to activate the inositol phosphate (IP) signaling system; stimulation with either lamprey GnRH-I or lamprey GnRH-III led to dose-dependent responses in transiently transfected COS7 cells. Furthermore, analyses of serially truncated lamprey GnRH receptor mutants indicate perturbations of the C-terminal tail disrupts IP accumulation, however, the tailless lamprey GnRH receptor was not only functional but was also capable of stimulating IP levels equal to wild type. Expression of the receptor transcript was demonstrated in the pituitary and testes using RT-PCR, whereas in situ hybridization showed expression and localization of the transcript in the proximal pars distalis of the pituitary. The phylogenetic placement and structural and functional features of this GnRH receptor suggest that it is representative of an ancestral GnRH receptor. In addition to having an important role in lamprey reproductive processes, the extensive C-terminal tail of this lamprey GnRH receptor may have great significance for understanding the evolutionary change of this vital structural feature within the GnRH receptor family.

Oestrogen-selective modulation of FSH and LH secretion by pituitary gland

Sir, We read the interesting article by Forward et al (2004) on the clinical and endocrine data for goserelin plus anastrozole in premenopausal women with advanced breast cancer. The authors presented interesting data regarding serum gonadotropin (FSH and LH) levels and androgen (testosterone, DHEAS and androstenedione) levels in 13 patients treated with the GnRH agonist, goserelin, plus the selective oestrogen receptor modulator, tamoxifen, followed by goserelin plus the aromatase inhibitor, anastrozole. The authors found that goserelin and tamoxifen led to a 90% fall in FSH levels down to 1.0 IU/L at 6 months (P<0.001). Once the therapy was changed to goserelin and anastrozole, there was a significant rise in the mean FSH level to 7.8 IU/l at 12 months (P<0.0001). Introduction of goserelin and tamoxifen resulted in an 89% reduction in mean LH levels (pretreatment vs 6 months=19.9 vs 0.3 IU/l; P=0.01), but with substitution of tamoxifen by anastrozole, no significant change in LH levels occurred. With regard to androgen levels, the authors found a significant fall in testosterone and androstenedione, but not DHEAS, levels in association with substitution of tamoxifen by anastrozole. In their explanation for these observations, the authors suggested that the rise in FSH was due to release of a negative feedback as a result of further reduction in oestradiol levels upon the introduction of an aromatase inhibitor, or a rebound phenomenon from coming off tamoxifen. Regarding the significant decline in androgen levels (testosterone and androstenedione), contrary to what is expected (a rise due to potential substrate accumulation secondary to inhibition of the aromatase enzyme), the authors provided little in the way of discussion. We would like to propose a possible mechanism for the endocrine changes found in this study. Regarding the differential changes in gonadotropins (rise in FSH levels while LH levels remained suppressed) with substitution of tamoxifen by the aromatase inhibitor, anastrozole, we believe that involvement of the activin/inhibin/follistatin system could explain these changes. Accumulating evidence suggests that oestrogen can modulate gonadotropin production through a direct effect on pituitary cells, independent of GnRH. This effect is thought to be mediated through the activin/inhibin/follistatin system (DePaolo, 1997; Nett et al, 2002; Welt et al, 2002; McNeilly et al, 2003). Activins are produced by a wide variety of tissues including the pituitary gland, and are specifically by gonadotropes (Roberts et al, 1989). Activins stimulate synthesis of FSH by a direct action on the gonadotropes (Mason et al, 1989). Follistatin, also produced by the pituitary gland, is an activin-binding protein and may decrease FSH synthesis by sequestering activin (Mather et al, 1993). Oestrogen was found to selectively suppress FSH production but not LH through an increase in follistatin/inhibin and a decrease in activin, independent of GnRH (Shupnik and Rosenzweig, 1991; DiGregorio and Nett, 1995; Baratta et al, 2001), mainly through activin suppression (Baratta et al, 2001). Testosterone has been found to have the opposite effect of oestrogen on pituitary FSH (but not LH) production, that is, an increase in FSH by the gonadotropes. This effect was found to be mediated through locally produced activins (Burger et al, 2004). Potentially, testosterone may accumulate at the tissue level as a result of aromatase inhibition even in the absence of measurable changes in the serum. Such locally accumulated testosterone could exert a stimulatory effect on FSH production by the anterior pituitary through an activin-mediated effect. The reason why a more suppressive effect on FSH production occurred with the antioestrogen tamoxifen is probably related to its selective oestrogen receptor modulation effect, known to exert antioestrogenic effects at the level of the hypothalamus, but oestrogenic effects at the level of the pituitary gland (Gonzalez et al, 2000). The significant decline in testosterone and androstenedione, but not DHEAS, in the face of aromatase inhibition is much harder to rationalise. It is possible that inhibition of oestrogen locally in the hypothalamus and pituitary by the aromatase inhibitor, together with GnRH receptor downregulation by goserelin, resulted in profound combined suppression of LH levels, especially at the level of bioactivity, which is not totally reflected in the immunoassay results. It is important to mention that the evidence presented above came mainly from animal studies. However, if we can extrapolate from the available animal data, it is pertinent here to point out another potential mechanism for aromatase inhibitors to enhance the efficacy of controlled ovarian hyperstimulation (COH) protocols, as we have recently reported (Mitwally and Casper, 2002, 2003, 2004). The ability of the aromatase inhibitors to increase endogenous FSH production by the anterior pituitary, even with pituitary GnRH receptor downregulation (as in the present publication by Forward et al), is an exciting approach to improve the outcome of ovarian stimulation particularly in poor responders in in vitro fertilisation protocols. We believe this area of aromatase inhibitor use warrants further investigation.

Regulation of FSHβ and GnRH receptor gene expression in activin receptor II knockout male mice

To examine in vivo, the local effects of inhibins and activins within the anterior pituitary, independent of their endocrine effects exerted from the gonad, in mediating FSH homeostasis, we used castrated knockout mice lacking either inhibin α or activin receptor II (ACVR2) alone or in combination. Compared to castrated wild-type (WT) mice, FSHβ mRNA levels in the pituitaries of Acvr2 null mice were significantly downregulated in the absence of gonadal feedback. FSHβ mRNA levels were not significantly higher in the pituitaries of castrated inhibin α null mice compared to those in Acvr2 null mice and remained the same in the pituitaries of castrated double mutant mice lacking both inhibin and ACVR2. In contrast to FSHβ mRNA expression changes, pituitary FSH content was significantly reduced in Acvr2 null mice whereas it was only slightly upregulated in inhibin α null mice. Combined absence of both ACVR2 signaling and inhibins caused a decrease in FSH content compared to that in the absence of inhibins alone. These changes in pituitary content were in parallel to those in serum FSH levels in these three groups of castrated mice, suggesting that the unopposed actions of locally produced inhibins are dominant over those effects mediated by ACVR2 signaling to regulate FSH biosynthesis and secretion. Thus, our in vivo results demonstrate that within the pituitary, locally produced activins and inhibins exert their actions at distinct phases of FSH homeostasis. In an independent set of experiments, we tested whether in vivo signaling via ACVR2 is necessary for hypothalamic GnRH biosynthesis and for GnRH receptor expression. Our results demonstrate that in contrast to previous in vitro studies, signaling through ACVR2 is neither required for hypothalamic synthesis of GnRH peptide nor for expression of GnRH receptors in the anterior pituitary. We conclude that within the hypothalamic–pituitary short loop, ACVR2 signaling is critical only for FSH homeostasis and not for GnRH biosynthesis or induction of pituitary GnRH receptor expression. Our studies confirm the importance of using in vivo genetic models for studying regulation of the hypothalamic–pituitary–gonadal axis.