The gonadotroph origin of null cell adenomas.

OBJECTIVE: The term "null cell" adenoma was first proposed in 1980 to designate pituitary adenomas lacking clinical, biochemical and morphological markers to disclose their cell origin. DESIGN: The aim of this study was to investigate the presence of α- and ß-gonadotropin subunits in clinically nonfunctioning pituitary tumors, which were initially immunonegative and thus diagnosed as null cell adenomas. For this reason, we reapplied immunohistochemistry using a more sensitive method comprising a tyramide signal amplification technique, combined with a polymer antibody immunohistochemical detection system. RESULTS: With this approach, all these previously negative tumors became positive for α- and ß-gonadotropin hormone subunits. CONCLUSIONS: Our results prove that so-called "null cell" adenomas produce α-SU or/and ß-FSH or ß-LH and therefore are gonadotrph adenomas in origin.

Proteomic analyses of recombinant human follicle-stimulating hormone and urinary-derived gonadotropin preparations.

OBJECTIVE: To understand the properties of each available gonadotropin preparation, especially in terms of the differences between urinary-derived and recombinant preparations. STUDY DESIGN: Human menopausal gonadotropin (hMG), highly purified urinary-derived follicle-stimulating hormone (uFSH-HP) and recombinant FSH (rFSH) were subjected to 2-dimensional gel electrophoresis (2-DE), and protein spots were visualized by silver-staining procedures. Major spots were analyzed by mass spectrometry. Fluorescent-labeled preparations were also subjected to 2-DE to evaluate the quantities of FSH isohormones contained in each preparation. RESULTS: 2-DE and mass spectrometry analyses of hMG identified many extracellular proteins as major impurities and several plasma membrane proteins including prion proteins. Both uFSH-HP and rFSH demonstrated slight impurities and showed several alpha and beta subunit isohormones. rFSH contained higher amounts of the basic isohormones of the alpha subunit than uFSH-HP, whereas the predominance of the basic isohormones was less significant in the beta subunit. CONCLUSION: Proteomic analyses demonstrated the detailed protein profiles of each preparation. Differences in the quantities of alpha subunit isohormones may contribute to the variations in FSH activity observed between recombinant and urinary-derived FSH preparations.

Gene expression and secretion of LH and FSH in relation to gene expression of GnRH receptors in the brushtail possum (Trichosurus vulpecula) demonstrates highly conserved mechanisms.

In eutherian mammals, the gonadotrophins (LH and FSH) are synthesized and stored in gonadotroph cells under the regulation of multiple mechanisms including GnRH. Very little is known about the regulation of gonadotrophin secretion and storage in pituitary glands of marsupials. This study revealed, using quantitative PCR and heterologous RIA techniques, that LHB mRNA expression levels remained constant over the oestrous cycle, regardless of the presence of a preovulatory LH surge, which is characteristic of a hormone secreted under regulation. Our sampling regime was unable to detect pulses of LH during the follicular phase, although GNRHR mRNA levels had increased at this time. Pulses of LH were, however, detected in the luteal phase of cycling females, in anoestrus females and in males. There was a positive correlation between gene expression of FSHB and plasma levels of FSH at different stages of the oestrous cycle and no pulses of FSH were detected at any time; all characteristics of a hormone secreted via the constitutive pathway. Using in situ hybridisation and immunohistochemistry methods, we determined that mRNA expression of LHB and FSHB, and protein storage of gonadotrophins exhibited a similar pattern of localisation within the pituitary gland. Additionally, sexual dimorphism of gonadotroph populations was evident. In summary, these findings are similar to that reported in eutherians and considering that marsupial evolution diverged from eutherians over 100 million years ago suggests that the regulation of gonadotrophins is highly conserved indeed.

Presence of beta-follicle-stimulating hormone and beta-luteinizing hormone transcripts in the brain of Cichlasoma dimerus (Perciformes: Cichlidae): effect of brain-derived gonadotropins on pituitary hormone release.

Follicle-stimulating hormone (FSH) and luteinizing hormone (LH) play key roles in vertebrate gametogenesis and steroidogenesis. They are mainly synthesized in the pituitary gland. While investigating the ontogeny of FSH and LH cells in the cichlid fish Cichlasoma dimerus by immunohistochemistry (IHC), we unexpectedly found immunoreactive neurons in the preoptic area, sending their projections through different brain areas and neurohypophysis. Our previous work using Western blot and IHC techniques applied to the adult brain confirmed these findings. To further demonstrate the extrapituitary expression of these hormones, we performed RT-PCR detecting sequences coding for beta-FSH and beta-LH subunits in the C. dimerus pituitary and brain (preoptic-hypothalamic area). The expression of these transcripts in both organs was consistent with their peptide expression showing a high sequence homology when compared with other phylogenetically related fish. An individual pituitary in vitro culture system was utilized to study the possible modulatory effect of brain-derived gonadotropins on pituitary hormone secretion. Pituitary explants were cultured with different concentrations of LH or FSH, and the culture media were analyzed by Western blot. Exogenous LH produced a dose-dependent increase in pituitary beta-LH, beta-FSH and somatolactin (SL) releases. No effect was observed on growth hormone (GH). The effect on prolactin (PRL) was not consistent among treatments. Exogenous FSH produced an inhibition in beta-LH release, dose-dependent increases in beta-FSH and SL releases, and no effect on PRL and GH releases. These findings support the concept of regulation of pituitary trophic hormones by brain-derived gonadotropins.

Development of real-time PCR assays in the study of gonadotropin subunits, follistatin and prolactin genes expression in the porcine anterior pituitary during the preovulatory period.

OBJECTIVES: Neural control of the anterior pituitary function consists of the interplay of neuropeptides action, gonadal steroid hormones and many other factors. The physiological effect of this regulatory action is the release and synthesis of protein hormones in the precise time and quantity. The main factor responsible for the gonadotropins release and synthesis is the gonadotropin-releasing hormone (GnRH). We must still study the modulation of the synthesis of the gonadotropins subunits - LHbeta, FSHbeta and alpha subunit by different forms of GnRH and by its analogs, in order to better understand the regulation of gonadotropin release and synthesis. THE AIM of this study was to develop real-time PCR assays of five candidate reference genes for normalization purposes in order to quantify target transcripts in anterior pituitary cells during the preovulatory period. Moreover, we focused on the influence of GnRH receptor antagonist (antide) treatment on mRNA expression levels of GPalpha, LHbeta, FSHbeta, FST(follistatin) and PRL(prolactin) genes in these cells. MATERIAL AND METHODS: Anterior pituitary cells were obtained from pituitary glands of four mature pigs at the preovulatory phase. Cells were incubated with or without antide and relative mRNA level of target genes was measured using the Applied Biosystems 7500 Real Time System. For an exact comparison of mRNA quantity, the stability of five reference genes, ACTB, B2M, GAPDH, RPL1, and TOP2B was evaluated to choose the most appropriate reference gene for qRT-PCR normalization in the pituitary cells. Expression stability of reference genes was calculated using the geNorm application. The developed method of PCR assay was applied to study gene expression in pig pituitary cells in short culture. RESULTS: The most stably expressed genes in the pituitary cells were GAPDH and TOP2B. The expression of ACTB, B2M and RPL1 appeared to be highly unstable. After normalization to the GAPDH/TOP2B, results showed that the mRNA expression of the FSHbeta gene was highest in comparison with LHbeta, GPalpha, FST and PRL genes (p<0.005). Pre-treatment of cells by the antide resulted in lower mRNA expression of these genes, while FSHbeta mRNA had a significantly lower expression (p<0.05) in comparison with control. CONCLUSIONS: Real-time PCR analysis of the expression of LHbeta, FSHbeta, alpha subunit, follistatin and prolactin genes in porcine anterior pituitary cells during the preovulatory period is suitable for the study of modulatory action of metal complexes with GnRH on the expression of these genes.

Menarquia extraordinariamente precoz como signo inicial de una pubertad precoz central idiopática en una niña de 15 meses / Extraordinarily early menarche as an initial sign of central idiopathic precocious puberty in a 15 month old child

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Assembly and structural characterization of an authentic complex between human follicle stimulating hormone and a hormone-binding ectodomain of its receptor.

Follicle stimulating hormone (FSH) is secreted from the pituitary gland to regulate reproduction in vertebrates. FSH signals through a G-protein coupled receptor (FSHR) on the target cell surface. We describe here the strategy to produce a soluble FSH-FSHR complex that involves the co-secretion of a truncated FSHR ectodomain (FSHR(HB)) and a covalently linked FSHalphabeta heterodimer from baculovirus-infected insect cells. FSH binds to FSHR(HB) with a high affinity comparable to that for the full-length receptor. The crystal structure of the FSH-FSHR(HB) complex provides explanations for the high affinity and specificity of FSH interaction with FSHR, and it shows an unexpected dimerization of these complexes. Here we also compare the crystal structure with theoretical models of the FSH-FSHR-binding mode. We conclude that the FSH-FSHR(HB) structure gives an authentic representation of FSH binding to intact FSHR.

All-or-none N-glycosylation in primate follicle-stimulating hormone beta-subunits.

Human FSH exists as two major glycoforms designated, tetra-glycosylated and di-glycosylated hFSH. The former possesses both alpha- and beta-subunit carbohydrates while the latter possesses only alpha-subunit carbohydrate. Western blotting differentiated the glycosylated, 24,000 M(r) hFSHbeta band from the non-glycosylated 21,000 M(r) FSHbeta band. Postmenopausal urinary hFSH preparations possessed 75-95% 24,000 M(r) hFSHbeta, while pituitary hFSH immunopurified from 21- to 43-year-old females and 21-43-year-old males possessed only 35-40% 24,000 M(r) hFSHbeta. The pituitary hFSH from a postmenopausal woman on estrogen replacement was 75% 21,000 M(r) hFSHbeta. Other immunopurified postmenopausal pituitary hFSH preparations possessed 50-60% 21,000 M(r) hFSHbeta. Gel filtration removed predominantly 21,000 M(r) free hFSHbeta and reduced its abundance to 13-22% in postmenopausal pituitary hFSH heterodimer preparations. A major regulatory mechanism for FSH glycosylation involves control of beta-subunit N-glycosylation, possibly by inhibition of oligosaccharyl transferase. Two primate species exhibited the same all-or-none pattern of pituitary FSHbeta glycosylation.

Continued improvements in the quality and consistency of follitropin alfa, recombinant human FSH.

The use of gonadotrophins for the treatment of infertility began in the 1930s following early work on the pituitary-ovarian axis and the discovery of FSH and LH. The technological development of pharmaceutical gonadotrophins over the last 40 years has shown improvements in specific activity, purity, degradation and impurities. Throughout these pharmaceutical developments the gonadotrophin content of both urinary and recombinant preparations has been assessed using an animal in-vivo bioassay. This paper reflects upon the manufacturing history of recombinant human FSH (r-hFSH) and follitropin alfa filled by mass (FbM), and evaluates the impact of introducing a pharmaceutical product that is formulated and assayed by a physicochemical method for r-hFSH protein content. It also compares the analytical consistency of follitropin alfa FbM with another commercially available r-hFSH, follitropin beta.

Molecular and expression characterization of three gonadotropin subunits common alpha, FSHbeta and LHbeta in groupers.

A SMART cDNA plasmid library was constructed from protogyous greasy grouper (Epinephelus coioides) pituitary, and the full-length cDNAs of three gonadotropin (GTH) subunits common alpha, FSHbeta and LHbeta were cloned and sequenced from the library. The nucleotide sequences of common alpha, FSHbeta and LHbeta subunit cDNAs are 647, 594 and 574 bp in length, and encode for mature peptides of 94, 99 and 115 aa, respectively. High homology was observed by amino acid sequence alignment and identity comparison of the grouper mature peptides of common alpha, FSHbeta and LHbeta with that of other fishes. Phylogenetic tree analyses of the three GTH mature subunits revealed similar phylogeny relationships among the studied fish species. Three polyclonal antibodies were prepared from the in vitro expressed common alpha, FSHbeta and LHbeta mature proteins, respectively. Western blot analysis and immunofluoresence localization were performed on two typical stages of ovarian development stages in red-spotted grouper. Significant differences in protein expression levels of three gonadotropin subunits were revealed between the two ovarian development stages. In the individuals with resting ovary, common alpha was almost not detected in pituitaries, and FSHbeta and LHbeta expression levels were very low. While in the individuals with developing ovary, the expression of all three gonadotropin subunits reached to a high level. Immunofluoresence localization indicated that the grouper FSHbeta cells mainly distributed in the middle area of PPD, while the LHbeta cells distributed more widely, including in the area similar to the FSHbeta cells and at the external periphery of pituitary near to the PI side. The common alpha might be expressed in both FSHbeta and LHbeta cells. Double immunofluoresence localization further demonstrated FSHbeta and LHbeta expression in distinct cells in the PPD area, although the FSHbeta and LHbeta cells were detected in the identical area of PPD.

Pituitary expression of LHbeta- and FSHbeta-subunit mRNA, cellular distribution of LHbeta-subunit mRNA and LH and FSH synthesis during and after treatment with a gonadotrophin-releasing hormone agonist in heifers.

The aim was to examine transcriptional and post-transcriptional regulation of LH and FSH biosynthesis. Female cattle were allocated to three groups: (i) Group 1, control (n = 6), synchronized to be at around Day 11 of the oestrous cycle on Day 31; (ii) Group 2 (n = 6), treated with gonadotrophin-releasing hormone (GnRH) agonist (deslorelin) for 31 days; and (iii) Group 3 (n = 6), treated with deslorelin for 28 days. All animals were slaughtered on Day 31. For animals in Group 2, pituitary content of LHbeta-subunit mRNA was suppressed 60% (P < 0.001) and LH 95% (P < 0.001), whereas FSHbeta-subunit mRNA was suppressed 25% (P > 0.05) and FSH 90% (P < 0.001). Three days after treatment with deslorelin (Group 3) LHbeta-subunit mRNA and LH remained suppressed (50% and 95%, respectively; P < 0.001). At the same time, FSHbeta-subunit mRNA did not differ from controls (P > 0.05) whereas FSH remained reduced by 80% (P < 0.001). The ratio of LHbeta-subunit mRNA present in the nucleus versus cytoplasm of gonadotroph cells was reduced (P < 0.05) in heifers during treatment with deslorelin (0.59 +/- 0.05) compared with the ratio in control heifers (1.31 +/- 0.22) and heifers 3 days after discontinuation of treatment (1.01 +/- 0.05). The findings indicated that treatment with GnRH agonist can suppress LHbeta-subunit mRNA expression without any significant effect on FSHbeta-subunit mRNA. As LH and FSH contents were suppressed to a greater degree than their beta-subunit mRNAs, it would appear that treatment with a GnRH agonist might influence gonadotrophin biosynthesis by a post-transcriptional mechanism(s). For LHbeta-subunit mRNA, this would appear not to be reduced export of message from the nucleus.

[Two cases of non-functional gonadotroph adenoma pituitary apoplexy following GnRH-agonist treatment revealing gonadotroph adenoma and pseudopituitary apoplexy after GnRH administration]. / Deux cas de macroadénomes gonadotropes silencieux: apoplexie hypophysaire au décours d'une première injection d'agoniste de la LH-RH révélant un adénome gonadotrope. Pseudoapoplexie hypophysaire d'un adénome gonadotrope au décours d'un test de stimulation.

We report here two cases of pituitary apoplexy or pseudoapoplexy revealing a gonadotroph adenoma. A 69-year-old man, who had just started antiandrogen treatment (Gn-RH agonist) for prostatic cancer, was admitted to neurosurgery emergency because of increasing headache and visual impairment. The CT-scan disclosed the presence of a large pituitary mass with lateral invasion of the left cavernous sinus. Hormonel testing showed panhypopituitarism. A few days later, diabetes insipidus appeared. The patient first received corticosteroid therapy and underwent surgical adenomectomy. Immunostaining of the tumor tissue was positive for FSHbeta, confirming the diagnosis of gonadotroph adenoma. Three months after surgery, the endocrine evaluation showed pituitary insufficiency. An 81-year-old man complained of mnemonic disorders. The CT-scan revealed a pituitary mass without extension. The Ophthalmological examination showed left temporal upper quadranopsia. Endocrinological tests with administration of GN-HR triggered headache and vomiting. A second CT-scan was unchanged. Hormone testing revealed increased serum levels of FSH and decreased serum levels of LH. Surgical management of the primary tumor was undertaken due to the visual field alteration. Immunohistochemical studies confirmed the diagnosis of gonadotroph FSHbeta adenoma.

Successful pregnancy after bromocriptine therapy in an anovulatory woman complicated with ovarian hyperstimulation caused by follicle-stimulating hormone-producing plurihormonal pituitary microadenoma.

Gonadotropin-producing pituitary adenomas are extremely rare in reproductive-age women. We report here a case of gonadotroph microadenoma with ovarian hyperstimulation. It was found in a 29-yr-old infertile Japanese woman with enlarged multicystic ovaries. The patient had an elevated basal serum estradiol level (up to 6755 pM, or 1840 pg/ml). Serum FSH and prolactin were mildly elevated (15.4 IU/liter, 1.4 nM or 31.4 ng/ml), whereas LH was low (0.5 IU/liter). The FSH level was paradoxically elevated in response to TRH administration. Dynamic magnetic resonance imaging revealed a pituitary microadenoma. Daily administration of bromocriptine, a dopamine agonist, normalized the ovarian size, and the patient ovulated naturally. She conceived after 3 months of bromocriptine therapy and delivered a normal child. She underwent elective transsphenoidal pituitary surgery, 3 yr after the delivery. Immunostaining of the resected tumor showed that 80% and less than 5% of the tumor cells stained for FSH-beta and prolactin, respectively. Furthermore, RT-PCR suggested that dopamine type 2 receptor was expressed in the adenoma. Gonadotroph microadenoma should be considered in women with spontaneous ovarian hyperstimulation, even if they have no neurological symptoms or marked pituitary enlargement. In such cases, bromocriptine therapy may be an alternative to pituitary surgery.

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

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

Androgen receptors in gonadotrophs in pituitary cultures from adult male monkeys and rats.

There is substantial evidence demonstrating that the principal feedback action of androgens to decrease LH secretion in male primates, including man, is to slow the GnRH pulse generator, whereas in male rats androgens not only decrease GnRH but also suppress LH synthesis and secretion through a direct pituitary effect. Previous experiments in our laboratory revealed that testosterone (T) suppresses LH secretion and decreases alpha-subunit mRNA levels in male rat pituitary cell cultures perifused with pulses of GnRH but not in pituitary cells from adult male monkeys. In the present study, we sought to determine whether the lack of responsiveness of gonadotrophs to androgens in the primate is androgen receptor (AR) related. Primary cultures were prepared from the anterior pituitary glands of adult male monkeys and rats. Cells were identified as gonadotrophs if they were immunoreactive for LH-beta or FSH-beta. Of these cells in the monkey, 80% contained both gonadotropins, 17% contained only LH-beta, and 3% contained only FSH-beta. AR immunoreactivity (IR) was nuclear in 22% and 15%, respectively, of monkey and rat FSH-beta-positive cells in the absence of T. Following T treatment, nuclear AR IR was identified in 79% of monkey and 81% of rat gonadotrophs. T treatment similarly intensified AR IR in mouse gonadotroph alphaT3-1 and LbetaT2 cells and in monkey and rat fibroblasts. Single-cell RT-PCR confirmed coexpression of LH-beta and AR mRNA as well as LH-beta and GH mRNA in monkey gonadotrophs. Our data reveal that most monkey, as well as rat, gonadotrophs are AR-positive with nuclear localization in the presence of T. GH expression is not required for AR expression in gonadotrophs. We conclude that the failure of T to inhibit LH secretion and decrease alpha-subunit mRNA expression in the male primate is not due a disturbance in AR nuclear shuttling.

Cushing's disease after surgical resection and radiation therapy for nonfunctioning pituitary adenoma.

OBJECTIVE: To describe a patient with an aggressive nonfunctioning pituitary adenoma in whom Cushing's disease developed after two resections of tumor and radiation therapy. METHODS: We present a case report, with serial laboratory and immunohistochemical data, and summarize information about similar patients described in the medical literature. RESULTS: A 48-year-old woman had irregular menstrual periods, decreased peripheral vision, headaches, and weight gain. Laboratory and radiologic investigation revealed a large nonfunctioning pituitary adenoma. Transsphenoidal subtotal resection of the tumor improved her vision. Results of immunohistochemical studies were positive for b-follicle-stimulating hormone and b-luteinizing hormone. She had radiation therapy 1 year postoperatively for rapidly enlarging residual tumor. Bifrontal craniotomy was done 3 months later because of worsening vision. The pituitary adenoma from the second surgical procedure stained negatively for all pituitary hormones. Postoperatively, she received tapering doses of prednisone for 4 months. Two months after the last dose of prednisone, she had signs and symptoms of hypercortisolism. Inferior petrosal sinus venous sampling studies for plasma corticotropin confirmed the presence of Cushing's disease. She did not tolerate medical therapy. Bilateral adrenalectomy led to remission of hypercortisolism. CONCLUSION: Nonfunctioning pituitary tumors often come to clinical attention when they are large and cause symptoms associated with hypopituitarism or invasion of parasellar structures. In contrast, functioning pituitary tumors may have few compressive symptoms if they manifest with complaints attributable to excessive pituitary hormones.