Early preantral follicles of the domestic cat express gonadotropin and sex steroid signaling potential.

Key biomolecular processes, which regulate primordial ovarian follicle dormancy and early folliculogenesis in mammalian ovaries, are not fully understood. The domestic cat is a useful model to study ovarian folliculogenesis and is the most relevant for developing in vitro growth methods to be implemented in wild felid conservation breeding programs. Previously, RNA-sequencing of primordial (PrF), primary (PF), and secondary follicle (SF) samples from domestic cat implicated ovarian steroidogenesis and steroid reception during follicle development. Here, we aimed to identify which sex steroid biosynthesis and metabolism enzymes, gonadotropin receptors, and sex steroid receptors are present and may be potential regulators. Differential gene expression, functional annotation, and enrichment analyses were employed and protein localization was studied too. Gene transcripts for PGR, PGRMC1, AR (steroid receptors), CYP11A1, CYP17A1, HSD17B1 and HSD17B17 (steroidogenic enzymes), and STS (steroid metabolizing enzyme) were significantly differentially expressed (Q values of ≤0.05). Differential gene expression increased in all transcripts during follicle transitions apart from AR which decreased by the secondary stage. Immunohistochemistry localized FSHR and LHCGR to oocytes at each stage. PGRMC1 immunostaining was strongest in granulosa cells, whereas AR was strongest in oocytes throughout each stage. Protein signals for steroidogenic enzymes were only detectable in SFs. Products of these significantly differentially expressed genes may regulate domestic cat preantral folliculogenesis. In vitro growth could be optimized as all early follicles express gonadotropin and steroid receptors meaning hormone interaction and response may be possible. Protein expression analyses of early SFs supported its potential for producing sex steroids.

Pharmacological characterization, localization and quantification of expression of gonadotropin receptors in Atlantic salmon (Salmo salar L.) ovaries.

The gonadotropins Fsh and Lh interact with their receptors (Fshr and Lhr, respectively) in a highly specific manner in mammals with little overlap in biological activities. In fish, the biological activities seem less clearly separated considering, for example, the steroidogenic potency of both Fsh and Lh. Important determinants of the biological activity are the specificity of hormone-receptor interaction and the cellular site of receptor expression. Here, we report the pharmacological characterization of Atlantic salmon Fshr and Lhr, identify receptor-expressing cells in the ovary, and validate receptor mRNA quantification systems. For the pharmacological studies, we used highly purified coho salmon gonadotropins and found that the Fshr preferentially responded to Fsh, but was also activated by approximately 6-fold higher levels of Lh. The Lhr was specific for Lh and did not respond to Fsh. Photoperiod manipulation was used to generate ovarian tissue samples with largely differing stages of maturation. Specific real-time, quantitative (rtq) PCR assays revealed up to 40-fold (fshr) and up to 350-fold (lhr) changes in ovarian expression levels, which correlated well with the differences in ovarian weight, histology, and circulating oestrogen levels recorded in January and June, respectively. Vitellogenic ovaries were used to localise receptor-expressing cells by in situ hybridization. Granulosa cells of small and large vitellogenic follicles were positive for both receptors. Also theca cells of small and large vitellogenic follicles expressed fshr mRNA, while only in large vitellogenic follicles theca cells were (weakly) positive for lhr mRNA. While only ovulatory Lh levels seem high enough to cross-activate the Fshr, expression by both receptors by granulosa and theca cells suggests that homologous ligand receptor interaction will prevail.

Leydig cells express follicle-stimulating hormone receptors in African catfish.

This report aimed to establish, using African catfish, Clarias gariepinus, as model species, a basis for understanding a well-known, although not yet clarified, feature of male fish reproductive physiology: the strong steroidogenic activity of FSHs. Assays with gonadotropin receptor-expressing cell lines showed that FSH activated its cognate receptor (FSHR) with an at least 1000-fold lower EC50 than when challenging the LH receptor (LHR), whereas LH stimulated both receptors with similar EC50s. In androgen release bioassays, FSH elicited a significant response at lower concentrations than those required to cross-activate of the LHR, indicating that FSH stimulated steroid release via FSHR-dependent mechanisms. LHR/FSHR-mediated stimulation of androgen release was completely abolished by H-89, a specific protein kinase A inhibitor, pointing to the cAMP/protein kinase A pathway as the main route for both LH- and FSH-stimulated steroid release. Localization studies showed that intratubular Sertoli cells express FSHR mRNA, whereas, as reported for the first time in a vertebrate, catfish Leydig cells express both LHR and FSHR mRNA. Testicular FSHR and LHR mRNA expression increased gradually during pubertal development. FSHR, but not LHR, transcript levels continued to rise between completion of the first wave of spermatogenesis at about 7 months and full maturity at about 12 months of age, which was associated with a previously recorded approximately 3-fold increase in the steroid production capacity per unit testis weight. Taken together, our data strongly suggest that the steroidogenic potency of FSH can be explained by its direct trophic action on FSHR-expressing Leydig cells.

Altura final em criancas com puberdade precoce central: a experiência de um serviço de referência / Final Height in Children with Central Precocious Puberty the experience of a Specialized Center The Experience of a Specialized Center

Introdução: A puberdade precoce central ocorre principalmente devido a ativação precoce do eixo hipotalâmico-hipofisário-gonadal e conseqüentemente ao aumento do hormônios gonadotróficos. A prematura ativação desse eixo não envolve apenas mudanças físicas precoces da puberdade, mas também aceleração do crescimento linear e aceleração da maturação óssea, que leva a fusão das epífises ósseas de maneira prematura e à diminuição da altura final. Objetivo: Identificar a altura final de pacientes que apresentaram Puberdade Precoce Central atendidos no Serviço de Endocrinologia Pediátrica do Hospital Infantil Joana de Gusmão. Métodos: Foram avaliados os registros de pacientes que haviam atingido a AF no período de 1997-2007. As variáveis analisadas foram: sexo, idade cronológica, idade óssea, idade ao diagnóstico, idade ao atingir a altura final, tempo de tratamento até altura final, tempo de acompanhamento até a altura final, tratamento utilizado, altura no início e término do tratamento, altura predita pelo método de Bayley – Pinneau, altura-alvo e altura final ( transformada em escore z). Resultados: Foram incluídos 56 pacientes, 96,4 % do sexo feminino e 90,75 % dos pacientes apresentavam PPC idiopática. Os pacientes masculinos foram tratados com análogo do hormônio liberador de gonadotrofinas por 2,7 anos em média, enquanto que as pacientes femininas foram tratadas durante 3,1 anos. A altura final foi alcançada aos 15,1 anos nos meninos e 14,2 anos nas meninas.Conclusões: A média de altura final foi 171,25 cm no sexo masculino e 160,77 cm no sexo feminino. O escore-z de AF foi de -0,55 desvios padrão da média nos meninos e 0,04 desvios padrão da média nas meninas. A diferença entre altura final e altura alvo foi de -5,25 cm nos meninos e 2,4 cm nas meninas.

Background: Central precocious puberty is mainly due to the precocious activation of hypothalamic-pituitary-gonadal axis leading to an increase of gonadotropic hormones. The premature activation of this axis it involves not only early physical changes of puberty, but also linear growth acceleration and acceleration of bone maturation, which leads to early epiphyseal fusion and short adult height. Objective: To identify final height in central precocious puberty patients treated at Pediatric Endocrinology Service of Hospital Infantil Joana de Gusmão. Methods: The study evaluated the registration of patients that had reached the final height between 1997-2007. Data included sex, chronological age, bone age, age at diagnosis, age at final height, duration of treatment, duration of accompaniment from the start of treatment to final height, treatment used, height at the start and at the end of treatment, predicted height by Bayley – Pinneau method, target height and final height (these are transformed in z-score). Results: Fifty six patients were involved. 96,4 % were female sex and 90,75 % had idiopathic central precocious puberty. The males were treated with Gonadotropin Releasing Hormone Analogue by 2,7 years and females were treated by 3,1 years. Final height was reached at 15,1 years in boys and 14,2 years in girls. Conclusions: Final height average was 171,25 cm in males and 160,77 cm in females. The z-score of final height was -0,55 standard deviation of average in boys and 0,04 standard deviation of average in girls. The difference between final height and target height were -5,25 cm in boys and 2,4 cm in girls.

Molecular characterization of two sea bass gonadotropin receptors: cDNA cloning, expression analysis, and functional activity.

The follicle-stimulating hormone (FSH) and the luteinizing hormone (LH) play central roles in vertebrate reproduction. They act through their cognate receptors to stimulate testicular and ovarian functions. The present study reports the cloning and characterization of two sea bass (Dicentrarchus labrax) cDNAs encoding a FSH receptor (sbsFSHR) and a LH receptor (sbsLHR). The mature proteins display typical features of the glycoprotein hormone receptor family members, but the sbsFSHR also contains some remarkable differences when compared with other fish or mammalian FSHRs. Among them, a distinct extracellular N-terminal cysteine domain as regards to its length and cysteine number, and the presence of an extra leucine-rich repeat. Expression analysis revealed that the sbsFSHR is exclusively expressed in gonadal tissues, specifically in the follicular wall of previtellogenic and early-vitellogenic follicles. On the contrary, sbsLHR mRNA was found to be widely distributed in sea bass somatic tissues. When stably expressed in mammalian cell lines, sbsFSHR was specifically stimulated by bovine FSH, while sbsLHR was activated by both bovine LH and FSH. Nevertheless, specific stimulation of the sbsLHR was observed when recombinant sea bass gonadotropins were used. The isolation of a FSHR and a LHR in sea bass opens new ways to study gonadotropin action in this species.

Gonadotropins and ovarian cancer.

Ovarian epithelial cancer (OEC) accounts for 90% of all ovarian cancers and is the leading cause of death from gynecological cancers in North America and Europe. Despite its clinical significance, the factors that regulate the development and progression of ovarian cancer are among the least understood of all major human malignancies. The two gonadotropins, FSH and LH, are key regulators of ovarian cell functions, and the potential role of gonadotropins in the pathogenesis of ovarian cancer is suggested. Ovarian carcinomas have been found to express specific receptors for gonadotropins. The presence of gonadotropins in ovarian tumor fluid suggests the importance of these factors in the transformation and progression of ovarian cancers as well as being prognostic indicators. Functionally, there is evidence showing a direct action of gonadotropins on ovarian tumor cell growth. This review summarizes the key findings and recent advances in our understanding of these peptide hormones in ovarian cancer development and progression and their role in potential future cancer therapy. We will first discuss the supporting evidence and controversies in the "gonadotropin theory" and the use of animal models for exploring the involvement of gonadotropins in the etiology of ovarian cancer. The role of gonadotropins in regulating the proliferation, survival, and metastasis of OEC is next summarized. Relevant data from ovarian surface epithelium, which is widely believed to be the precursor of OEC, are also described. Finally, we will discuss the clinical applications of gonadotropins in ovarian cancer and the recent progress in drug development.

Hormonal therapy in ovarian cancer.

Ovarian carcinoma continues to be the leading cause of death due to gynecological malignancy. Epidemiologic studies indicate that steroid hormones play roles in ovarian carcinogenesis. Gonadotropins, estrogen, and androgen may be causative factors, while gonadotropin-releasing hormone and progesterone may be protective factors in ovarian cancer pathogenesis. Experimental studies have shown that hormonal receptors are expressed in ovarian cancer cells and mediate the growth-stimulatory or growth-inhibitory effects of the hormones on these cells. Hormonal therapeutic agents have been evaluated in several clinical trials. Most of these trials were conducted in patients with recurrent or refractory ovarian cancer, with modest efficacy and few side effects. Better understanding of the mechanisms through which hormones affect cell growth may improve the efficacy of hormonal therapy. Molecular markers that can reliably predict major clinical outcomes should be investigated further in well-designed trials.

Mutations along the pituitary-gonadal axis affecting sexual maturation: novel information from transgenic and knockout mice.

During the last 10 years, numerous activating and inactivating mutations have been detected in the genes encoding the two gonadotrophins, luteinising hormone (LH) and follicle-stimulating hormone (FSH), as well as their cognate receptors (R), LHR and FSHR. Because activation of the hypothalamic-pituitary-gonadal axis is a crucial event in the onset and progression of puberty, mutations affecting gonadotrophin action have major influence on this developmental process. Many of the phenotypic effects observed have been expected on the basis of the existing information about gonadotrophin action (e.g. delayed puberty), but also many unexpected findings have been made, including the lack of phenotype in women with activating LHR mutations, and the discrepancy in phenotypes of men with inactivating mutations of FSHbeta (azoospermia and infertility) and FSHR (oligozoospermia and subfertility). Some of the possible mutations, such as inactivating LHbeta and activating FSHR mutations in women, have not yet been detected. Genetically modified mice provide relevant phenocopies for the human mutations and serve as good models for studies on molecular pathogenesis of these conditions. They may also predict phenotypes of the mutations that have not yet been detected in humans. We review here briefly the effects of gonadotrophin subunit and receptor mutations on puberty in humans and contrast the information with findings on genetically modified mice with similar mutations.

Zebrafish as a model for vertebrate reproduction: characterization of the first functional zebrafish (Danio rerio) gonadotropin receptor.

Vertebrate reproduction is tightly regulated by conserved glycoprotein hormones produced by the pituitary gland. Follicle-stimulating hormone (FSH) in tetrapods and gonadotropic hormone I (GTH-I) in fishes are orthologous glycoprotein hormones that control the timing of egg production and the number of eggs produced. Zebrafish, a well-established genetic model for developmental biology, also offers potential advantages for studies of reproductive toxicology, especially for modeling the impact of pollutants on fish reproductive processes. To facilitate these studies we have identified, expressed, and characterized the zebrafish GTH-I receptor. This receptor (zfGTHR-I)exhibits strong sequence similarity to the tetrapod FSH receptors and to GTHR-I from salmon and catfish. Human 293 cells transfected with zfGTHR-I exhibit increased cAMP levels after treatment with carp pituitary extracts or human FSH, but not when treated with a ligand to a related receptor (human chorionic gonadotropin). Northern blotting and RT-PCR analyses indicate that zfGTHR is expressed in ovaries from sexually mature fish, but not in immature fish. Several alternative splice variants of the receptor affecting putative exons 2-4 that encode dramatically shortened receptor fragments lacking the transmembrane domain as well as regions previously implicated in ligand binding were identified by RT-PCR. The zfGTHR-I sequence opens the way to study effects of genetic mutations or chemicals on ovarian zfGTHR-I expression and function in zebrafish.

Does full-term pregnancy at a young age protect women against breast cancer through hCG?

Recent studies found that human and animal breast tissues and human breast cell lines contain low levels of receptors that bind hCG and its structural and functional homologue, LH. Those gonadotropins exert numerous anticancer effects in breast cancer models and cells, which might explain decreased breast cancer incidence in women who complete full-term pregnancies at a young age. The new findings also imply that premature chronic elevations of LH levels might contribute to decreased breast cancer incidence in women with early menopause, and elevated LH levels might contribute to a better prognosis after ovariectomy. Those findings predict that breast cancer risk might be reduced by early hCG treatment of women who plan to delay their first pregnancies; prophylactic hCG treatment might help women with family histories of breast cancer or oncogene mutations that predict breast cancer; and better prognoses might result when hCG is administered to breast cancer tissue.

Abnormal melatonin secretion in male patients with hypogonadism.

Recently we have demonstrated that melatonin secretion is increased in untreated male patients with GnRH deficiency. Testosterone administration to these patients decreased melatonin secretion to normal levels. These data, however, did not exclude a gonadotropic effect on melatonin secretion. To further elucidate whether gonadal steroids and/or gonadotropins modulate melatonin secretion in humans we compared untreated young males with hypogonadotropic hypogonadism (IGD, n = 6), and hypergonadotropic hypogonadism caused by KlinEfelter's syndrome (KS, n = 11) to normal pubertal male controls (n = 7). KS patients were subdivided into two groups: KS-1, with low testosterone; and KS-2, with normal testosterone levels. Serum samples for melatonin concentrations were obtained every 15 min from 7 PM to 7 AM in a controlled light-dark environment with simultaneous sleep recordings. All KS patients had elevated gonadotropin levels and decreased melatonin levels. Mean (+/- SD) dark-time nocturnal melatonin levels in KS-1 were 92 +/- 21 pmol/L and were 146 +/- 46 pmol/L in KS-2 compared with 178 +/- 64 pmol/L in controls. Integrated nocturnal melatonin secretion values (AUC) were 64 +/- 14 pmol/min x L x 10(3) in KS-1 and 96 +/- 29 pmol/min x L x 10(3) in KS-2 compared with 116 +/- 42 pmol/min x L x 10(3) in controls. All IGD patients had low gonadotropin and testosterone levels. Their dark-time melatonin levels (286 +/- 26 pmol/L) and the AUC values (184 +/- 15 pmol/min/L x 10(3)) were increased. These data indicate that melatonin secretion is increased in male patients with GnRH deficiency and decreased in low testosterone hypergonadotropic hypogonadal patients. Taken together, our results suggest that both gonadotropins and gonadal steroids modulate melatonin secretion in humans.

Agonist activity of mammalian gonadotropin-releasing antagonists in chicken gonadotropes reflects marked differences in vertebrate gonadotropin-releasing receptors.

The pharmacology of mammalian and avian gonadotropin-releasing (GnRH) receptors differs for agonist analogues. We have therefore compared the activities of mammalian-based GnRH antagonists in sheep and chicken gonadotropes to further elucidate the different structural requirements of the receptors. The antagonist activities of ten GnRH analogues were compared in cultured sheep and chicken pituitary cells by determining the dose required to cause a 50% inhibition of luteinizing hormone secretion (IC50) induced by GnRH at its half-maximal concentration (EC50). Nine analogues showed high antagonist activity in the sheep bioassay. Analogue IC50s varied between half and twice ((1.22-6.06) x 10(-10) M) the GnRH EC50 (3 x 10(-10) M). One of these peptides exhibited partial agonist activity. In contrast, eight of the analogues showed low antagonist activity in chicken pituitary cells, with IC50s varying from 46 to 1490 times ((1.4-44.7) x 10(-7) M) the GnRH EC50 (3 x 10(-9) M) and had a different order of potencies compared with that in the sheep. Furthermore, two analogues did not display antagonist activity at all in the chicken bioassay, but acted as pure agonists, stimulating LH secretion. These findings demonstrate marked differences in pharmacology between the avian and mammalian pituitary GnRH receptors and emphasize that GnRH antagonists, selected for their efficacy in mammals, cannot necessarily be used for physiological studies in non-mammalian vertebrates. The distinctly different pharmacology of the receptors and structural requirements of analogues for agonist/antagonist activity establish a basis for identifying receptor features involved in ligand-induced signal propagation using chimaeras of cloned sheep and chicken receptors.

Regulation of gonadotropin receptor gene expression.

The receptors for the gonadotropins differ from the other G protein-coupled receptors by having a large extracellular hormone-binding domain, encoded by nine or ten exons. Alternative splicing of the large pre-mRNA of approximately 100 kb can result in mRNA species that encode truncated receptor proteins. In this review we discuss the regulation of gonadotropin receptor mRNA expression and the possible roles of alternative splicing in gonadotropin receptor function.

[Gonadotropin receptor system and its signal transduction pathway].

Recent advances in genetic studies have elucidated structure of gonadotropin receptors. This transmembrane receptor contains seven transmembrane domains and induces multiple biological changes in granulosa/theca cells resulting in follicular maturation in women. The signal transduction involves G protein mediated systems, cAMP mediated systems and phospholipase C systems. This paper reviews recent advances in gonadotropin receptor system and its signal transduction pathway.

Phospholipase D treatment enhances gonadotrophin receptor-coupled adenylate cyclase activity in isolated bovine luteal cells.

LH-stimulated adenylate cyclase activity in membrane preparations of bovine luteal cells could be enhanced by treating the cells with either phospholipase D or its hydrolysis product, phosphatidic acid. Similar augmentary effects were also produced following treatment of the cells with EGF. Moreover, EGF could stimulate the formation of [3H]phosphatidic acid in [3H]myristic acid preloaded cells, suggesting that EGF is able to activate cellular phospholipase D. Also, PMA was able to increase the phosphatidic acid formation with a parallel increase in the adenylate cyclase activity. We propose, therefore, that phosphatidic acid may act as an intracellular second messenger linking EGF-mediated activation of phospholipase D with the sensitization of LH receptor-coupled adenylate cyclase signalling system.

Significance of the glycan moiety of the rat ovarian luteinizing hormone/chorionic gonadotropin (CG) receptor and human CG for receptor-hormone interaction.

The role of the glycan moiety of the rat ovarian LH/CG receptor and human CG (hCG) in high-affinity receptor-hormone interaction was investigated by cross-linking and quantitative binding experiments. hCG and its derivatives, desialylated hCG and deglycosylated hCG were labeled either to the alpha-subunit (125I) or the beta-subunit (3H). The ligands were attached to ovarian membrane particles, which were treated with neuraminidase or peptide-N-glycosidase F to remove terminal sialic acids or N-linked oligosaccharides of the receptor, respectively, and the complexes formed were solubilized, cross-linked with glutaraldehyde, and analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. All of the ligands produced similar autoradiographic patterns with the native or glycosidase-treated receptor, and only the receptor-(alpha)hCG and receptor-(alpha, beta)hCG complexes were detected. Moreover, quantitative binding studies indicated that all of the hormone derivatives had similar affinities for the native or glycosidase-treated receptor. In addition, the orientation of the carbohydrate side chains on the receptor-hormone complex was studied by digesting the complex with the glycosidases. The molecular weight of the receptor, evidenced by ligand blotting, was reduced to the same extent, whether the membrane-bound free receptor or receptor-hormone complex was treated with the glycosidases, suggesting that the oligosaccharide side chains of the receptor are apart from the hormone binding region. As peptide-N-glycosidase F treatment reduced the size of the Mr 90,000 receptor first to about Mr 67,000 and finally to about Mr 62,000, there may possibly be 2 N-linked carbohydrate chains per receptor polypeptide. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis of the glycosidase-treated receptor-[125I]hCG complex also revealed that neuraminidase was able to remove the sialic acids from both subunits of the receptor-bound hormone. In conclusion, the results suggest that hCG interacts with the polypeptide backbone of its ovarian receptor mainly through the peptide core of its alpha-subunit. Moreover, the carbohydrate side chains of both subunits of hCG are positioned on the outward face of the receptor-hormone complex.

Ontogeny of gonadotrophin receptors and gonadotrophin-stimulated cyclic AMP production in the neonatal rat ovary.

The sequence of appearance of FSH and LH receptors, and response of cyclic AMP (cAMP) production to these hormones and cholera toxin, were studied in the fetal and neonatal rat ovary. Specific binding of radiolabelled human (h)FSH and chorionic gonadotrophin (CG) to ovarian homogenates was first detectable on day 7 of life. The content of FSH receptors per ovary increased tenfold between days 7 and 16, and that of LH receptors 27-fold. A significant response of cAMP production in vitro to FSH appeared on day 4 of life, but no significant effect of hCG on cAMP was achieved until day 7. In contrast, cholera toxin had a marked effect on cAMP production by day 17 of fetal life. Although both FSH and LH receptors were detectable in the neonatal rat ovary by day 7, the present findings indicate that the FSH responsiveness of the ovary appears earlier than that of LH. The post-receptor machinery of cAMP production is already functional in the fetal ovary as shown by the experiments with cholera toxin. The appearance of the receptor may therefore be the last link in the ontogeny of the gonadotrophin signal transduction system in the ovary. To study the hormone dependence of the appearance of gonadotrophin responsiveness, neonatal female rats were treated on days 1-6 or 1-9 of life with a potent gonadotrophin-releasing hormone antagonist, and killed on the following day. In both treatment groups, the pituitary LH and FSH contents were suppressed.(ABSTRACT TRUNCATED AT 250 WORDS)

Hypogonadotropic hypogonadism in severe beta-thalassemia: effect of chelation and pulsatile gonadotropin-releasing hormone therapy.

We studied pituitary-gonadal function in 11 male and 5 female patients, aged 12-30 yr, with severe beta-thalassemia and chronic iron overload. All had normal basal serum cortisol, T4, and PRL concentrations and normal serum cortisol and GH responses to insulin-induced hypoglycemia and TSH responses to TRH. Of the 11 male patients (all over 17 yr of age), only 3 attained full pubertal development and 4 had subnormal serum LH and FSH responses to GnRH. As a group, their mean basal serum testosterone (T) level was low [11.7 +/- 4.9 (+/- SE) nmol/L; normal, 10-40 nmol/L], and 9 of the 11 male patients responded to hCG with a rise in serum T. Two of the 3 female patients over 17 yr of age were prepubertal with undetectable serum estradiol (E2) levels and absent serum LH and FSH responses to GnRH; the other female patient had regular menstrual cycles and normal serum E2 levels and LH and FSH responses to GnRH. Six of the prepubertal patients (4 males and 2 females, aged 17-30 yr) were studied serially for 3 yr after the start of chelation therapy. Despite a fall of median serum ferritin from 11,910 to 1,303 pmol/L, there was no progression of puberty, and their basal and GnRH-stimulated serum LH and FSH and serum T or E2 levels did not change. Three of these patients (1 male and 2 female) then received pulsatile sc GnRH therapy in addition to chelation therapy for 6 months with no improvement. We conclude that chronic iron overload in patients with severe thalassemia leads to variable degrees of hypogonadotropic hypogonadism, which do not respond to chelation therapy given late in the course of the disease. The hypogonadism in most patients was due to pituitary hyporesponsiveness to GnRH.